The development of multicellular organisms requires the generation of complex three-dimensional structures from simple sheets of cells. Formation of the mammalian brain involves dynamic changes in cell shape and organization that transform an epithelial sheet into the closed neural tube that is essential for the development of the brain and spinal cord (

Wilde et al., 2014

;

Juriloff and Harris, 2018

). Disruptions in neural tube closure are among the most common causes of birth defects, affecting around 1 in 2,000 births, representing a significant challenge in human health (

Wallingford et al., 2013

;

Zaganjor et al., 2016

). Neural tube closure requires the integration of multiple cell behaviors, including apical constriction, cell division, and cell rearrangements, which promote epithelial reorganization, bending, and fusion (

Nikolopoulou et al., 2017

;

Vijayraghavan and Davidson, 2017

;

Matsuda and Sokol, 2021

). These processes are driven by spatiotemporally regulated actomyosin networks that are predicted to alter the physical environment experienced by individual cells and create mechanical stresses that could disrupt cell adhesion and epithelial integrity. However, the mechanical forces that drive mammalian neural tube closure, and how cells maintain tissue integrity in the presence of these forces, are not understood.

Biophysical studies have identified a number of force-induced changes in protein conformation and activity that could allow cells to maintain cell adhesion and tissue structure under tension. Mechanical forces promote structural changes in E-cadherin, α-catenin, and filamentous actin (F-actin) that are predicted to stabilize protein interactions within junctional complexes and strengthen connections between adherens junctions and the actin cytoskeleton (

Yonemura et al., 2010

;

Rakshit et al., 2012

;

Buckley et al., 2014

;

Huang et al., 2017

;

Sun et al., 2020

). In addition, mechanical forces modulate interactions between subunits of cell-matrix adhesions (

Burridge and Guilluy, 2016

) and epithelial tight junctions (

Spadaro et al., 2017

;

Citi 2019

;

Citi et al., 2024

) that could provide additional strategies to reinforce cell-matrix anchoring and epithelial barrier function. Studies of epithelial remodeling

in vivo

have identified tricellular junctions—where three cells meet at a single point or vertex—as sites of increased tension that engage a variety of force responses in epithelial cells (

Higashi and Miller, 2017

;

Bosveld et al., 2018

). Tricellular adherens junctions recruit several proteins that are required to stabilize cell adhesion under tension, both in culture and in epithelial tissues

in vivo

(

le Duc et al., 2010

;

Sawyer et al., 2011

;

Thomas et al., 2013

;

Choi et al., 2016

;

Razzell et al., 2018

;

Rauskolb et al., 2019

;

Yu and Zallen, 2020

;

Perez-Vale et al., 2021

;

Cavanaugh et al., 2022

;

Sheppard et al., 2023

). In addition, components of tricellular tight junctions are required to maintain epithelial barrier function under tension in cultured cells and in the

Xenopus

embryo (

Oda et al., 2014

;

Stephenson et al., 2019

;

Cho et al., 2022

;

van den Goor et al., 2024

). However, how epithelial cells maintain cell adhesion at tricellular junctions in the presence of the physiological forces required to drive epithelial remodeling during mammalian embryogenesis is poorly understood, in part due to the challenges of directly visualizing dynamic processes in mutant embryos.

An excellent candidate for mediating force responses in mammalian tissues is the junction-actin linker protein Vinculin. Vinculin has been shown to be recruited by mechanical forces to cell-matrix adhesions, adherens junctions, and tight junctions across a broad array of cell types (

Goldmann, 2016

;

Bays and DeMali, 2017

;

Citi, 2019

), and is required to reinforce connections between cell adhesion complexes and the actin cytoskeleton in response to mechanical forces

in vitro

(

Maddugoda et al., 2007

;

Peng et al., 2010

;

le Duc et al., 2010

;

Yonemura et al., 2010

;

Taguchi et al., 2011

;

Huveneers et al., 2012

;

Peng et al., 2012

;

Huang et al., 2017

). In addition to linking junctions to actin, Vinculin also regulates myosin II activity and actin organization at adherens junctions through interactions with actin regulatory proteins (

Wen et al., 2009

;

Le Clainche et al., 2010

;

le Duc et al., 2010

;

Leerberg et al., 2014

;

Ito et al., 2017

). Vinculin has essential

in vivo

roles in regulating heart development and function (

Xu et al., 1998

;

Zemljic-Harpf et al., 2007

;

Cheng et al., 2016

;

Fukuda et al., 2019

), vascular remodeling (

Carvalho et al., 2019

;

Kotini et al., 2022

), stem cell proliferation (

Biswas et al., 2021

;

Bohere et al, 2022

), and epithelial and endothelial barrier function (

Higashi et al., 2016

;

van der Stoel et al., 2023

;

van den Goor et al., 2024

;

Landino et al., 2025

). Furthermore, mouse embryos lacking

Vinculin

display a fully penetrant failure of cranial neural tube closure (

Xu et al., 1998

;

Marg et al., 2010

) and mutations in human Vinculin are associated with increased risk of neural tube defects (

Wang et al., 2021

). However, the mechanisms by which Vinculin regulates mammalian neural tube closure, and how Vinculin influences junctional organization and cell behavior during this process, are unknown.

Here we used a high-throughput embryonic stem cell-based method to efficiently generate large numbers of mutant embryos in order to analyze the role of Vinculin during mouse cranial neural tube closure. Using biophysical and live imaging approaches, we show that mechanical forces in the developing midbrain are anisotropic and increase as neural tube closure proceeds. Mechanical forces are generated correctly in

Vinculin

mutants, but neuroepithelial cells fail to maintain cell adhesion at high-tension junctions as forces increase during neural fold elevation, resulting in a failure of cranial neural tube closure. The defects in

Vinculin

mutants were most pronounced at adherens junctions, whereas tight junctions remained largely intact, and resulted in a progressive disruption of cell adhesion, cell division, and apical constriction behaviors during cranial neural tube closure. These results demonstrate that Vinculin is necessary to stabilize cell adhesion in the presence of endogenous forces in the mouse cranial neural plate and suggest that this function is necessary to convert mechanical forces into changes in cranial neural tube structure.

Results

Mechanical forces increase during cranial neural fold elevation in the developing mouse midbrain

Formation of the mouse cranial neural tube requires the sequential elevation, apposition, and fusion of the cranial neural folds on either side of the midline (

Figure 1A

). The spatiotemporal forces that drive these changes, and how neuroepithelial integrity is maintained in the presence of these forces, are not well understood. To address these questions, we analyzed the spatiotemporal distribution of forces during neural fold elevation in the developing mouse midbrain. The active form of the myosin II regulatory light chain (phosphorylated MRLC or pMRLC) is enriched at interfaces parallel to the mediolateral axis during midbrain elevation, suggesting that forces are spatially regulated during closure (

McGreevy et al., 2015

;

Brooks et al., 2020

;

Bogart and Brooks, 2025

). We found that pMRLC planar polarity was present in late, but not early, stages of neural fold elevation, suggesting that these forces may also be temporally regulated (

Figure 1B-1D

,

Figure 1—figure supplement 1A

). To investigate this possibility, we performed laser ablation experiments to measure relative contractile forces in the lateral midbrain, using myosin IIB-GFP to target individual cell edges for ablation (

Bao et al., 2007

). The peak recoil velocity in response to laser ablation is estimated to be proportional to the tension acting on the edge immediately prior to ablation (

Hutson et al., 2003

;

Farhadifar et al., 2007

). We found that peak recoil velocities were significantly higher at edges aligned with the mediolateral axis than at edges aligned with the anterior-posterior axis (

Figure 1E-L

, see

Supplementary File 1

for a summary of all n values and statistical analyses), consistent with the effects of larger, tissue-scale ablations (

De La O et al., 2025

). The peak recoil velocities at anterior-posterior and mediolateral junctions were both significantly higher in late elevation compared to similarly oriented edges in early elevation (

Figure 1E-L

). Treatment of embryos expressing the adherens junction marker GFP-Plekha7 (

Shioi et al., 2017

) with the Rho-kinase inhibitor Y-27632 to reduce actomyosin contractility eliminated the response to ablation, indicating that these forces require myosin II activity (

Figure 1—figure supplement 1B-D

). Together, these results demonstrate that mechanical forces at cell-cell junctions are planar polarized in the mouse cranial neural plate and significantly increase during neural fold elevation.

Using embryonic stem cell-derived embryos to study neural tube closure

These results raised the question of how epithelial integrity is maintained as mechanical forces increase during midbrain neural fold elevation. We hypothesized that active mechanisms could be required to maintain cell and tissue structure in the presence of the forces required for cranial closure. Identifying key components of these processes requires direct live imaging of cell behavior in mutant embryos lacking critical force-response mechanisms. However, studying dynamic behaviors in mutant mouse embryos is challenging, as failure of cranial neural tube closure typically results in embryonic lethality, limiting the number of mutant embryos that can be obtained through genetic crossing strategies. To circumvent this limitation, we took advantage of established methods for generating mouse lines derived from mouse embryonic stem cells (ESCs) (

Bradley et al., 1984

;

Beddington and Robertson, 1989

;

Nagy et al., 1990

;

Nagy et al., 1993

;

Hadjantonakis et al., 1998

;

Nagy and Rossant, 2001

;

Tam and Rossant, 2003

). Injection of genetically modified mouse ESCs at the blastocyst stage (embryonic day 3.5, or E3.5) or aggregation with morula stage embryos are conventional methods for producing chimeric animals for generating stable mouse lines. By contrast, injection of mouse ESCs into host embryos at E2.5 (8-cell to morula stages) tends to produce embryos and animals in which nearly all epiblast lineages are derived from the injected ESCs, which can be used for immediate analysis (

Lallemand and Brulet, 1990

;

Yagi et al., 1993

;

Poueymirou et al., 2007

;

Kiyonari et al., 2010

;

Ukai et al., 2017

). Therefore, we sought to apply this system to generate embryos homozygous for lethal mutations to study the mechanisms of cranial neural tube closure.

To evaluate the reproducibility of this method for routine production of large numbers of genetically modified embryos with strong contributions from the injected ESCs, we performed a series of control experiments. First, GFP-negative parental ESCs (HK3i) (

Kiyonari et al., 2010

) were injected into E2.5 host embryos expressing Histone-H2B-GFP from the

ROSA26 (R26)

locus (

Abe et al., 2011

). Injected embryos were transferred to surrogate females and recovered for analysis at E8.5-9.5 to assess the contribution of the injected ESCs to the developing embryo during cranial closure (

Figure 2A

). As expected, the majority of embryos obtained with this method (90%) were derived nearly entirely from the GFP-negative ESCs in the embryonic lineages (

Figure 2B

). Host-derived cells expressing Histone-H2B-GFP were primarily observed in the gut tube, which retains a small number of extraembryonic cells at this stage (

Kwon et al., 2008

). These results confirm that mouse ESC injections provide an efficient strategy for generating embryos in which the embryonic lineages are nearly fully derived from the injected ESCs.

To validate the use of this method for studies of cranial neural tube closure, we tested whether ESC-derived embryos reproduce the phenotypes of known closure mutants. Using CRISPR/Cas9 genome editing (

Mulas et al., 2019

), we generated ESCs homozygous for a null mutation in

Shroom3

(

Figure 2—figure supplement 1A-C

, Materials and methods), which encodes an actin-binding protein that is essential for cranial neural tube closure in mouse, frog, and chick embryos (

Hildebrand and Soriano, 1999

;

Haigo et al., 2003

;

Nishimura and Takeichi, 2008

;

Massarwa and Niswander, 2013

;

McGreevy et al., 2015

). The use of ESC injections to generate

Shroom3

^ESC


embryos produced an average of 6

Shroom3

^ESC


embryos per surrogate female, a significantly increased yield of mutant embryos compared to heterozygote crosses. Embryos generated from unedited parental ESCs or from ESCs mutant for the

tyrosinase

coat color gene, referred to collectively as Control

^ESC

embryos, completed cranial closure normally, indicating that the parental ESC line and the ESC embryo generation protocol do not interfere with wild-type closure (

Figure 2C

). By contrast,

Shroom3

^ESC


embryos displayed a complete failure of cranial closure, resulting in fully penetrant exencephaly (79/79

Shroom3

^ESC


embryos compared to 0/41 Control

^ESC

embryos). In addition,

Shroom3

^ESC


embryos showed significantly reduced apical constriction (

Figure 2D-F

), a Shroom3-dependent cell behavior that is essential for neural fold elevation (

Haigo et al., 2003

;

McGreevy et al., 2015

;

Brooks et al., 2020

;

Baldwin et al., 2022

). These results demonstrate that ESC-derived embryos recapitulate the cell behaviors and tissue-level closure defects characteristic of

Shroom3

mutants.

Next, we tested if this ESC injection approach could be used to generate embryos for live imaging. To visualize cell junctions, we derived ESCs from

R26-PHA7-EGFP

embryos that constitutively express the adherens junction marker GFP-Plekha7 from the

R26

locus (

Shioi et al., 2017

) and the established ESCs were injected into GFP-negative host embryos (Materials and methods). Imaging of the recovered embryos revealed widespread expression of GFP-Plekha7 throughout the cranial neural plate and confirmed that GFP-Plekha7 displayed the expected colocalization with N-cadherin (

Figure 2—figure supplement 1D

and E). A subset of embryos went on to produced viable and fertile adults that displayed germline transmission (

Figure 2—figure supplement 1F

). Together, these results demonstrate the utility of this method for generating numerically robust cohorts of genetically modified embryos for studying cellular and molecular mechanisms of cranial neural tube closure.

Vinculin is required for cranial neural fold elevation

To determine how epithelial cells maintain cell adhesion and tissue integrity during cranial closure, we used this ESC-derived embryo method to generate embryos lacking the actin-binding protein Vinculin. Vinculin is recruited by mechanical forces to a variety of structures in cells, including adherens junctions, tight junctions, and cell-matrix adhesions (

Goldmann, 2016

;

Bays and DeMali, 2017

;

Citi, 2019

). Disruption of Vinculin expression or actin-binding activity leads to impaired cranial closure in the mouse embryo (

Xu et al., 1998

;

Marg et al., 2010

); however, how Vinculin influences neural tube closure is unknown. To elucidate the role of Vinculin in cranial closure, we used CRISPR/Cas9 gene editing to generate ESCs lacking Vinculin (

Figure 3—figure supplement 1A

and B). Homozygous

Vinculin

mutant ESCs were injected into 8-cell stage host embryos to generate

Vinculin

^ESC


embryos that lack Vinculin in the embryonic lineages, producing an average of 8 mutant embryos per surrogate female. In addition, ESCs heterozygous for a similar

Vinculin

deletion were injected to produce functionally wild-type Control

^ESC

embryos that retain Vinculin protein (

Figure 3—figure supplement 1C

). To test if these ESC-derived embryos reproduce the phenotypes of

Vinculin

mutants, we compared

Vinculin

^ESC


embryos to conditional mutants lacking

Vinculin

in the embryonic lineages generated by combining a conditional

Vinculin

^flox


allele (

Zemljic-Harpf et al., 2007

) with an epiblast-specific Sox2-Cre driver (

Hayashi et al., 2002

), hereafter referred to as

Vinculin

^ΔEpi


embryos.

Vinculin

^ESC


and

Vinculin

^ΔEpi


embryos displayed fully penetrant exencephaly at embryonic day E9.5, were slightly smaller than Control

^ESC

or littermate controls, and exhibited fully penetrant lethality by E10.5 (

Figure 3—figure supplement 1D

and E), recapitulating several known phenotypes of

Vinculin

mutants (

Xu et al., 1998

;

Marg et al., 2010

). Therefore, we used both

Vinculin

^ESC


and

Vinculin

^ΔEpi


embryos to study the effects of disrupting Vinculin activity in embryonic lineages on cranial neural tube closure.

To determine how Vinculin influences neural tube closure, we first examined when cranial neural plate defects arise in

Vinculin

^ESC


embryos. The midbrain neural folds elevate between E8.0 and E8.5, resulting in a decrease in the ratio of the apical span to the basal span of the tissue (

Figure 3A-C

). Neural fold elevation initiated normally in

Vinculin


^ESC

embryos, but the neural folds failed to progress to late elevation and apposition, resulting in an arrest of neural tube closure (

Figure 3A-C

). The cell behaviors that drive neural tube closure require apical-basal polarity, which orients apical constriction and apical-basal elongation behaviors that are required for neural fold elevation (

Grego-Bessa et al., 2015

;

McGreevy et al., 2015

;

Grego-Bessa et al., 2016

;

Brooks et al., 2020

;

Baldwin et al., 2022

). F-actin enrichment at the apical cell contacts and basal localization of the extracellular matrix protein laminin occurred normally in

Vinculin

^ESC


embryos, indicating that Vinculin is not required to establish apical-basal polarity (

Figure 3A

). Moreover, although apical-basal elongation was slightly delayed in

Vinculin

^ESC


embryos, the final cell heights were comparable to Control

^ESC

embryos, demonstrating that Vinculin is also dispensable for apical-basal elongation (

Figure 3—figure supplement 2A

). The distribution of apical cell areas in

Vinculin

^ESC


embryos was indistinguishable from Control

^ESC

embryos in early elevation, indicating that apical constriction behaviors initiate normally (

Figure 3D-G

). By contrast, apical cell profiles in

Vinculin

^ESC


embryos were significantly larger in late elevation, suggesting a defect in the progression of apical constriction (

Figure 3E and H

). No differences in the frequency of mitosis or apoptosis were observed between

Vinculin

^ESC


and Control

^ESC

embryos, indicating that these defects are not due to a disruption of cell proliferation or survival (

Figure 3—figure supplement 2B-E

). Together, these results demonstrate that Vinculin is not required for cell proliferation, cell survival, apical-basal polarity, or apical-basal elongation in the midbrain neural plate, but Vinculin is required to maintain the apical constriction behaviors that are necessary for neural fold elevation.

Vinculin is dispensable for force generation but is recruited to cell-cell junctions in a tension-dependent manner

As

Vinculin

^ESC


embryos display defects in neural tube closure as mechanical forces increase during neural fold elevation, we hypothesized that Vinculin could be required to generate or respond to the forces that drive neural tube closure. Consistent with a potential role in generating forces, Vinculin has been shown to regulate actomyosin localization at cell-cell junctions in epithelial cells in culture (

le Duc et al., 2010

;

Leerberg et al., 2014

) as well as in zebrafish cardiomyocytes and

Xenopus

embryos

in vivo

(

Fukuda et al., 2019

;

van den Goor et al., 2024

). Alternatively, Vinculin could stabilize cell adhesion in response to mechanical forces, as Vinculin is recruited to tricellular junctions that are predicted to be under increased tension (

Cho et al., 2022

;

van den Goor et al., 2024

) and acts to reinforce cell adhesion in the presence of experimentally applied forces

in vitro

and in the

Xenopus

embryo (

Yonemura et al., 2010

;

Huveneers et al., 2012

;

Thomas et al., 2013

;

Kannan and Tang, 2015

;

Cho et al., 2022

;

van den Goor et al., 2024

). However, whether Vinculin is required to generate or respond to endogenous forces during mouse neural tube closure is unknown.

To investigate whether Vinculin is required to generate mechanical forces during neural tube closure, we analyzed the localization of myosin IIB and filamentous actin (F-actin) in

Vinculin

^ESC


and

Vinculin

^ΔEpi


embryos. Myosin IIB and F-actin exhibit strong enrichment at cell-cell junctions in wild type embryos, including at bicellular junctions where two cells meet, tricellular junctions where three cells meet, and multicellular junctions where four or more cells meet (

Figure 4A and B

). By contrast, myosin IIB and F-actin were both present at cell-cell junctions in

Vinculin

^ΔEpi


embryos, but myosin IIB often appeared to dissociate from the membrane at tricellular and multicellular junctions, accompanied by a strong central accumulation of F-actin (

Figure 4A and C

). Similar results were observed in

Vinculin

^ESC


embryos (

Figure 4— figure supplement 1A

and

1B

). Myosin IIB and F-actin localization was less strongly affected at bicellular junctions, although myosin IIB at these structures was more diffusely localized (

Figure 4—figure supplement 1C

and D). These results indicate that Vinculin is not required to recruit actomyosin networks to cell-cell junctions, but is necessary for their organization at these structures.

To test if Vinculin is required for the generation of actomyosin forces during neural fold elevation, we performed laser ablation of individual cell edges in wild-type and mutant embryos. As laser ablation requires live imaging of embryos before phenotypic differences between wild-type and mutant embryos become apparent, it can be challenging to obtain sufficient numbers of mutant embryos through traditional genetic crosses. Therefore, we took advantage of newly isolated ESCs expressing the adherens junction marker GFP-Plekha7 and used the same CRISPR/Cas9 strategy to generate a

Vinculin

null mutant ESC line expressing GFP-Plekha7 (

Figure 4—figure supplement 2A

). Mutant ESCs were injected into GFP-negative host embryos to generate

Vinculin

^ESC


embryos expressing GFP-Plekha7, which produced an average of 7 mutant embryos per surrogate female. The resulting embryos lacked Vinculin protein and displayed fully penetrant exencephaly, compared to Control

^ESC

embryos generated by injection of unedited GFP-Plekha7 cells, indicating that

Vinculin

^ESC


embryos expressing GFP-Plekha7 recapitulate the defects of

Vinculin

null mutants (

Figure 4—figure supplement 2B

and C). Moreover, analysis of relative forces using laser ablation revealed wild-type recoil velocities at mediolateral cell edges in

Vinculin

^ESC


embryos, with no differences compared to Control

^ESC

embryos in early or late elevation (

Figure 4D-G

). These results indicate that mechanical forces in the elevating neural plate are generated correctly in the absence of Vinculin.

As Vinculin is not required to generate forces in the cranial neural plate, we next asked if it is necessary to reinforce cell adhesion in response to mechanical forces during elevation. If Vinculin stabilizes cell adhesion under tension, then it is predicted to be recruited to high-tension structures within cells. To test this, we generated ESC-derived embryos that constitutively express N-terminally tagged GFP-Vinculin from the

R26

locus. GFP-Vinculin localized to cell-cell junctions in live-imaged embryos during early and late elevation and was enriched at tricellular junctions that are predicted to be under increased tension (

Figure 4H-J

). GFP-Vinculin intensity at tricellular junctions continued to increase during late elevation (

Figure 4K

), correlating with increasing mechanical forces, though its relative enrichment became less pronounced at these stages, likely due to increased recruitment to bicellular junctions. These changes were not due to changes in gene expression, as GFP-Vinculin levels did not change significantly during elevation (

Figure 4— figure supplement 3

). To test if GFP-Vinculin localization at tricellular junctions is regulated by force, we treated mouse embryos for 2 h with 200 μM of the Rho-kinase inhibitor Y-27632 to inhibit actomyosin contractility. Treatment with the Rho-kinase inhibitor abolished GFP-Vinculin localization at all junctions, indicating that Vinculin localization requires myosin activity (

Figure 4L

). These results demonstrate that Vinculin is recruited to cell-cell junctions in response to actomyosin contractility in the cranial neural plate, with the strongest localization observed at tricellular junctions that are under increased tension.

Vinculin is required to maintain adherens junctions during elevation, but plays a minimal role in tight junction organization

The tension-dependent recruitment of Vinculin to cell-cell junctions, as well as the essential role of Vinculin in neural fold elevation, suggest that Vinculin may participate in functionally important force responses in the mouse cranial neural plate. Vinculin has well-known roles in stabilizing adherens junctions in the developing heart, skin, and vasculature (

Zemljic-Harpf et al., 2007

;

Biswas et al., 2021

;

Kotini et al., 2022

) and is also required for tight junction-mediated epithelial barrier function

in vitro

(

Konishi et al., 2019

) and in the

Xenopus

embryo (

Higashi et al., 2016

;

van den Goor et al., 2024

;

Landino et al., 2025

). However, whether Vinculin is required to regulate adherens junctions, tight junctions, or both during mouse neural tube closure is not known.

To investigate whether Vinculin regulates adherens junction localization in the cranial neural plate, we analyzed the localization of adherens junction proteins in Control

^ESC

and

Vinculin

^ESC


embryos (

Figure 5A-D

). The adherens junction protein N-cadherin was generally continuously localized along bicellular junctions, with few gaps in N-cadherin localization detected in either Control

^ESC

or

Vinculin

^ESC


embryos (

Figure 5—figure supplement 1A

and B). By contrast, gaps in N-cadherin localization were frequently observed at tricellular and multicellular junctions in

Vinculin

^ESC


embryos, particularly in late elevation when cells are under increased tension (35±4 gaps in

Vinculin

^ESC


and 18±5 gaps/region in Control

^ESC

, mean±SD), whereas fewer defects in adherens junction localization were detected in early elevation when mechanical forces are lower (12±3 gaps/region in

Vinculin

^ESC


and 4±2 gaps/region in Control

^ESC

) (

Figure 5A-F

,

Figure 5— figure supplement 1C and D

). Similar defects were observed in

Vinculin

^ESC


and

Vinculin

^ΔEpi


embryos, and mutant embryos generated with both methods displayed a loss of both N-cadherin and the adherens junction marker GFP-Plekha7 (

Figure 6A and B

,

Figure 5—figure supplement 1E-G

). To test if Vinculin is specifically required to regulate adherens junction localization under force, we analyzed protein localization at adherens junctions that are predicted to be under different levels of tension. Defects in GFP-Plekha7 localization were more frequent at higher-order junctions, with gaps detected at 18% of 3-cell junctions, 49% of 4-cell junctions, and 78% of 5+ cell junctions in

Vinculin

^ESC


embryos, compared to 5% of 3-cell junctions, 14% of 4-cell junctions, and 32% of 5+ cell junctions in Control

^ESC

embryos (

Figure 5G-I

). These results demonstrate that adherens junction defects increase during elevation and are most pronounced at junctions that sustain the highest forces, consistent with an essential role of Vinculin in maintaining cell adhesion under tension.

As Vinculin is required to maintain adherens junction localization as mechanical forces increase during elevation, we next examined if Vinculin is required for the localization of tight junction proteins in the cranial neural plate. To investigate this possibility, we compared the distribution of GFP-Plekha7 with the tight junction-associated protein ZO-1. In contrast to adherens junctions, which displayed frequent gaps in GFP-Plekha7 localization in the absence of Vinculin, significantly fewer gaps were detected in ZO-1 localization (5±3 gaps in

Vinculin

^ESC


embryos and 1±1 gaps in Control

^ESC

embryos in late elevation) (

Figure 6A-C

,

Figure 6—figure supplement 1A-C

). These results indicate that tight junction gaps are less frequently detected than adherens junction gaps in both Control

^ESC

and

Vinculin

^ESC


embryos. Gaps in ZO-1 localization only occurred at sites where adherens junction localization were already defective, and were only present at a small subset of these sites (56/498 adherens junction gaps in

Vinculin

^ESC


embryos and 9/150 adherens junction gaps in Control

^ESC

embryos), and gaps in ZO-1 localization, when present, were consistently smaller than the gap in the corresponding adherens junction (

Figure 6D and E

). To better visualize these structures, we performed high-resolution Airyscan imaging and deconvolution of the apical junctional domain in Control

^ESC

and

Vinculin

^ESC


embryos. These results revealed that ZO-1 was continuously associated with the membrane at moderately sized gaps in adherens junction localization, whereas localized interruptions in ZO-1 signal were visible in regions where adherens junctions were more severely disrupted (

Figure 6F and G

). Despite these differences, ZO-1 signal remained slightly apical to GFP-Plekha7 at bicellular junctions in Control

^ESC

and

Vinculin

^ESC


embryos, indicating that these structures are correctly spatially delineated along the apical-basal axis (

Figure 6F and G

,

Figure 6—figure supplement 1D

and E). Together, these results indicate that adherens junctions are strongly disrupted in the absence of Vinculin, whereas tight junctions display only minor defects.

Vinculin is required to maintain tissue integrity in response to force-intensive behaviors during neural tube closure

The findings that Vinculin is required to stabilize adherens junctions under tension, and that

Vinculin

mutants display fully penetrant defects in cranial neural tube closure, indicate that Vinculin regulates critical cell behaviors that drive structural changes in the mouse cranial neural plate. Multiple behaviors contribute to neural tube closure in mouse, chick, and

Xenopus

embryos, including apical constriction (

Haigo et al., 2003

;

McGreevy et al., 2015

;

Chu et al., 2018

;

Brooks et al., 2020

;

Baldwin et al., 2022

;

Matsuda et al., 2023

;

Bogart and Brooks, 2025

;

Itoh et al., 2025

), cell rearrangement (

Davidson and Keller, 1999

;

Nishimura and Takeichi, 2008

;

Nishimura et al., 2012

;

Williams et al., 2014

;

Ossipova et al., 2015

), and cell division (

Morriss-Kay, 1981

;

Jacobson and Tam, 1982

;

Schoenwolf and Smith, 1990

;

Alvarez and Schoenwolf, 1991

;

Bogart and Brooks, 2025

). However, how Vinculin influences cell behavior during cranial closure is unknown, in part due to the difficulty of directly visualizing dynamic cell behaviors in mutant embryos.

To address this question, we took advantage of the availability of Control

^ESC

and

Vinculin

^ESC


embryos expressing GFP-Plekha7 and used these embryos to perform time-lapse imaging of cell behavior in the lateral midbrain (Materials and methods). Control

^ESC

and

Vinculin

^ESC


embryos were mounted immediately before and during early elevation and imaged every six minutes for three hours (Videos 1-3). Control

^ESC

embryos displayed a 17±2% (mean±SEM) decrease in apical area after 1.5 hours and a 22±1% overall decrease in area after 3 hours (

Figure 7A

,

Figure 7—figure supplement 1A

and B). A similar change in area was observed after 1.5 hours in

Vinculin

^ESC


embryos, although junctional defects precluded an analysis of apical area at later time points in 3 of 5 mutant embryos (

Figure 7A

,

Figure 7—figure supplement 1A

and B). These results indicate that apical remodeling initiates correctly in the absence of Vinculin, but is often accompanied by defects in cell adhesion.

To investigate whether additional cell behaviors contribute to the junctional defects in mutant embryos, we analyzed higher-order junctions where 5 or more cells meet, also known as rosettes (

Blankenship et al., 2006

), which are thought to experience the highest forces in the tissue. Tracking of GFP-Plekha7 localization at these junctions revealed that 29/31 rosettes in Control

^ESC

embryos formed through cell rearrangement (the remaining 2 could not be determined), indicative of active remodeling. As expected based on our results in fixed embryos, few rosettes displayed central gaps in Control

^ESC

embryos (11±7%, mean±SEM, n= 5/47 rosettes), and the gaps that did form were either rapidly repaired (1/5 rosettes) or remained relatively small, with little or no further expansion in area during the imaging period (4/5 rosettes) (

Figure 7A, B

, and D). Rosettes were present in similar numbers in

Vinculin

^ESC


embryos, indicating that these structures form correctly in the absence of Vinculin (

Figure 7—figure supplement 1C

). However, the majority of rosette structures displayed gaps in GFP-Plekha7 signal (71±8%, n=33/48 rosettes) (

Figure 7D

), similar to the defects observed in fixed embryos (

Figure 5I

). Tracking rosettes over time in mutant embryos revealed that most gaps were either quickly repaired (20/33) or persisted but did not increase in size (7/33). However, a subset of gaps at rosette centers (6/33) expanded and merged with other gaps in the tissue (

Figure 7A, B

, and D). These results demonstrate that defects in GFP-Plekha7 localization in rosettes are more frequent in

Vinculin

^ESC


embryos, but are only occasionally associated with a widespread disruption of cell adhesion.

As the mislocalization of GFP-Plekha7 at higher-order junctions was only infrequently associated with a broader disruption of cell adhesion in the absence of Vinculin, we examined whether other cell behaviors could contribute to the defects in

Vinculin

^ESC


mutants. Cell division is known to exert mechanical forces on epithelial sheets through mitotic cell rounding (

Kondo and Hayashi, 2013

;

Monster et al., 2021

) and contraction of the cytokinetic ring (

Founounou et al., 2013

;

Guillot and Lecuit, 2013

;

Herszterg et al., 2013

). Vinculin is required to reinforce cell adhesion during these processes in MDCK cells and in the

Xenopus

embryo (

Higashi et al., 2016

;

Monster et al., 2021

;

Landino et al., 2025

). To test if Vinculin is required to maintain cell adhesion during cell division in the mouse cranial neural plate, we analyzed GFP-Plekha7 localization in dividing cells, which were identified by the presence of an ingressing cleavage furrow and tracked until the appearance of GFP-Plekha7 at the new vertex or interface (

Figure 7A and C

). Similar numbers of dividing cells were detected in Control

^ESC

and

Vinculin

^ESC


movies (

Figure 7—figure supplement 1D

), indicating that Vinculin is not required to initiate cell division. However, in contrast to cells in Control

^ESC

embryos, which maintained continuous GFP-Plekha7 localization throughout division (

Figure 7C and E

), dividing cells in

Vinculin

^ESC


embryos frequently displayed reduced GFP-Plekha7 signal at contacts with neighboring cells (73±9% of divisions in

Vinculin

^ESC


embryos compared to 8±5% of divisions in Control

^ESC

embryos). In addition, nearly half of the cells that began ingression in

Vinculin

^ESC


embryos failed to establish a new GFP-Plekha7-positive vertex or interface within 90 minutes (49±11% of divisions compared to 1±1% of divisions in Control

^ESC

embryos) (

Figure 7C and E

) and those that did took significantly longer to establish adhesion (

Figure 7F

,

Figure 7–figure supplement 1E

). Strikingly, defects in dividing cells often appeared to be focal points that expanded to produce larger regions of disrupted GFP-Plekha7 signal. In particular, sites of GFP-Plekha7 mislocalization in dividing cells often merged with each other and with defective rosettes, resulting in widespread regions of junctional disruption in

Vinculin

^ESC


embryos (

Figure 7A

). Together, these live imaging studies demonstrate that Vinculin is required to maintain cell adhesion in the presence of multiple force-intensive cell behaviors during cranial neural tube closure.

Discussion

Mechanical forces are essential for tissue remodeling, but how cells maintain cell adhesion in the presence of the dynamic forces that drive epithelial morphogenesis in mammals is not well understood. Here we use gene targeting and ESC-derived embryo generation to elucidate how cells respond to mechanical forces during mouse cranial neural tube closure. We show that actomyosin-mediated contractile forces increase during neural fold elevation, but Vinculin is not required for the generation of mechanical forces during this process. Instead, Vinculin is recruited to tricellular and multicellular junctions under stress and is required to maintain cell adhesion at high-tension sites as forces increase during neural tube closure. Live imaging of mutant embryos reveals that adhesion defects frequently initiate at high-order adherens junctions and in dividing cells, where mechanical forces are predicted to be highest, whereas tight junctions remain largely intact. Together, these results show that Vinculin is required to maintain cell adhesion during the force-intensive epithelial remodeling events that promote cranial neural tube closure, and demonstrate an essential role for Vinculin in transducing mechanical forces into the critical cell- and tissue-scale changes that shape mammalian tissue structure.

A wide range of molecular and biophysical functions have been ascribed to Vinculin based on studies

in vitro

and in cultured cells (

Goldmann, 2016

;

Bays and DeMali, 2017

;

Citi, 2019

), but the critical roles of Vinculin in cells exposed to physiological forces

in vivo

are just beginning to be elucidated. Our findings that Vinculin is required to maintain cell adhesion under tension in the mouse cranial neural plate are reminiscent of previous studies showing that Vinculin is required to maintain epithelial integrity in MDCK cells (

Monster et al., 2021

) and in the

Xenopus

gastrula (

Higashi et al., 2016

; van den Goor et al., 2023;

Landino et al., 2025

). However, Vinculin knockdown in the

Xenopus

embryo results in a depletion of F-actin and pMRLC at tricellular junctions (

van den Goor et al., 2024

), whereas Vinculin is dispensable for cortical actomyosin localization and force-generating activity in the mouse cranial neural plate. Moreover, Vinculin functions primarily at tricellular junctions in the

Xenopus

embryo (

van den Goor et al., 2024

), whereas loss of Vinculin in the mouse cranial neural plate causes more striking defects at multicellular junctions that are predicted be under increased force load. Additionally, Vinculin is required to maintain cell adhesion under endogenous forces in the mouse embryo, whereas ectopic forces are necessary to induce junctional defects in the

Xenopus

embryo (

van den Goor et al., 2024

), consistent with an increased mechanical demand on cells during neural fold elevation.

Notably, we observed frequent interruptions in adherens junction localization in the wild-type mouse neural plate, which affected over 10% of 4-cell junctions and nearly a third of rosettes during elevation. This suggests that cells continually repair transient gaps in adhesion that arise during the course of normal development. How cells achieve epithelial homeostasis in order to maintain tissue integrity during closure is opaque. In one model, Vinculin and other actin-junction linkers could reinforce adherens junctions under tension in the cranial neural plate, similar to the combinatorial roles of junction-actin linkers in maintaining cell adhesion under tension in the

Drosophila

embryo (

Sawyer et al., 2011

;

Razzell et al., 2018

;

Rauskolb et al., 2019

;

Yu and Zallen, 2020

;

Perez-Vale et al., 2021

;

Sheppard et al., 2023

). Alternatively, the presence of a continuous tight junction network could maintain epithelial integrity when adherens junctions are locally disrupted, facilitating ongoing junctional repair. Although tight junctions do not typically support strong adhesion (

Citi, 2019

), they have been shown to promote mechanical force responses that could actively maintain epithelial integrity (

Spadaro et al., 2017

;

Otani et al., 2019

;

Cho et al., 2022

;

Nguyen et al., 2024

). The mouse cranial neural plate may be a uniquely high-tension setting in which multiple force-intensive behaviors—including apical constriction, cell division, and junctional remodeling—are coordinately regulated in time and space across large populations of cells. The requirement for active mechanisms to reinforce cell adhesion as mechanical forces escalate during cranial closure may be among the reasons why cranial neural fold elevation is a frequent point of failure in mouse neural tube mutants (Harris and Juriloff, 2018).

Mouse ESCs have long been used as a powerful strategy for creating genetically modified mouse lines (

Capecchi, 2005

). The ability to generate embryos and animals that are nearly completely derived from ESCs unlocks the potential to perform immediate analysis of mutant phenotypes (

Poueymirou et al., 2007

;

Lai et al., 2015

), generate fluorescent embryos and animals for live imaging (

Hadjantonakis et al., 1998

;

Wang et al., 2016

), and create postnatal models of complex human diseases such as cancer (

Dow and Lowe, 2012

;

Alonso-Curbelo et al., 2021

;

Burdziak et al., 2023

). Here we use this method to bypass the constraints of Mendelian genetics in order to generate large numbers of lethal mutant embryos for phenotypic analysis. ESC-derived embryos complete embryogenesis and develop into viable and fertile animals, demonstrating that this pipeline does not interfere with neural tube closure or other essential processes, providing an excellent platform for studying embryonic and post-embryonic development. This approach significantly accelerates the generation of desired genotypes, is well-suited to investigating dynamic cell behaviors by live imaging, and can be used to produce theoretically unlimited yields of mutant embryos. This approach has several advantages over standard genetic crossing strategies. Early ESC injections yield mutant embryos at a rate that is several times higher than conventional genetic crosses without the need for often difficult and time-consuming crossing strategies, significantly reducing the animal burden without sacrificing the monumental power of mouse genetics to elucidate the causes of, and innovate treatments for, human disease. Additionally, increasingly powerful genome editing technologies (

Wang et al., 2013

) can be harnessed to generate complex genotypes in mouse ESCs in culture followed by functional analyses

in vivo

, making it possible to rapidly test targeted mutations

in vivo

with a level of control previously limited to gastruloid and

in vitro

models. By comparison, fully

ex vivo

embryo-like models share this flexibility and go even further in reducing animal use (

Amadei et al., 2022

;

Tarazi et al., 2022

;

Yilmaz et al., 2025

;

Jorgensen et al., 2025

), but currently do not complete embryonic development, with few embryos surviving to neural tube closure stages. ESC-derived embryos can be used to rapidly characterize candidate genes, protein sequences, and disease variants in a physiological context, perform live imaging of mutants with genetically encoded fluorophores and biosensors, and disrupt multiple genes simultaneously to study combinatorial gene functions and model diseases with a multifactorial etiology, as is the case for many neural tube defects (

Wilde et al., 2014

;

Juriloff and Harris, 2018

;

Lee and Gleeson, 2020

). Combining the power of genome engineering with high-throughput

in vivo

studies of cell dynamics will accelerate exploration of the large and interconnected network of factors that modulate cell interactions during mammalian development.

Materials and methods

Mouse strains

The following published strains were used in this study:

Vcl

^flox


(

Vcl

^tm1Ross


) (

Zemljic-Harpf et al., 2007

) (Jackson Laboratory stock #028451), Sox2-Cre (

Edil3

^{Tg(Sox2-cre)1Amc}


) (

Hayashi et al., 2002

) (Jackson Laboratory stock 008454),

R26-Plekha7-GFP

(

R26-PHA7-EGFP

) (

Shioi et al., 2017

) (RIKEN Accession # CDB0261K), and Myosin IIB-GFP (

Bao et al., 2007

). All stocks used for genetic crosses were maintained on an FVB/N background (Jackson Laboratory stock 001800).

R26-Histone2B-GFP (R26-H2B-EGFP)

(

Abe et al., 2011

) (RIKEN Accession # CDB0238K), C57BL/6J (Jackson Laboratory stock #000664), and C57BL/6N (Jackson Laboratory stock #005304 or Charles River stock #027) were used to generate host embryos for ESC injection.

Vcl

^ΔEpi


embryos were generated by crossing Sox2-Cre;

Vcl

^Δ/+


males to

Vcl

^flox/flox


females to generate Sox2-Cre;

Vcl


<sup>Δ

/-</sup>


mutant embryos. Controls for

Vcl

^ΔEpi


embryos were stage-matched wild type (

Vcl

^+/+


or

Vcl

^flox/+


) or heterozygous (

Vcl


<sup>Δ

/+</sup>


or

Vcl

^-/+


) littermate controls. Embryos were harvested at E7.75-E10.0 and timed matings were confirmed by the presence of a vaginal plug, with noon of the day of the plug considered E0.5. Embryos were staged by somite number, corresponding to E8.0 (pre-elevation, 0-3 somites), E8.25 (early elevation, 4-6 somites), E8.5 (late elevation, 7-8 somites), and E8.75 (apposition, 9+ somites). For

Vcl

^ΔEpi


embryos and littermate controls, embryos were genotyped by PCR using the following primers to test for the

Vcl

^flox


and

Vcl


^Δ

alleles: 5’-
CCTGCGCGGGATTACCTCATTGAC
-3’ (Vcl flox Fwd), 5’-
TTACGCCTAGCACTTGAA
-3’ (Vcl Δ Fwd), 5’-
TGCTCACCTGGCCCAAGATTCTTT
-3’ (Vcl common rev). The following primers were used to test for Sox2-Cre: 5’-
CTTGTGTAGAGTGATGGCTTGA
-3’ (WT Sox2 Fwd), 5’-
CCAGTGCAGTGAAGCAAATC
-3’ (Sox2-Cre Fwd), 5’-
TAGTGCCCCATTTTTGAAGG
-3’ (Common Rev).

All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health and approved Institutional Animal Care and Use Committee protocols (15-08-013 and 90-12-033) of Memorial Sloan Kettering Cancer Center.

Mouse embryonic stem cell (ESC) culture

ESC modifications were performed on the HK3i mouse ESC line derived from a C57BL/6N background (

Kiyonari et al., 2010

) or wild-type Plekha7-GFP ESC clone A3 derived from

R26-PHA7-GFP

embryos in C56BL/6N (

Shioi et al., 2017

). ESCs were grown on 0.1% gelatin-coated tissue culture plates in Cellartis 3i mES/iPSC culture medium (Takara Bio) containing ERK/MEK inhibitor, GSK3β inhibitor, FGFR-TK inhibitor, and 1000 U/mL ESGRO recombinant mouse LIF protein (EMD Millipore), referred to as 3i/LIF medium. HK3i mouse ESCs (

Kiyonari et al., 2010

) were grown in a humidified incubator at 37 °C with 5% CO

₂

on mouse embryonic fibroblast (MEF) feeder cells or drug-resistant MEF feeder cells (DR-4) (

Tucker et al., 1997

) that were mitotically inactivated by 4,000 cGy X-ray irradiation or 10 ug/mL mitomycin C for 2-3 hr. Feeder cells were plated in medium containing 1x Knockout DMEM (Gibco), 5-15% FBS (GeminiBio and Millipore Sigma), 2 mM GlutaMAX Supplement (Gibco), and 1 mM MEM non-essential amino acids (Gibco). ESC clones or non-clonal, pooled cell populations for DNA analyses to assess the occurrence of knock-in insertions or indel mutations were grown without feeder cells in medium containing 1x KO DMEM (Gibco), 15% KO serum replacement (Gibco), 2 mM GlutaMAX Supplement (Gibco), 1 mM MEM non-essential amino acids (Gibco), 1000 U/mL ESGRO recombinant mouse LIF protein (EMD Millipore), 1 uM 2-mercaptoethanol (Gibco), 1 uM PD0325901 (amsbio), and 3 uM CHIR99021 (amsbio), referred to as KSR/2i medium, in a humidified incubator at 37°C with 5% CO

₂

.

Plasmid vectors for genome editing in ESCs

To select the gRNA sequences to generate

Shroom3

and

Vinculin

knockout alleles, the following design tools were used: IDT, CRISPOR (

Concordet and Haeussler, 2018

), GuideScan2 (

Perez et al., 2017

;

Schmidt et al., 2025

), and CHOPCHOP v3 (

Labun et al., 2019

). DNA sequence information and chromosomal coordinates were obtained from Ensembl (Cunningham et al., 2021; Martin et al., 2022) and when a genome could be selected,

Mus musculus

GRCm39/mm39 was used. To generate plasmids expressing sgRNAs for ESC modification, DNA inserts containing sequences corresponding to two crRNAs, each of which was flanked by the U6 promoter and gRNA scaffold sequences, were

in vitro

synthesized and subcloned into the

pUC57

vector (GenScript). The 2x tandem U6-sgRNA fragment was then cloned into the

pPuro-Dest-Cas9

plasmid, a derivative of

pX330.puro

(

pX330.puro

was a gift from Sandra Martha Gomes Dias, Addgene plasmid #110403). The following gRNA target sites were selected for an ∼2,710 bp deletion within exon 5 of

Shroom3

: 5’ target
GTACTCGAAGATGCTCGAAC
and 3’ target
TTCGCCAGCGGACGCCTAGT
. The gRNA target sites to induce an ∼536 bp deletion with breakpoints within introns 2 and 3 flanking

Vinculin

exon 3 were 5’ (intron 2)
GGCCAAGTCCTGTTATAAAT
and 3’ (intron 3)
CCGTTAAAATTGCACTTTAG
-3’.

To generate the

pR26-GFP-4xGSS-Vcl-201

targeting vector, the

pR26-GFP-4xGSS

backbone containing the N-terminal GFP and an

in vitro

synthesized cDNA fragment containing the coding sequence from the

Vcl-201

isoform preceded by a 4xGSS linker sequence (Genscript) was first subcloned into the

pUC57

vector (

pUC57-4xGSS-Vcl-201

). The

4xGSS-Vcl

insert was excised and cloned into the

pR26-GFP

targeting vector backbone containing a 1,083 bp 5’ homology arm, an adenovirus major late transcript splice acceptor (

Friedrich and Soriano, 1991

), EGFP, a poly-A signal from the bovine growth hormone gene, a human Ubiquitin C gene promoter-driven Neo-R cassette, and a 811 bp 3’ homology arm.

Generation of

Shroom3

mutant,

Vinculin

mutant, and

R26-GFP-Vinculin

ESC lines

Genome editing was performed using methods adopted from published protocols (

Ukai et al., 2017

;

Mulas et al., 2019

) in the HK3i cell line derived from a C57BL/6N background (

Kiyonari et al., 2010

) or a wild-type GFP-Plekha7 ESC line (clone A3) derived in-house from

R26-PHA7-EGFP

embryos (

Shioi et al., 2017

) according to previously described methods (

Kiyonari et al., 2010

).

Shroom3

mutant ESC clones 15, 21, 33, and 38 carried biallelic deletions of exon 5 of the

Shroom3-203

isoform, producing a frameshift mutation in downstream exons (

Figure 2—figure supplement 1B

). Control embryos for

Shroom3

^ESC


were generated from wild-type HK3i ESCs (

Kiyonari et al., 2010

) or

tyrosinase (Tyr)

mutant ESCs (clones 32 and 41) derived from HK3i generated by CRISPR/Cas9 genome editing.

Vinculin

^ESC


embryos were generated from the homozygous mutant

Vinculin

ESC clone 58, which carried biallelic deletions of exon 3 of the

Vinculin-201

isoform, producing a frameshift mutation in downstream exons that alters the protein sequence after amino acid 79 of 1,066 (

Figure 3—figure supplement 1B

,

Figure 4—figure supplement 2A

).

Control

^ESC


embryos were generated from the heterozygous

Vinculin

ESC clone 15, which carried a monoallelic deletion.

Vinculin

^ESC


embryos expressing GFP-Plekha7 from the

R26

locus were generated from

Vinculin

biallelic mutant ESC clone 41 and control embryos were generated from wild-type

R26-PHA7-EGFP

ESC clone A3.

To generate the

Shroom3

and

Vinculin

ESC lines, HK3i or R26-GFP-Plekha7 cells were transfected using Lipofectamine™ LTX Reagent with PLUS™ Reagent (Invitrogen) with

pPuro-(Dest-sgRNA)-Cas9

constructs that express the Puromycin resistance gene, Cas9, and corresponding sgRNAs per the manufacturer’s instructions. Lipofected cells were plated at 3 x 10

⁴

to 1 x 10

⁵

cells onto 60 mm tissue culture-treated dishes coated with 0.1% gelatin pre-seeded with DR-4 MEF feeder cells. Lipofected cells were subjected to puromycin selection (2 mg/ml) (Gibco) for 24 hr starting 18 to 24 hr after lipofection. Following puromycin removal, cells were cultured for an additional 8-12 days in non-selective 3i/LIF medium. Individual colonies were isolated into 96-well plates, dissociated, and expanded to generate the original frozen stocks (passage 0, P0 generation) and for DNA isolation. DNA extraction was performed using the DNeasy Blood and Tissue Kit (Qiagen). Candidate biallelic and monoallelic deletion mutant clones at each locus were identified by PCR screening using the indicated primers. Selected clones were expanded to P2 to P5 generations for embryo production by injection. Fractions of individual clones were also cultured in non-adhesive, floating cell clump-forming conditions for elimination of feeder cells. DNA isolated from floating ESC clumps (DNeasy Blood and Tissue Kit) was PCR amplified for deep sequencing of amplicons around the deletion (for sequencing mutant alleles) or around the 5’ and 3’ CRISPR cut sites (for sequencing heterozygous controls), with 97,000-850,000 reads obtained/amplicon (Sloan Kettering Integrated Genomics Operation Facility) using the indicated primers.

Primers used to screen for

Shroom3

deletions were: A (forward)
CCCTGCCATCTCCTTTCTCCTG
, B (reverse)
CCGTCTTGACGTAGCGGATGTC
, G (forward)
CCCTGCCATCTCCTTTCTCCTG
, and D (reverse)
CCCTGCCATCTCCTTTCTCCTG
. Primer pair A and B amplifies across the 5’ gRNA target site and is predicted to generate an amplicon of ∼232 bp for the wild-type allele only. Primer pair G and D amplifies across the 3’ gRNA target site and is predicted to generate an amplicon of ∼246 bp for the wild-type allele only. Primer pair A and D amplifies across the deletion and is predicted to generate an amplicon of ∼269 bp for deletion alleles only.

Primers used to screen for

Vinculin

deletions were: A (forward)
TCACAAGCCTAGTGCACAGAG
, B (reverse)
TCACAAGCCTAGTGCACAGAG
, C (forward)
GTGGGTGCCAAGAACCAAAC
, and E (reverse)
GTTTCACTGTGTAGCCTTGGC
. Primer pair A and B amplifies across the 5’ gRNA target site and is predicted to generate an amplicon of ∼238 bp for the wild-type allele only. Primer pair C and E amplifies across the 3’ gRNA target site and is predicted to generate an amplicon of ∼236 bp for the wild-type allele only. Primer pair A and E amplifies across the deletion and is predicted to generate an amplicon of ∼241 bp for deletion alleles and ∼777 bp for the wild-type allele.

To generate the

R26-GFP-Vinculin

ESC knock-in line, HK3i ESCs were transfected using Lipofectamine™ LTX Reagent with PLUS™ Reagent (Invitrogen) with the

pDonor-MCS-ROSA26

vector (Addgene 37200) (

Perez-Pinera et al., 2012

) containing ∼0.8 kb left and right homology arms (chromosome 6: GRCm39:6:113052181:113053829) for targeted insertion of a GFP-Vinculin cDNA and a neomycin cassette flanked by FRT sites into the

R26

locus. The donor plasmid was transfected with a

pPuro-(Dest-sgRNA)- Cas9

vector (sgRNA R26-T2) (

Nakao et al., 2016

) to induce a double-strand break in the

R26

locus. Lipofected cells were plated at 1 x 10

⁵

onto 60 mm tissue culture-treated dishes coated with 0.1% gelatin containing DR-4 MEF feeder cells. Cells were subjected to puromycin selection (2 mg/ml) (Gibco) for 24 hr starting 18-24 hr after lipofection. Following puromycin removal, cells were treated with 200 ug/uL Geneticin (G418) (Gibco) for 48 hr and this was decreased to 150 ug/uL G418 for the following 6-10 days. Individual colonies were isolated into 96-well plates, dissociated, and expanded to generate the original stocks and for DNA isolation. DNA extraction was performed using the DNeasy Blood and Tissue Kit (Qiagen) or the 96-well plate method according to a protocol from the McManus lab (

) modified in-house for PCR tube strips. Targeted ESC clones were identified by PCR screening using the indicated primers and copy number analysis using droplet digital PCR (Thermo Scientific) was performed to assess potential multi-copy integration and random integration events.

R26-GFP-Vinculin

clone 27 was expanded to P2 to P4 generations and used for embryo production by injection.

Primers used to screen for

R26

insertions were: RosaP1 (forward)
CTCAGAGAGCCTCGGCTAGGTAGG
, EGFPSQ1 (reverse)
AGCTCCTCGCCCTTGCTCACC
, iNeoSQ1 (forward)
AGGAACTTCGTTGGTACCGTACG
, and RosaP4 (reverse)
GGAGACATCCACCTGGAAACCATTAATGG
. Primer pair RosaP1 and EGFPSQ1 amplifies across the 5’ knock-in insertion junction and is predicted to generate an amplicon of 1,442 bp for the knock-in allele. Primer pair iNeoSQ1 and RosaP4 amplifies across the 3’ knock-in insertion junction and is predicted to generate an amplicon of 903 bp for the knock-in allele.

Generation of ESC-derived embryos

Embryos were generated from selected clones through ESC injection into E2.5 (8-cell/morula stage) host embryos, followed by same-day transfer into E0.5 pseudopregnant females or next-day transfer into E2.5 pseudopregnant females according to standard methods (

Behringer et al., 2013

). E7.75-E10 embryos were recovered for analysis. Homozygous R26-Histone-H2B-GFP (R26-H2B-EGFP) males (

Abe et al., 2011

) and C57BL/6 females were mated to produce host embryos for ESC injection (

Behringer et al., 2013

). GFP-negative ESCs were injected into R26-Histone-H2B-GFP host embryos (

Abe et al., 2011

) and embryos with no GFP-positive host cells detected outside of the gut tube were analyzed. GFP-positive ESCs were injected into E2.5 wild-type C57BL/6 host embryos and embryos with widespread GFP expression in the cranial neural plate were analyzed. B6CBAF1 female mice were used to generate pseudopregnant surrogates. The analysis of the contribution of Histone-H2B-GFP-expressing host cells in

Figure 2B

was performed in

Vinculin

^ESC


and Control

^ESC

embryos.

Whole-mount immunofluorescence

Embryos were dissected in ice cold phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde (4% PFA, Electron Microscopy Services) for 1-2 hr at room temperature or overnight at 4 °C or with Dent’s fixative (4:1 methanol:DMSO) and fixed overnight at 4 °C. Dent’s-fixed embryos were rehydrated for 30 minutes each in solutions of 75:25, 50:50, and 25:75 methanol:PBS at room temperature. After fixation, embryos were rinsed three times in PBS + 0.1% Triton-X100 (PBS-Tr) and washed 3 x 30 min in PBS-Tr at room temperature. Embryos were then incubated in blocking solution (PBS + 0.1% Triton-X100, 3% BSA) for 1 hr at room temperature and incubated overnight at 4 °C in primary antibody diluted in antibody staining solution (PBS + 0.1% Triton-X100, 1.5% BSA). Primary antibodies used for staining of paraformaldehyde-fixed embryos were chicken anti-GFP to visualize Histone-H2B-GFP (Abcam ab13970, 1:2000), rabbit anti-N-cadherin (Cell Signaling 13116, 1:500), rabbit anti-nonmuscle Myosin IIB heavy chain (BioLegend 909901, 1:500), mouse anti-phospho-histone H3 (Ser10) (Cell Signaling 95777, 1:500), rabbit anti-phosphomyosin light chain (Thr18/Ser19) (Cell Signaling 95777, 1:100), rabbit anti-Shroom3 (UPT132, 1:100) (

Hildebrand, 2005

), and mouse anti-ZO1 (Invitrogen 33-9100, 1:500). Th rat anti-ZO-1 primary antibody (DSHB R26.4C, 1:100) was used to stain embryos fixed with Dent’s fixative (

Figure 2D-F

). Embryos were washed 3 x 30 min in PBS-Tr, incubated with AlexaFluor-conjugated secondary antibodies (1:500), AlexaFluor-546 Phalloidin (Thermo Fisher, 1:500), and/or Hoechst 33342 (1 mg/mL, Invitrogen, 1:1000) in antibody solution for 90 min at room temperature, washed 3 x 30 min in PBS-Tr, and stored in PBS-Tr at 4 °C until imaging. Embryos were mounted for imaging in Attofluor cell chambers (ThermoFisher A7816) by slightly compressing the embryos under pieces of coverglass held in place by vacuum grease (Dow Corning) on a circular 25 mm, #1.5 coverslip (Fisher NC1272770).

Cryosectioning

Fixed embryos were equilibrated in 15% sucrose in PBS, then 30% sucrose in PBS for 30 minutes each with gentle rocking, mounted in blocks of OCT (Tissue-Tek) with the cranial flexure up, then tilted at an angle of 25-40° with the forebrain facing up to obtain transverse sections of the posterior midbrain. OCT blocks were frozen and 12 µm sections were acquired on SuperFrost Plus slides (Fisher Scientific) with a Leica CM3050S cryostat and kept frozen at -80 °C until analysis. For staining, slides were defrosted and sections were rehydrated with PBS + 0.1% Tween-20 (Sigma) (PBS-Tw) for 10 min, permeabilized with 0.5% PBS-Tr for 30 mins, and washed 5 x 5 min in PBS-Tw. Sections were incubated in blocking solution (PBS-Tw with 2% BSA) for 10 min at room temperature and incubated in primary antibody diluted in blocking solution overnight at 4°C. Primary antibodies were chicken anti GFP to visualize Histone-H2B-GFP (Abcam ab13970, 1:2000) and rabbit anti-laminin (Sigma L9393, 1:1000). Sections were washed 5 x 10 min with PBS-Tw, re-blocked for an additional 10 min in blocking solution, and incubated with AlexaFluor-conjugated secondary antibodies (1:500) with AlexaFluor-546 Phalloidin (Thermo Fisher, 1:500) and Hoechst 33342 (1 mg/mL, Invitrogen, 1:1000) in blocking solution for 30 min at room temperature. Embryos were washed 3 x 5 min with PBS-Tw and mounted under a #1.5 coverslip (Corning 2980-245) in fluorescence mounting media (Dako).

Live embryo culture

All steps were performed in media equilibrated to 37 °C and 5% CO

₂

. Timed pregnant females were euthanized and uterine horns were dissected and transferred into DMEM/F-12, GlutaMAX (DMEM) (ThermoFisher). Intact egg cylinders were removed from the decidua and transferred to 1:1 DMEM and whole embryo culture rat serum (Envigo). The parietal yolk sac and Reichert’s membrane were carefully dissected, maintaining an intact ectoplacental cone. The yolk sac and amnion were removed from around the neural folds, being careful to avoid damage to embryonic tissue. For live imaging, embryos were dissected immediately before and during early elevation (2-5 somites), mounted with the midbrain and hindbrain neural folds facing down in a 35 mm Lumox culture dish (Sarstedt) and immobilized with cut pieces of 70 µm cell strainer (Falcon) held in place with vacuum grease. Mineral oil (Sigma) was added to culture media to prevent evaporation. Embryos were incubated in a stage-top incubator (Pecon) at 37°C and 5% CO

₂

while imaging. For Rho-kinase inhibitor treatments, intact egg cylinders were treated with 200 µm Y-27632 (Sigma) or an equal volume of ultrapure H

₂

O, pre-warmed in 1:1 DMEM:rat serum, and cultured at 37 °C and 5% CO

₂

in a 24-well Lumox culture plate (Sarstedt) for 2 hr before laser ablation or imaging.

Microscopy

Cryosections were imaged on a Zeiss LSM700 or Zeiss LSM900 confocal microscope using a 20x/0.8 Plan-Apochromat objective with a 0.5x optical zoom and z-stacks of 8-14 µm were obtained with 2 µm z-slices and 1 µm z-steps. Whole mount fixed imaging was performed on a Zeiss LSM900 using a 20x/0.8 Plan-Apochromat objective or a Plan-Apochromat 40x/1.3 oil immersion objective. Z-stacks of 50-210 µm were obtained with 1.6-2.0 µm z-slices and 0.8-1.0 µm z-steps with an optical zoom of 0.45-0.5x for the 20x objective. Z-stacks of 10-100 µm were obtained with 0.3-0.5 µm z-slices and 0.6-1.0 µm z-steps with an optical zoom of 0.5-2.0x for the 40x objective. Tiled images were computationally stitched with 10% overlap using Zen-Blue software (Zeiss). Airyscan imaging (

Wu and Hammer, 2021

) was performed on a Zeiss LSM900 confocal microscope equipped with an Airyscan2 detector using a Plan-Apochromat 63x/1.4 oil immersion objective with an optical zoom of 1.7x. Z-stacks of 5-8 µm were obtained with 0.13 µm z-steps. Airyscan images were deconvolved using Huygens Professional Software (Scientific Volume Imaging). Live imaging was performed on a Zeiss LSM900 using a Plan-Apochromat 40x/1.3 oil immersion objective with an optical zoom of 0.5X. Z-stacks of 25-55 µm were obtained with 0.3-0.8 µm z-slices and 0.6-0.8 µm z-steps. Images were acquired at 6 min intervals for 3 hours. Light micrographs were acquired on a Zeiss Stemi 508 stereomicroscope with an attached Canon EOS T7i camera.

Laser ablations

Laser ablation experiments were performed on a spinning disk confocal with a Yokogawa CSU X1 scan head and an Excelitas PCO.Edge 4.2 sCMOS camera on a Zeiss Observer Z1 microscope with a Zeiss 40X Plan NeoFluor 1.3-NA objective using an iLas ablation system (Gataca Systems, Massy, France). Images of the lateral midbrain were acquired as z-stacks of five z-slices with a step size of 0.5 μm using a Zeiss 40X Plan NeoFluor 1.3-NA oil-immersion objective and a PCO Edge 4.2 camera. To ablate a cell interface, 10 pulses of 355-nm light were focused at a single cell interface visualized with myosin IIB-GFP or GFP-Plekha7, along a 16-pixel line perpendicular to the cell interface. Pre-and post-ablation images were acquired every 2 s. The distance between the two tricellular junctions attached to the cut interface was measured immediately before and up to 8 s after ablation and the instantaneous velocity was measured at every time point. The peak velocity, which is predicted to correlate with tension at the junction prior to ablation, was used for analysis. Calibration of the iLas ablation system was performed prior to every experiment. Ablations that damaged edges other than the targeted edge or showed no measurable recoil were discarded. A maximum of 5 ablations were performed/neural fold and ablations were performed on both neural folds.

Western blots

Single embryos staged between E8.0-9.5 were lysed for 10 min in ice cold RIPA buffer with Protease/Phosphatase Inhibitor cocktail (Cell Signaling) and manually homogenized with a pestle. Protein concentration was determined by BCA (Thermo Fisher). 30 µg of protein was boiled for 10 min in SDS sample buffer, separated on a PVDF membrane (Millipore), blocked in 5% milk in TBST, and immunoblotted with mouse anti-vinculin (Sigma, V9131, 1:1000) or mouse anti-β-catenin (BD Bioscience, 610153, 1:2000) antibodies overnight at 4 °C in 5% BSA, TBS-Tw. Membranes were washed 6 times for 5 mins with TBS-Tw, blotted with HRP-conjugated secondary antibodies (Jackson Laboratory, 1:5000) in 5% milk, TBST for 30 min at room temperature, and developed with Amersham ECL Western Blot Detection Reagent (Fisher Scientific).

Image analysis and quantification

Maximum intensity projections were created from acquired image z-stacks using ZEN-Blue software (Zeiss) or FIJI (

Schindelin et al., 2012

). All confocal images and analyses are based on maximum intensity projections of the entire z-stack, except for

Figure 4A-C

,

Figure 4–figure supplement 1A

, and

Figure 6F and G

, which are maximum intensity projections of 1.8-7.6 µm in the apical junctional domain to highlight junctional myosin IIB signal. Drift that occurred while acquiring z-stacks of GFP-Vinculin in live embryos was corrected using the StackReg plugin in FIJI (

Thévenaz et al., 1998

).

ZO-1, myosin IIB, and pMRLC planar polarity measurements (

Figure 1C and D

) were measured in 50 µm x 50 µm regions using SIESTA software (

Fernandez-Gonzalez and Zallen, 2011

). Planar polarity was calculated by dividing the mean intensity of ML junctions (oriented at 0-15° relative to the mediolateral axis) by the mean intensity of AP junctions (oriented at 75-90° relative to the mediolateral axis) after subtracting background signal. Background signal was calculated as the average of the mean pixel intensities in 20-40 circular cytoplasmic regions drawn in FIJI.

Apical cell areas (

Figure 2D-F

,

Figure 3D-H

,

Figure 7—figure supplement 1A

and B) were measured in a 100 µm x 100 µm lateral region of the midbrain. N-cadherin was used to label the apical borders of cells in early and late elevation embryos and ZO-1 was used to label the apical borders of cells in pre-elevation embryos, as N-cadherin expression is low at pre-elevation stages (

Brooks et al., 2025

). GFP-Plekha7 was used to label the apical adherens junctions in time-lapse movies. Cell segmentation was performed using SeedWater Segmenter (

Mashburn et al., 2012

), and apical cell areas were quantified and heat maps were generated using the MorphLibJ plugin in FIJI (

Legland et al., 2016

).

The apical and basal spans of the midbrain neuroepithelium (

Figure 3C

) were measured by manually drawing lines along the apical and basal borders of the neural plate from the non-neural ectoderm-neuroepithelial border of one neural fold to the other using the segmented line tool in FIJI. Cell height (

Figure 3–Supplemental Figure 2A

) was determined by measuring the distance between the apical and basal borders of the neural plate, roughly halfway between the non-neural ectoderm-neuroepithelial border and the midline for each neural fold.

The percentage of mitotic cells (

Figure 3—figure supplement 2B

and C) was calculated by dividing the number of phospho-histone H3-positive nuclei by the total number of cells in a 100 µm x 100 µm region of the midbrain in 7-9 somite wild-type and

Vinculin

^ΔEpi


embryos, using ZO-1 or GFP-Plekha7 to visualize apical cell outlines. Total cell counts were determined by segmenting cells using SeedWater Segmenter (

Mashburn et al., 2012

).

GFP-Vinculin intensity at tricellular junctions (TCJ intensity) was measured in FIJI as the mean intensity of a circular region of interest (ROI) centered on the tricellular junction (

Figure 4K

). The GFP-Vinculin and GFP-Plekha7 TCJ ratios (

Figure 4H and I

) were calculated in FIJI by dividing by dividing the TCJ intensity by the average mean intensities of lines drawn along the three connected bicellular junctions after subtracting background signal as calculated above.

Intensity profiles of the width of the myosin IIB and F-actin signal at bicellular junctions (

Figure 4—figure supplement 1C

and D) were measured along 3 µm-long lines perpendicular to the bicellular junction and centered on the myosin IIB signal in FIJI. An average value was obtained for 10 bicellular junctions/neural fold after subtracting background signal as calculated above and normalized to the maximum intensity for each line (8 neural folds were analyzed in 4 embryos).

Western blots (

Figure 4—figure supplement 3A-C

) were quantified by measuring the mean grey value of a box drawn around each band. The same sized box was used for each band of a particular protein and band intensities were normalized to the intensity of the β-catenin loading control.

The number of adherens junction and tight junction gaps (

Figure 5E and F

,

Figure 6B and C

,

Figure 5— figure supplement 1G

,

Figure 6—figure supplement 1B

and C) was quantified by manually counting regions of aberrant loss of junctional signal in 50 µm x 50 µm regions of the lateral midbrain. The percentage of 3, 4, and 5+ cell junctions with gaps (

Figure 5G-I

) was determined by dividing the number of gaps at each type of junction by the total number of that type of junction in each image. The areas of adherens junction and tight junction gaps (

Figure 6B-E

,

Figure 6—figure supplement 1B

and C) were determined by drawing circular ROIs over gaps in signal, with the diameter of the ROI representing the furthest points of lost signal. Places where ROIs for multiple gaps overlapped were treated as one large gap and a new circular ROI was redrawn to cover the overlapping gaps. The average gap area at tight junctions was measured at each site in which there was an adherens junction gap, and scored as a value of 0 if no corresponding tight junction gap was detected. Of the adherens junction gaps observed, 141/150 were not associated with tight junction gaps in Control

^ESC

and 442/498 were not associated with tight junction gaps in

Vinculin

^ESC


. Gaps at bicellular junctions (plotted in

Figure 5—figure supplement 1A

and B) were not included in the other adherens junction gap measurements in

Figure 5

,

Figure 6

,

Figure 5—figure supplement 1G

, and

Figure 6—figure supplement 1

.

Intensity profiles along orthogonal XZ reslices of AiryScan z-stacks at bicellular junctions (

Figure 6—figure supplement 1D

and E) were measured along 3 µm-long lines drawn from apical to basal and centered on the ZO-1 signal. An average value was obtained for 10 bicellular junctions/neural fold and normalized to the maximum intensity for each line and (4 neural folds/embryo were analyzed in 2 embryos).

For analysis of time-lapse movies (

Figure 7

,

Figure 7—figure supplement 1

), maximum intensity projections were registered in XY using the Correct 3D drift plugin in FIJI. Cell behavior was analyzed in 100 µm x 100 µm regions in the lateral midbrain. Junctions where 5 or more cells meet were scored as defective when there was a clear loss of GFP-Plekha7 signal at the central high-order vertex. Dividing cells were identified by the presence an ingressing cleavage furrow, which generates a distinctive figure-8 morphology, and t=0 was defined as one time point before the cleavage furrow started to ingress. Dividing cells were tracked every 6 min until a clear GFP-Plekha7-positive 4-cell vertex, a new interface between daughter cells, or a new interface between neighboring cells that separated the two daughter cells was detected. Cell divisions were scored as having a gap in GFP-Plekha7 signal at neighbor contacts if a significant reduction in GFP-Plekha7 signal was detected at the interface between the dividing cell and surrounding cells that was not restored within 90 minutes (

Figure 7E

, left). Cell divisions were scored as having a gap in GFP-Plekha7 signal at new contacts if GFP-Plekha7 was not detected at a new vertex or interface within 90 minutes after the onset of ingression (

Figure 7E

, right). Only cell divisions that began within the first 90 minutes of the 3-hour movie were analyzed.

Statistics and figure preparation

Statistical analysis and graph preparation were performed with GraphPad Prism. Statistical analyses used were the unpaired t-test with Welch’s correction, which does not assume equal standard deviations, and the Kolmogorov-Smirnov test to compare pooled distributions. See

Supplementary File 1

for all n and p values. Figures were assembled using Adobe Illustrator.

Figure supplements

Data availability

All data are available in the manuscript or supplementary materials.

Acknowledgements

The authors thank Corey Elowsky, Kazi Hossain, Lila Neahring, Carlos Patiño Descovich, Nathan Shugarts-Devanapally, Masako Tamada, and Carolyn Ton for helpful feedback on the manuscript. We are grateful to LARGE, RIKEN BDR (Kobe, Japan) for technical advice and provision of the HK3i ESC line, the

R26-H2B-EGFP

mouse line (

Abe et al., 2011

; RIKEN accession No. CDB0238K), and the

R26-PHA7-EGFP

mouse line (

Shioi et al., 2017

) (RIKEN accession No. CDB0261K). Use of services by MSK Core Facilities, including animal housing and veterinary services, mouse genome engineering, and DNA sequencing, was supported by NIH/NCI Cancer Center Support Grant P30 CA008748. ERB was supported by NIH/NINDS F32 fellowship NS098832 and startup funds from North Carolina State University. JAZ is an investigator of the Howard Hughes Medical Institute.

Additional information

Funding

Howard Hughes Medical Institute to Jennifer A Zallen, National Cancer Institute P30 CA008748 to Yas Furuta and Jennifer A Zallen, National Institute of Neurological Disorders and Stroke NS098832 to Eric R Brooks, North Carolina State University to Eric R Brooks.

Author contributions

Ian S Prudhomme – Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review and editing; Eric R Brooks – Conceptualization, Investigation, Methodology, Writing – review and editing; Nilay Taneja – Formal analysis, Investigation, Methodology, Visualization, Writing – review and editing; Bhaswati Bhattacharya – Investigation, Methodology; Brian J LaFleche – Methodology; Yas Furuta – Conceptualization and optimization of ESC-based methodology, Writing – review and editing; Jennifer A Zallen – Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing – review and editing.

Ethics

All animal experiments were conducted in accordance with PHS guidelines and the NIH Guide for the Care and Use of Laboratory Animals and approved Institutional Animal Care and Use Committee protocols (15-08-013 and 90-12-033) of Memorial Sloan Kettering Cancer Center.

Funding

Howard Hughes Medical Institute (HHMI)

• 
  

  
  Jennifer A Zallen
  

  
  

HHS | NIH | National Institute of Neurological Disorders and Stroke (NINDS) (NS098832)

• 
  

  
  Eric R Brooks
  

  
  

HHS | NIH | National Cancer Institute (NCI) (P30 CA008748)

• 
  

  
  Yasuhide Furuta
  

  
  


• 
  

  
  Jennifer A Zallen
  

  
  

North Carolina State University (NCSU)

• 
  

  
  Eric R Brooks
  

  
  

Additional files

Supplemental Movie 1.

Time-lapse movie of a Control

^ESC

embryo expressing GFP-Plekha7.
Images were acquired every 6 min for 3 hr. Maximum intensity projections, anterior up. Bar, 10 μm.

Supplemental Movie 2.

Time-lapse movie of a moderately defective

Vinculin

^ESC


embryo expressing GFP-Plekha7.
Images were acquired every 6 min for 3 hr. Maximum intensity projections, anterior up. Bar, 10 μm.

Supplemental Movie 3.

Time-lapse movie of a severely defective

Vinculin

^ESC


embryo expressing GFP-Plekha7.
Images were acquired every 6 min for 3 hr. Maximum intensity projections, anterior up. Bar, 10 μm.

References

• 
  
  
  
  1. 
     Abe T
     
  
  
  2. 
     Kiyonari H
     
  
  
  3. 
     Shioi G
     
  
  
  4. 
     Inoue K-I
     
  
  
  5. 
     Nakao K
     
  
  
  6. 
     Aizawa S
     
  
  
  7. 
     Fujimori T
     
  
  
  
  
  2011
  
  
  
  Establishment of conditional reporter mouse lines at ROSA26 locus for live cell imaging
  
  
  
  
  Genesis
  
  
  49
  
  :579–590
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Alonso-Curbelo D
     
  
  
  2. 
     Ho Y-J
     
  
  
  3. 
     Burdziak C
     
  
  
  4. 
     Maag JLV
     
  
  
  5. 
     Morris JP
     
  
  
  6. 
     Chandwani R
     
  
  
  7. 
     Chen H-A
     
  
  
  8. 
     Tsanov KM
     
  
  
  9. 
     Barriga FM
     
  
  
  10. 
      Luan W
      
  
  
  11. 
      Tasdemir N
      
  
  
  12. 
      Livshits G
      
  
  
  13. 
      Azizi E
      
  
  
  14. 
      Chun J
      
  
  
  15. 
      Wilkinson JE
      
  
  
  16. 
      Mazutis L
      
  
  
  17. 
      Leach SD
      
  
  
  18. 
      Koche R
      
  
  
  19. 
      Pe’er D
      
  
  
  20. 
      Lowe SW
      
  
  
  
  
  2021
  
  
  
  A gene-environment-induced epigenetic program initiates tumorigenesis
  
  
  
  
  Nature
  
  
  590
  
  :642–648
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Alvarez IS
     
  
  
  2. 
     Schoenwolf GC
     
  
  
  
  
  1991
  
  
  
  Patterns of neurepithelial cell rearrangement during avian neurulation are determined prior to notochordal inductive interactions
  
  
  
  
  Dev Biol
  
  
  143
  
  :78–92
  
  
  
  https://doi.org/10.1016/0012-1606(91)90056-9
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Amadei G
     
  
  
  2. 
     Handford CE
     
  
  
  3. 
     Qiu C
     
  
  
  4. 
     De Jonghe J
     
  
  
  5. 
     Greenfeld H
     
  
  
  6. 
     Tran M
     
  
  
  7. 
     Martin BK
     
  
  
  8. 
     Chen D-Y
     
  
  
  9. 
     Aguilera-Castrejon A
     
  
  
  10. 
      Hanna JH
      
  
  
  11. 
      Elowitz MB
      
  
  
  12. 
      Hollfelder F
      
  
  
  13. 
      Shendure J
      
  
  
  14. 
      Glover DM
      
  
  
  15. 
      Zernicka-Goetz M
      
  
  
  
  
  2022
  
  
  
  Embryo model completes gastrulation to neurulation and organogenesis
  
  
  
  
  Nature
  
  
  610
  
  :143–153
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Baldwin AT
     
  
  
  2. 
     Kim JH
     
  
  
  3. 
     Seo H
     
  
  
  4. 
     Wallingford JB
     
  
  
  
  
  2022
  
  
  
  Global analysis of cell behavior and protein dynamics reveals region-specific roles for Shroom3 and N-cadherin during neural tube closure
  
  
  
  
  eLife
  
  
  11
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Bao J
     
  
  
  2. 
     Ma X
     
  
  
  3. 
     Liu C
     
  
  
  4. 
     Adelstein RS
     
  
  
  
  
  2007
  
  
  
  Replacement of nonmuscle myosin II-B with II-A rescues brain but not cardiac defects in mice
  
  
  
  
  J Biol Chem
  
  
  282
  
  :22102–22111
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Bays JL
     
  
  
  2. 
     DeMali KA
     
  
  
  
  
  2017
  
  
  
  Vinculin in cell-cell and cell-matrix adhesions
  
  
  
  
  Cell Mol Life Sci
  
  
  74
  
  :2999–3009
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Beddington RS
     
  
  
  2. 
     Robertson EJ
     
  
  
  
  
  1989
  
  
  
  An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo
  
  
  
  
  Development
  
  
  105
  
  :733–737
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Behringer R
     
  
  
  2. 
     Gertsenstein M
     
  
  
  3. 
     Nagy K
     
  
  
  4. 
     Nagy A
     
  
  
  
  
  2013
  
  
  Manipulating the mouse embryo: A laboratory manual, fourth edition
  
  
  New York, NY
  :
  Cold Spring Harbor Laboratory Press
  
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Biswas R
     
  
  
  2. 
     Banerjee A
     
  
  
  3. 
     Lembo S
     
  
  
  4. 
     Zhao Z
     
  
  
  5. 
     Lakshmanan V
     
  
  
  6. 
     Lim R
     
  
  
  7. 
     Le S
     
  
  
  8. 
     Nakasaki M
     
  
  
  9. 
     Kutyavin V
     
  
  
  10. 
      Wright G
      
  
  
  11. 
      Palakodeti D
      
  
  
  12. 
      Ross RS
      
  
  
  13. 
      Jamora C
      
  
  
  14. 
      Vasioukhin V
      
  
  
  15. 
      Jie Y
      
  
  
  16. 
      Raghavan S
      
  
  
  
  
  2021
  
  
  
  Mechanical instability of adherens junctions overrides intrinsic quiescence of hair follicle stem cells
  
  
  
  
  Dev Cell
  
  
  56
  
  :761–780
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Blankenship JT
     
  
  
  2. 
     Backovic ST
     
  
  
  3. 
     Sanny JSP
     
  
  
  4. 
     Weitz O
     
  
  
  5. 
     Zallen JA
     
  
  
  
  
  2006
  
  
  
  Multicellular rosette formation links planar cell polarity to tissue morphogenesis
  
  
  
  
  Dev Cell
  
  
  11
  
  :459–470
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Bogart AH
     
  
  
  2. 
     Brooks ER
     
  
  
  
  
  2025
  
  
  
  Canonical Wnt pathway modulation is required to correctly execute multiple independent cellular dynamic programs during cranial neural tube closure
  
  
  
  
  Dev Biol
  
  
  523
  
  :115–131
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Bohere J
     
  
  
  2. 
     Eldridge-Thomas BL
     
  
  
  3. 
     Kolahgar G
     
  
  
  
  
  2022
  
  
  
  Vinculin recruitment to α-catenin halts the differentiation and maturation of enterocyte progenitors to maintain homeostasis of the Drosophila intestine
  
  
  
  
  eLife
  
  
  11
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Bosveld F
     
  
  
  2. 
     Wang Z
     
  
  
  3. 
     Bellaïche Y
     
  
  
  
  
  2018
  
  
  
  Tricellular junctions: a hot corner of epithelial biology
  
  
  
  
  Curr Opin Cell Biol
  
  
  54
  
  :80–88
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Bradley A
     
  
  
  2. 
     Evans M
     
  
  
  3. 
     Kaufman MH
     
  
  
  4. 
     Robertson E
     
  
  
  
  
  1984
  
  
  
  Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines
  
  
  
  
  Nature
  
  
  309
  
  :255–256
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Brooks ER
     
  
  
  2. 
     Islam MT
     
  
  
  3. 
     Anderson KV
     
  
  
  4. 
     Zallen JA
     
  
  
  
  
  2020
  
  
  
  Sonic hedgehog signaling directs patterned cell remodeling during cranial neural tube closure
  
  
  
  
  eLife
  
  
  9
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Brooks ER
     
  
  
  2. 
     Moorman AR
     
  
  
  3. 
     Bhattacharya B
     
  
  
  4. 
     Prudhomme IS
     
  
  
  5. 
     Land M
     
  
  
  6. 
     Alcorn HL
     
  
  
  7. 
     Sharma R
     
  
  
  8. 
     Pe’er D
     
  
  
  9. 
     Zallen JA
     
  
  
  
  
  2025
  
  
  
  A single-cell atlas of spatial and temporal gene expression in the mouse cranial neural plate
  
  
  
  
  eLife
  
  
  13
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Buckley CD
     
  
  
  2. 
     Tan J
     
  
  
  3. 
     Anderson KL
     
  
  
  4. 
     Hanein D
     
  
  
  5. 
     Volkmann N
     
  
  
  6. 
     Weis WI
     
  
  
  7. 
     Nelson WJ
     
  
  
  8. 
     Dunn AR
     
  
  
  
  
  2014
  
  
  
  Cell adhesion. The minimal cadherin-catenin complex binds to actin filaments under force
  
  
  
  
  Science
  
  
  346
  
  :1254211
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Burdziak C
     
  
  
  2. 
     Alonso-Curbelo D
     
  
  
  3. 
     Walle T
     
  
  
  4. 
     Reyes J
     
  
  
  5. 
     Barriga FM
     
  
  
  6. 
     Haviv D
     
  
  
  7. 
     Xie Y
     
  
  
  8. 
     Zhao Z
     
  
  
  9. 
     Zhao CJ
     
  
  
  10. 
      Chen H-A
      
  
  
  11. 
      Chaudhary O
      
  
  
  12. 
      Masilionis I
      
  
  
  13. 
      Choo Z-N
      
  
  
  14. 
      Gao V
      
  
  
  15. 
      Luan W
      
  
  
  16. 
      Wuest A
      
  
  
  17. 
      Ho Y-J
      
  
  
  18. 
      Wei Y
      
  
  
  19. 
      Quail DF
      
  
  
  20. 
      Koche R
      
  
  
  21. 
      Mazutis L
      
  
  
  22. 
      Chaligné R
      
  
  
  23. 
      Nawy T
      
  
  
  24. 
      Lowe SW
      
  
  
  25. 
      Pe’er D
      
  
  
  
  
  2023
  
  
  
  Epigenetic plasticity cooperates with cell-cell interactions to direct pancreatic tumorigenesis
  
  
  
  
  Science
  
  
  380
  
  :eadd5327
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Burridge K
     
  
  
  2. 
     Guilluy C
     
  
  
  
  
  2016
  
  
  
  Focal adhesions, stress fibers and mechanical tension
  
  
  
  
  Exp Cell Res
  
  
  343
  
  :14–20
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Capecchi MR
     
  
  
  
  
  2005
  
  
  
  Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century
  
  
  
  
  Nat Rev Genet
  
  
  6
  
  :507–512
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Carvalho JR
     
  
  
  2. 
     Fortunato IC
     
  
  
  3. 
     Fonseca CG
     
  
  
  4. 
     Pezzarossa A
     
  
  
  5. 
     Barbacena P
     
  
  
  6. 
     Dominguez-Cejudo MA
     
  
  
  7. 
     Vasconcelos FF
     
  
  
  8. 
     Santos NC
     
  
  
  9. 
     Carvalho FA
     
  
  
  10. 
      Franco CA
      
  
  
  
  
  2019
  
  
  
  Non-canonical Wnt signaling regulates junctional mechanocoupling during angiogenic collective cell migration
  
  
  
  
  eLife
  
  
  8
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Cavanaugh KE
     
  
  
  2. 
     Staddon MF
     
  
  
  3. 
     Chmiel TA
     
  
  
  4. 
     Harmon R
     
  
  
  5. 
     Budnar S
     
  
  
  6. 
     Yap AS
     
  
  
  7. 
     Banerjee S
     
  
  
  8. 
     Gardel ML
     
  
  
  
  
  2022
  
  
  
  Force-dependent intercellular adhesion strengthening underlies asymmetric adherens junction contraction
  
  
  
  
  Curr Biol
  
  
  32
  
  :1986–2000
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Cheng F
     
  
  
  2. 
     Miao L
     
  
  
  3. 
     Wu Q
     
  
  
  4. 
     Gong X
     
  
  
  5. 
     Xiong J
     
  
  
  6. 
     Zhang J
     
  
  
  
  
  2016
  
  
  
  Vinculin b deficiency causes epicardial hyperplasia and coronary vessel disorganization in zebrafish
  
  
  
  
  Development
  
  
  143
  
  :3522–3531
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Cho Y
     
  
  
  2. 
     Haraguchi D
     
  
  
  3. 
     Shigetomi K
     
  
  
  4. 
     Matsuzawa K
     
  
  
  5. 
     Uchida S
     
  
  
  6. 
     Ikenouchi J
     
  
  
  
  
  2022
  
  
  
  Tricellulin secures the epithelial barrier at tricellular junctions by interacting with actomyosin
  
  
  
  
  J Cell Biol
  
  
  221
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Choi W
     
  
  
  2. 
     Acharya BR
     
  
  
  3. 
     Peyret G
     
  
  
  4. 
     Fardin M-A
     
  
  
  5. 
     Mège R-M
     
  
  
  6. 
     Ladoux B
     
  
  
  7. 
     Yap AS
     
  
  
  8. 
     Fanning AS
     
  
  
  9. 
     Peifer M
     
  
  
  
  
  2016
  
  
  
  Remodeling the zonula adherens in response to tension and the role of afadin in this response
  
  
  
  
  J Cell Biol
  
  
  213
  
  :243–260
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Chu C-W
     
  
  
  2. 
     Xiang B
     
  
  
  3. 
     Ossipova O
     
  
  
  4. 
     Ioannou A
     
  
  
  5. 
     Sokol SY
     
  
  
  
  
  2018
  
  
  
  The Ajuba family protein Wtip regulates actomyosin contractility during vertebrate neural tube closure
  
  
  
  
  J Cell Sci
  
  
  131
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Citi S
     
  
  
  
  
  2019
  
  
  
  The mechanobiology of tight junctions
  
  
  
  
  Biophys Rev
  
  
  11
  
  :783–793
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Citi S
     
  
  
  2. 
     Fromm M
     
  
  
  3. 
     Furuse M
     
  
  
  4. 
     González-Mariscal L
     
  
  
  5. 
     Nusrat A
     
  
  
  6. 
     Tsukita S
     
  
  
  7. 
     Turner JR
     
  
  
  
  
  2024
  
  
  
  A short guide to the tight junction
  
  
  
  
  J Cell Sci
  
  
  137
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Concordet J-P
     
  
  
  2. 
     Haeussler M
     
  
  
  
  
  2018
  
  
  
  CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens
  
  
  
  
  Nucleic Acids Res
  
  
  46
  
  :W242–W245
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Cunningham F
     
  
  
  2. 
     Allen JE
     
  
  
  3. 
     Allen J
     
  
  
  4. 
     Alvarez-Jarreta J
     
  
  
  5. 
     Amode MR
     
  
  
  6. 
     Armean IM
     
  
  
  7. 
     Austine-Orimoloye O
     
  
  
  8. 
     Azov AG
     
  
  
  9. 
     Barnes I
     
  
  
  10. 
      Bennett R
      
  
  
  11. 
      Berry A
      
  
  
  12. 
      Bhai J
      
  
  
  13. 
      Bignell A
      
  
  
  14. 
      Billis K
      
  
  
  15. 
      Boddu S
      
  
  
  16. 
      Brooks L
      
  
  
  17. 
      Charkhchi M
      
  
  
  18. 
      Cummins C
      
  
  
  19. 
      Da Rin Fioretto L
      
  
  
  20. 
      Davidson C
      
  
  
  21. 
      Dodiya K
      
  
  
  22. 
      Donaldson S
      
  
  
  23. 
      El Houdaigui B
      
  
  
  24. 
      El Naboulsi T
      
  
  
  25. 
      Fatima R
      
  
  
  26. 
      Giron CG
      
  
  
  27. 
      Genez T
      
  
  
  28. 
      Martinez JG
      
  
  
  29. 
      Guijarro-Clarke C
      
  
  
  30. 
      Gymer A
      
  
  
  31. 
      Hardy M
      
  
  
  32. 
      Hollis Z
      
  
  
  33. 
      Hourlier T
      
  
  
  34. 
      Hunt T
      
  
  
  35. 
      Juettemann T
      
  
  
  36. 
      Kaikala V
      
  
  
  37. 
      Kay M
      
  
  
  38. 
      Lavidas I
      
  
  
  39. 
      Le T
      
  
  
  40. 
      Lemos D
      
  
  
  41. 
      Marugán JC
      
  
  
  42. 
      Mohanan S
      
  
  
  43. 
      Mushtaq A
      
  
  
  44. 
      Naven M
      
  
  
  45. 
      Ogeh DN
      
  
  
  46. 
      Parker A
      
  
  
  47. 
      Parton A
      
  
  
  48. 
      Perry M
      
  
  
  49. 
      Piližota I
      
  
  
  50. 
      Prosovetskaia I
      
  
  
  51. 
      Sakthivel MP
      
  
  
  52. 
      Salam AIA
      
  
  
  53. 
      Schmitt BM
      
  
  
  54. 
      Schuilenburg H
      
  
  
  55. 
      Sheppard D
      
  
  
  56. 
      Pérez-Silva JG
      
  
  
  57. 
      Stark W
      
  
  
  58. 
      Steed E
      
  
  
  59. 
      Sutinen K
      
  
  
  60. 
      Sukumaran R
      
  
  
  61. 
      Sumathipala D
      
  
  
  62. 
      Suner M-M
      
  
  
  63. 
      Szpak M
      
  
  
  64. 
      Thormann A
      
  
  
  65. 
      Tricomi FF
      
  
  
  66. 
      Urbina-Gómez D
      
  
  
  67. 
      Veidenberg A
      
  
  
  68. 
      Walsh TA
      
  
  
  69. 
      Walts B
      
  
  
  70. 
      Willhoft N
      
  
  
  71. 
      Winterbottom A
      
  
  
  72. 
      Wass E
      
  
  
  73. 
      Chakiachvili M
      
  
  
  74. 
      Flint B
      
  
  
  75. 
      Frankish A
      
  
  
  76. 
      Giorgetti S
      
  
  
  77. 
      Haggerty L
      
  
  
  78. 
      Hunt SE
      
  
  
  79. 
      IIsley GR
      
  
  
  80. 
      Loveland JE
      
  
  
  81. 
      Martin FJ
      
  
  
  82. 
      Moore B
      
  
  
  83. 
      Mudge JM
      
  
  
  84. 
      Muffato M
      
  
  
  85. 
      Perry E
      
  
  
  86. 
      Ruffier M
      
  
  
  87. 
      Tate J
      
  
  
  88. 
      Thybert D
      
  
  
  89. 
      Trevanion SJ
      
  
  
  90. 
      Dyer S
      
  
  
  91. 
      Harrison PW
      
  
  
  92. 
      Howe KL
      
  
  
  93. 
      Yates AD
      
  
  
  94. 
      Zerbino DR
      
  
  
  95. 
      Flicek P
      
  
  
  
  
  2022
  
  
  
  Ensembl 2022
  
  
  
  
  Nucleic Acids Res
  
  
  50
  
  :D988–D995
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Davidson LA
     
  
  
  2. 
     Keller RE
     
  
  
  
  
  1999
  
  
  
  Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation and convergent extension
  
  
  
  
  Development
  
  
  126
  
  :4547–4556
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     De La O J
     
  
  
  2. 
     Okeke C
     
  
  
  3. 
     Galea GL
     
  
  
  4. 
     Martin AC
     
  
  
  
  
  2025
  
  
  
  An actomyosin-mediated mechanical mechanism for brain neural tube elevation
  
  
  
  
  bioRxiv
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Dow LE
     
  
  
  2. 
     Lowe SW
     
  
  
  
  
  2012
  
  
  
  Life in the fast lane: mammalian disease models in the genomics era
  
  
  
  
  Cell
  
  
  148
  
  :1099–1109
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Farhadifar R
     
  
  
  2. 
     Röper J-C
     
  
  
  3. 
     Aigouy B
     
  
  
  4. 
     Eaton S
     
  
  
  5. 
     Jülicher F
     
  
  
  
  
  2007
  
  
  
  The influence of cell mechanics, cell-cell interactions, and proliferation on epithelial packing
  
  
  
  
  Curr Biol
  
  
  17
  
  :2095–2104
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Fernandez-Gonzalez R
     
  
  
  2. 
     Zallen JA
     
  
  
  
  
  2011
  
  
  
  Oscillatory behaviors and hierarchical assembly of contractile structures in intercalating cells
  
  
  
  
  Phys Biol
  
  
  8
  
  :045005
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Founounou N
     
  
  
  2. 
     Loyer N
     
  
  
  3. 
     Le Borgne R
     
  
  
  
  
  2013
  
  
  
  Septins regulate the contractility of the actomyosin ring to enable adherens junction remodeling during cytokinesis of epithelial cells
  
  
  
  
  Dev Cell
  
  
  24
  
  :242–255
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Friedrich G
     
  
  
  2. 
     Soriano P
     
  
  
  
  
  1991
  
  
  
  Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice
  
  
  
  
  Genes Dev
  
  
  5
  
  :1513–1523
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Fukuda R
     
  
  
  2. 
     Gunawan F
     
  
  
  3. 
     Ramadass R
     
  
  
  4. 
     Beisaw A
     
  
  
  5. 
     Konzer A
     
  
  
  6. 
     Mullapudi ST
     
  
  
  7. 
     Gentile A
     
  
  
  8. 
     Maischein H-M
     
  
  
  9. 
     Graumann J
     
  
  
  10. 
      Stainier DYR
      
  
  
  
  
  2019
  
  
  
  Mechanical forces regulate cardiomyocyte myofilament maturation via the VCL-SSH1-CFL axis
  
  
  
  
  Dev Cell
  
  
  51
  
  :62–77
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Goldmann WH
     
  
  
  
  
  2016
  
  
  
  Role of vinculin in cellular mechanotransduction
  
  
  
  
  Cell Biol Int
  
  
  40
  
  :241–256
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Grego-Bessa J
     
  
  
  2. 
     Bloomekatz J
     
  
  
  3. 
     Castel P
     
  
  
  4. 
     Omelchenko T
     
  
  
  5. 
     Baselga J
     
  
  
  6. 
     Anderson KV
     
  
  
  
  
  2016
  
  
  
  The tumor suppressor PTEN and the PDK1 kinase regulate formation of the columnar neural epithelium
  
  
  
  
  eLife
  
  
  5
  
  :e12034
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Grego-Bessa J
     
  
  
  2. 
     Hildebrand J
     
  
  
  3. 
     Anderson KV
     
  
  
  
  
  2015
  
  
  
  Morphogenesis of the mouse neural plate depends on distinct roles of cofilin 1 in apical and basal epithelial domains
  
  
  
  
  Development
  
  
  142
  
  :1305–1314
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Guillot C
     
  
  
  2. 
     Lecuit T
     
  
  
  
  
  2013
  
  
  
  Adhesion disengagement uncouples intrinsic and extrinsic forces to drive cytokinesis in epithelial tissues
  
  
  
  
  Dev Cell
  
  
  24
  
  :227–241
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Hadjantonakis A-K
     
  
  
  2. 
     Gertsenstein M
     
  
  
  3. 
     Ikawa M
     
  
  
  4. 
     Okabe M
     
  
  
  5. 
     Nagy A
     
  
  
  
  
  1998
  
  
  
  Generating green fluorescent mice by germline transmission of green fluorescent ES cells
  
  
  
  
  Mech Dev
  
  
  76
  
  :79–90
  
  
  
  https://doi.org/10.1016/s0925-4773(98)00093-8
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Haigo SL
     
  
  
  2. 
     Hildebrand JD
     
  
  
  3. 
     Harland RM
     
  
  
  4. 
     Wallingford JB
     
  
  
  
  
  2003
  
  
  
  Shroom induces apical constriction and is required for hingepoint formation during neural tube closure
  
  
  
  
  Curr Biol
  
  
  13
  
  :2125–2137
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Hayashi S
     
  
  
  2. 
     Lewis P
     
  
  
  3. 
     Pevny L
     
  
  
  4. 
     McMahon AP
     
  
  
  
  
  2002
  
  
  
  Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain
  
  
  
  
  Mech Dev
  
  
  119
  
  :S97–S101
  
  
  
  https://doi.org/10.1016/s0925-4773(03)00099-6
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Herszterg S
     
  
  
  2. 
     Leibfried A
     
  
  
  3. 
     Bosveld F
     
  
  
  4. 
     Martin C
     
  
  
  5. 
     Bellaiche Y
     
  
  
  
  
  2013
  
  
  
  Interplay between the dividing cell and its neighbors regulates adherens junction formation during cytokinesis in epithelial tissue
  
  
  
  
  Dev Cell
  
  
  24
  
  :256–270
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Higashi T
     
  
  
  2. 
     Arnold TR
     
  
  
  3. 
     Stephenson RE
     
  
  
  4. 
     Dinshaw KM
     
  
  
  5. 
     Miller AL
     
  
  
  
  
  2016
  
  
  
  Maintenance of the epithelial barrier and remodeling of cell-cell junctions during cytokinesis
  
  
  
  
  Curr Biol
  
  
  26
  
  :1829–1842
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Higashi T
     
  
  
  2. 
     Miller AL
     
  
  
  
  
  2017
  
  
  
  Tricellular junctions: how to build junctions at the TRICkiest points of epithelial cells
  
  
  
  
  Mol Biol Cell
  
  
  28
  
  :2023–2034
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Hildebrand JD
     
  
  
  
  
  2005
  
  
  
  Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network
  
  
  
  
  J Cell Sci
  
  
  118
  
  :5191–5203
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Hildebrand JD
     
  
  
  2. 
     Soriano P
     
  
  
  
  
  1999
  
  
  
  Shroom, a PDZ Domain–Containing Actin-Binding Protein, Is Required for Neural Tube Morphogenesis in Mice
  
  
  
  
  Cell
  
  
  99
  
  :485–497
  
  
  
  https://doi.org/10.1016/s0092-8674(00)81537-8
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Huang DL
     
  
  
  2. 
     Bax NA
     
  
  
  3. 
     Buckley CD
     
  
  
  4. 
     Weis WI
     
  
  
  5. 
     Dunn AR
     
  
  
  
  
  2017
  
  
  
  Vinculin forms a directionally asymmetric catch bond with F-actin
  
  
  
  
  Science
  
  
  357
  
  :703–706
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Hutson MS
     
  
  
  2. 
     Tokutake Y
     
  
  
  3. 
     Chang M-S
     
  
  
  4. 
     Bloor JW
     
  
  
  5. 
     Venakides S
     
  
  
  6. 
     Kiehart DP
     
  
  
  7. 
     Edwards GS
     
  
  
  
  
  2003
  
  
  
  Forces for morphogenesis investigated with laser microsurgery and quantitative modeling
  
  
  
  
  Science
  
  
  300
  
  :145–149
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Huveneers S
     
  
  
  2. 
     Oldenburg J
     
  
  
  3. 
     Spanjaard E
     
  
  
  4. 
     van der Krogt G
     
  
  
  5. 
     Grigoriev I
     
  
  
  6. 
     Akhmanova A
     
  
  
  7. 
     Rehmann H
     
  
  
  8. 
     de Rooij J
     
  
  
  
  
  2012
  
  
  
  Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling
  
  
  
  
  J Cell Biol
  
  
  196
  
  :641–652
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Ito S
     
  
  
  2. 
     Okuda S
     
  
  
  3. 
     Abe M
     
  
  
  4. 
     Fujimoto M
     
  
  
  5. 
     Onuki T
     
  
  
  6. 
     Nishimura T
     
  
  
  7. 
     Takeichi M
     
  
  
  
  
  2017
  
  
  
  Induced cortical tension restores functional junctions in adhesion-defective carcinoma cells
  
  
  
  
  Nat Commun
  
  
  28
  
  :1834
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Itoh K
     
  
  
  2. 
     Ossipova O
     
  
  
  3. 
     Sokol SY
     
  
  
  
  
  2025
  
  
  
  Myocardin-related transcription factor regulates actomyosin contractility and apical junction remodeling during vertebrate neural tube closure
  
  
  
  
  Development
  
  
  152
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Jacobson AG
     
  
  
  2. 
     Tam PP
     
  
  
  
  
  1982
  
  
  
  Cephalic neurulation in the mouse embryo analyzed by SEM and morphometry
  
  
  
  
  Anat Rec
  
  
  203
  
  :375–396
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Jorgensen V
     
  
  
  2. 
     Bao M
     
  
  
  3. 
     Junyent S
     
  
  
  4. 
     Häfelfinger CM
     
  
  
  5. 
     Amaya L
     
  
  
  6. 
     Liao Z
     
  
  
  7. 
     Williams BA
     
  
  
  8. 
     Chen D-Y
     
  
  
  9. 
     Wu A
     
  
  
  10. 
      Thomson M
      
  
  
  11. 
      Zernicka-Goetz M
      
  
  
  
  
  2025
  
  
  
  Efficient stem cell-derived mouse embryo models for environmental studies
  
  
  
  
  Dev Cell
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Juriloff DM
     
  
  
  2. 
     Harris MJ
     
  
  
  
  
  2018
  
  
  
  Insights into the Etiology of Mammalian Neural Tube Closure Defects from Developmental, Genetic and Evolutionary Studies
  
  
  
  
  J Dev Biol
  
  
  6
  
  :22
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Kannan N
     
  
  
  2. 
     Tang VW
     
  
  
  
  
  2015
  
  
  
  Synaptopodin couples epithelial contractility to α-actinin-4-dependent junction maturation
  
  
  
  
  J Cell Biol
  
  
  211
  
  :407–434
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Kiyonari H
     
  
  
  2. 
     Kaneko M
     
  
  
  3. 
     Abe S-I
     
  
  
  4. 
     Aizawa S
     
  
  
  
  
  2010
  
  
  
  Three inhibitors of FGF receptor, ERK, and GSK3 establishes germline-competent embryonic stem cells of C57BL/6N mouse strain with high efficiency and stability
  
  
  
  
  Genesis
  
  
  48
  
  :317–327
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Kondo T
     
  
  
  2. 
     Hayashi S
     
  
  
  
  
  2013
  
  
  
  Mitotic cell rounding accelerates epithelial invagination
  
  
  
  
  Nature
  
  
  494
  
  :125–129
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Konishi S
     
  
  
  2. 
     Yano T
     
  
  
  3. 
     Tanaka H
     
  
  
  4. 
     Mizuno T
     
  
  
  5. 
     Kanoh H
     
  
  
  6. 
     Tsukita K
     
  
  
  7. 
     Namba T
     
  
  
  8. 
     Tamura A
     
  
  
  9. 
     Yonemura S
     
  
  
  10. 
      Gotoh S
      
  
  
  11. 
      Matsumoto H
      
  
  
  12. 
      Hirai T
      
  
  
  13. 
      Tsukita S
      
  
  
  
  
  2019
  
  
  
  Vinculin is critical for the robustness of the epithelial cell sheet paracellular barrier for ions
  
  
  
  
  Life Sci Alliance
  
  
  2
  
  :e201900414
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Kotini MP
     
  
  
  2. 
     van der Stoel MM
     
  
  
  3. 
     Yin J
     
  
  
  4. 
     Han MK
     
  
  
  5. 
     Kirchmaier B
     
  
  
  6. 
     de Rooij J
     
  
  
  7. 
     Affolter M
     
  
  
  8. 
     Huveneers S
     
  
  
  9. 
     Belting H-G
     
  
  
  
  
  2022
  
  
  
  Vinculin controls endothelial cell junction dynamics during vascular lumen formation
  
  
  
  
  Cell Rep
  
  
  39
  
  :110658
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Kwon GS
     
  
  
  2. 
     Viotti M
     
  
  
  3. 
     Hadjantonakis A-K
     
  
  
  
  
  2008
  
  
  
  The endoderm of the mouse embryo arises by dynamic widespread intercalation of embryonic and extraembryonic lineages
  
  
  
  
  Dev Cell
  
  
  15
  
  :509–520
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Labun K
     
  
  
  2. 
     Montague TG
     
  
  
  3. 
     Krause M
     
  
  
  4. 
     Torres Cleuren YN
     
  
  
  5. 
     Tjeldnes H
     
  
  
  6. 
     Valen E
     
  
  
  
  
  2019
  
  
  
  CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing
  
  
  
  
  Nucleic Acids Res
  
  
  47
  
  :W171–W174
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Lai KMV
     
  
  
  2. 
     Gong C
     
  
  
  3. 
     Atanasio A
     
  
  
  4. 
     Rojas J
     
  
  
  5. 
     Quispe J
     
  
  
  6. 
     Posca J
     
  
  
  7. 
     White D
     
  
  
  8. 
     Huang M
     
  
  
  9. 
     Fedorova D
     
  
  
  10. 
      Grant C
      
  
  
  11. 
      Miloscio L
      
  
  
  12. 
      Droguett G
      
  
  
  13. 
      Poueymirou WT
      
  
  
  14. 
      Auerbach W
      
  
  
  15. 
      Yancopoulos GD
      
  
  
  16. 
      Frendewey D
      
  
  
  17. 
      Rinn J
      
  
  
  18. 
      Valenzuela DM
      
  
  
  
  
  2015
  
  
  
  Diverse phenotypes and specific transcription patterns in twenty mouse lines with ablated lincRNAs
  
  
  
  
  PLoS One
  
  
  10
  
  :e0125522
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Lallemand Y
     
  
  
  2. 
     Brûlet P
     
  
  
  
  
  1990
  
  
  
  An in situ assessment of the routes and extents of colonisation of the mouse embryo by embryonic stem cells and their descendants
  
  
  
  
  Development
  
  
  110
  
  :1241–1248
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Landino J
     
  
  
  2. 
     Misterovich E
     
  
  
  3. 
     van den Goor L
     
  
  
  4. 
     Adhikary B
     
  
  
  5. 
     Chumki S
     
  
  
  6. 
     Davidson LA
     
  
  
  7. 
     Miller AL
     
  
  
  
  
  2025
  
  
  
  Neighbor cells restrain furrowing during Xenopus epithelial cytokinesis
  
  
  
  
  Dev Cell
  
  
  60
  
  :2139–2148
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Le Clainche C
     
  
  
  2. 
     Dwivedi SP
     
  
  
  3. 
     Didry D
     
  
  
  4. 
     Carlier M-F
     
  
  
  
  
  2010
  
  
  
  Vinculin is a dually regulated actin filament barbed end-capping and side-binding protein
  
  
  
  
  J Biol Chem
  
  
  285
  
  :23420–23432
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     le Duc Q
     
  
  
  2. 
     Shi Q
     
  
  
  3. 
     Blonk I
     
  
  
  4. 
     Sonnenberg A
     
  
  
  5. 
     Wang N
     
  
  
  6. 
     Leckband D
     
  
  
  7. 
     de Rooij J
     
  
  
  
  
  2010
  
  
  
  Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II-dependent manner
  
  
  
  
  J Cell Biol
  
  
  189
  
  :1107–1115
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Lee S
     
  
  
  2. 
     Gleeson JG
     
  
  
  
  
  2020
  
  
  
  Closing in on Mechanisms of Open Neural Tube Defects
  
  
  
  
  Trends Neurosci
  
  
  43
  
  :519–532
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Leerberg JM
     
  
  
  2. 
     Gomez GA
     
  
  
  3. 
     Verma S
     
  
  
  4. 
     Moussa EJ
     
  
  
  5. 
     Wu SK
     
  
  
  6. 
     Priya R
     
  
  
  7. 
     Hoffman BD
     
  
  
  8. 
     Grashoff C
     
  
  
  9. 
     Schwartz MA
     
  
  
  10. 
      Yap AS
      
  
  
  
  
  2014
  
  
  
  Tension-sensitive actin assembly supports contractility at the epithelial zonula adherens
  
  
  
  
  Curr Biol
  
  
  24
  
  :1689–1699
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Legland D
     
  
  
  2. 
     Arganda-Carreras I
     
  
  
  3. 
     Andrey P
     
  
  
  
  
  2016
  
  
  
  MorphoLibJ: integrated library and plugins for mathematical morphology with ImageJ
  
  
  
  
  Bioinformatics
  
  
  32
  
  :3532–3534
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Maddugoda MP
     
  
  
  2. 
     Crampton MS
     
  
  
  3. 
     Shewan AM
     
  
  
  4. 
     Yap AS
     
  
  
  
  
  2007
  
  
  
  Myosin VI and vinculin cooperate during the morphogenesis of cadherin cell cell contacts in mammalian epithelial cells
  
  
  
  
  J Cell Biol
  
  
  178
  
  :529–540
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Marg S
     
  
  
  2. 
     Winkler U
     
  
  
  3. 
     Sestu M
     
  
  
  4. 
     Himmel M
     
  
  
  5. 
     Schönherr M
     
  
  
  6. 
     Bär J
     
  
  
  7. 
     Mann A
     
  
  
  8. 
     Moser M
     
  
  
  9. 
     Mierke CT
     
  
  
  10. 
      Rottner K
      
  
  
  11. 
      Blessing M
      
  
  
  12. 
      Hirrlinger J
      
  
  
  13. 
      Ziegler WH
      
  
  
  
  
  2010
  
  
  
  The vinculin-DeltaIn20/21 mouse: characteristics of a constitutive, actin-binding deficient splice variant of vinculin
  
  
  
  
  PLoS One
  
  
  5
  
  :e11530
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Martin FJ
     
  
  
  2. 
     Amode MR
     
  
  
  3. 
     Aneja A
     
  
  
  4. 
     Austine-Orimoloye O
     
  
  
  5. 
     Azov AG
     
  
  
  6. 
     Barnes I
     
  
  
  7. 
     Becker A
     
  
  
  8. 
     Bennett R
     
  
  
  9. 
     Berry A
     
  
  
  10. 
      Bhai J
      
  
  
  11. 
      Bhurji SK
      
  
  
  12. 
      Bignell A
      
  
  
  13. 
      Boddu S
      
  
  
  14. 
      Branco Lins PR
      
  
  
  15. 
      Brooks L
      
  
  
  16. 
      Ramaraju SB
      
  
  
  17. 
      Charkhchi M
      
  
  
  18. 
      Cockburn A
      
  
  
  19. 
      Da Rin Fiorretto L
      
  
  
  20. 
      Davidson C
      
  
  
  21. 
      Dodiya K
      
  
  
  22. 
      Donaldson S
      
  
  
  23. 
      El Houdaigui B
      
  
  
  24. 
      El Naboulsi T
      
  
  
  25. 
      Fatima R
      
  
  
  26. 
      Giron CG
      
  
  
  27. 
      Genez T
      
  
  
  28. 
      Ghattaoraya GS
      
  
  
  29. 
      Martinez JG
      
  
  
  30. 
      Guijarro C
      
  
  
  31. 
      Hardy M
      
  
  
  32. 
      Hollis Z
      
  
  
  33. 
      Hourlier T
      
  
  
  34. 
      Hunt T
      
  
  
  35. 
      Kay M
      
  
  
  36. 
      Kaykala V
      
  
  
  37. 
      Le T
      
  
  
  38. 
      Lemos D
      
  
  
  39. 
      Marques-Coelho D
      
  
  
  40. 
      Marugán JC
      
  
  
  41. 
      Merino GA
      
  
  
  42. 
      Mirabueno LP
      
  
  
  43. 
      Mushtaq A
      
  
  
  44. 
      Hossain SN
      
  
  
  45. 
      Ogeh DN
      
  
  
  46. 
      Sakthivel MP
      
  
  
  47. 
      Parker A
      
  
  
  48. 
      Perry M
      
  
  
  49. 
      Piližota I
      
  
  
  50. 
      Prosovetskaia I
      
  
  
  51. 
      Pérez-Silva JG
      
  
  
  52. 
      Salam AIA
      
  
  
  53. 
      Saraiva-Agostinho N
      
  
  
  54. 
      Schuilenburg H
      
  
  
  55. 
      Sheppard D
      
  
  
  56. 
      Sinha S
      
  
  
  57. 
      Sipos B
      
  
  
  58. 
      Stark W
      
  
  
  59. 
      Steed E
      
  
  
  60. 
      Sukumaran R
      
  
  
  61. 
      Sumathipala D
      
  
  
  62. 
      Suner M-M
      
  
  
  63. 
      Surapaneni L
      
  
  
  64. 
      Sutinen K
      
  
  
  65. 
      Szpak M
      
  
  
  66. 
      Tricomi FF
      
  
  
  67. 
      Urbina-Gómez D
      
  
  
  68. 
      Veidenberg A
      
  
  
  69. 
      Walsh TA
      
  
  
  70. 
      Walts B
      
  
  
  71. 
      Wass E
      
  
  
  72. 
      Willhoft N
      
  
  
  73. 
      Allen J
      
  
  
  74. 
      Alvarez-Jarreta J
      
  
  
  75. 
      Chakiachvili M
      
  
  
  76. 
      Flint B
      
  
  
  77. 
      Giorgetti S
      
  
  
  78. 
      Haggerty L
      
  
  
  79. 
      Ilsley GR
      
  
  
  80. 
      Loveland JE
      
  
  
  81. 
      Moore B
      
  
  
  82. 
      Mudge JM
      
  
  
  83. 
      Tate J
      
  
  
  84. 
      Thybert D
      
  
  
  85. 
      Trevanion SJ
      
  
  
  86. 
      Winterbottom A
      
  
  
  87. 
      Frankish A
      
  
  
  88. 
      Hunt SE
      
  
  
  89. 
      Ruffier M
      
  
  
  90. 
      Cunningham F
      
  
  
  91. 
      Dyer S
      
  
  
  92. 
      Finn RD
      
  
  
  93. 
      Howe KL
      
  
  
  94. 
      Harrison PW
      
  
  
  95. 
      Yates AD
      
  
  
  96. 
      Flicek P
      
  
  
  
  
  2023
  
  
  
  Ensembl 2023
  
  
  
  
  Nucleic Acids Res
  
  
  51
  
  :D933–D941
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Mashburn DN
     
  
  
  2. 
     Lynch HE
     
  
  
  3. 
     Ma X
     
  
  
  4. 
     Hutson MS
     
  
  
  
  
  2012
  
  
  
  Enabling user-guided segmentation and tracking of surface-labeled cells in time-lapse image sets of living tissues
  
  
  
  
  Cytometry A
  
  
  81
  
  :409–418
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Massarwa R
     
  
  
  2. 
     Niswander L
     
  
  
  
  
  2013
  
  
  
  In toto live imaging of mouse morphogenesis and new insights into neural tube closure
  
  
  
  
  Development
  
  
  140
  
  :226–236
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Matsuda M
     
  
  
  2. 
     Rozman J
     
  
  
  3. 
     Ostvar S
     
  
  
  4. 
     Kasza KE
     
  
  
  5. 
     Sokol SY
     
  
  
  
  
  2023
  
  
  
  Mechanical control of neural plate folding by apical domain alteration
  
  
  
  
  Nat Commun
  
  
  14
  
  :8475
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Matsuda M
     
  
  
  2. 
     Sokol SY
     
  
  
  
  
  2021
  
  
  
  Xenopus neural tube closure: A vertebrate model linking planar cell polarity to actomyosin contractions
  
  
  
  
  Curr Top Dev Biol
  
  
  145
  
  :41–60
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     McGreevy EM
     
  
  
  2. 
     Vijayraghavan D
     
  
  
  3. 
     Davidson LA
     
  
  
  4. 
     Hildebrand JD
     
  
  
  
  
  2015
  
  
  
  Shroom3 functions downstream of planar cell polarity to regulate myosin II distribution and cellular organization during neural tube closure
  
  
  
  
  Biol Open
  
  
  4
  
  :186–196
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Monster JL
     
  
  
  2. 
     Donker L
     
  
  
  3. 
     Vliem MJ
     
  
  
  4. 
     Win Z
     
  
  
  5. 
     Matthews HK
     
  
  
  6. 
     Cheah JS
     
  
  
  7. 
     Yamada S
     
  
  
  8. 
     de Rooij J
     
  
  
  9. 
     Baum B
     
  
  
  10. 
      Gloerich M
      
  
  
  
  
  2021
  
  
  
  An asymmetric junctional mechanoresponse coordinates mitotic rounding with epithelial integrity
  
  
  
  
  J Cell Biol
  
  
  220
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Morriss-Kay GM
     
  
  
  
  
  1981
  
  
  
  Growth and development of pattern in the cranial neural epithelium of rat embryos during neurulation
  
  
  
  
  J Embryol Exp Morphol
  
  
  65 Suppl
  
  :225–241
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Mulas C
     
  
  
  2. 
     Kalkan T
     
  
  
  3. 
     von Meyenn F
     
  
  
  4. 
     Leitch HG
     
  
  
  5. 
     Nichols J
     
  
  
  6. 
     Smith A
     
  
  
  
  
  2019
  
  
  
  Defined conditions for propagation and manipulation of mouse embryonic stem cells
  
  
  
  
  Development
  
  
  146
  
  :dev173146
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Nagy A
     
  
  
  2. 
     Gócza E
     
  
  
  3. 
     Diaz EM
     
  
  
  4. 
     Prideaux VR
     
  
  
  5. 
     Iványi E
     
  
  
  6. 
     Markkula M
     
  
  
  7. 
     Rossant J
     
  
  
  
  
  1990
  
  
  
  Embryonic stem cells alone are able to support fetal development in the mouse
  
  
  
  
  Development
  
  
  110
  
  :815–821
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Nagy A
     
  
  
  2. 
     Rossant J
     
  
  
  
  
  2001
  
  
  
  Chimaeras and mosaics for dissecting complex mutant phenotypes
  
  
  
  
  Int J Dev Biol
  
  
  45
  
  :577–582
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Nagy A
     
  
  
  2. 
     Rossant J
     
  
  
  3. 
     Nagy R
     
  
  
  4. 
     Abramow-Newerly W
     
  
  
  5. 
     Roder JC
     
  
  
  
  
  1993
  
  
  
  Derivation of completely cell culture-derived mice from early-passage embryonic stem cells
  
  
  
  
  Proc Natl Acad Sci U S A
  
  
  90
  
  :8424–8428
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Nakao H
     
  
  
  2. 
     Harada T
     
  
  
  3. 
     Nakao K
     
  
  
  4. 
     Kiyonari H
     
  
  
  5. 
     Inoue K
     
  
  
  6. 
     Furuta Y
     
  
  
  7. 
     Aiba A
     
  
  
  
  
  2016
  
  
  
  A possible aid in targeted insertion of large DNA elements by CRISPR/Cas in mouse zygotes
  
  
  
  
  Genesis
  
  
  54
  
  :65–77
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Nguyen TP
     
  
  
  2. 
     Otani T
     
  
  
  3. 
     Tsutsumi M
     
  
  
  4. 
     Kinoshita N
     
  
  
  5. 
     Fujiwara S
     
  
  
  6. 
     Nemoto T
     
  
  
  7. 
     Fujimori T
     
  
  
  8. 
     Furuse M
     
  
  
  
  
  2024
  
  
  
  Tight junction membrane proteins regulate the mechanical resistance of the apical junctional complex
  
  
  
  
  J Cell Biol
  
  
  223
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Nikolopoulou E
     
  
  
  2. 
     Galea GL
     
  
  
  3. 
     Rolo A
     
  
  
  4. 
     Greene NDE
     
  
  
  5. 
     Copp AJ
     
  
  
  
  
  2017
  
  
  
  Neural tube closure: cellular, molecular and biomechanical mechanisms
  
  
  
  
  Development
  
  
  144
  
  :552–566
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Nishimura T
     
  
  
  2. 
     Honda H
     
  
  
  3. 
     Takeichi M
     
  
  
  
  
  2012
  
  
  
  Planar cell polarity links axes of spatial dynamics in neural-tube closure
  
  
  
  
  Cell
  
  
  149
  
  :1084–1097
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Nishimura T
     
  
  
  2. 
     Takeichi M
     
  
  
  
  
  2008
  
  
  
  Shroom3-mediated recruitment of Rho kinases to the apical cell junctions regulates epithelial and neuroepithelial planar remodeling
  
  
  
  
  Development
  
  
  135
  
  :1493–1502
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Oda Y
     
  
  
  2. 
     Otani T
     
  
  
  3. 
     Ikenouchi J
     
  
  
  4. 
     Furuse M
     
  
  
  
  
  2014
  
  
  
  Tricellulin regulates junctional tension of epithelial cells at tricellular contacts through Cdc42
  
  
  
  
  J Cell Sci
  
  
  127
  
  :4201–4212
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Ossipova O
     
  
  
  2. 
     Kim K
     
  
  
  3. 
     Sokol SY
     
  
  
  
  
  2015
  
  
  
  Planar polarization of Vangl2 in the vertebrate neural plate is controlled by Wnt and Myosin II signaling
  
  
  
  
  Biol Open
  
  
  4
  
  :722–730
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Otani T
     
  
  
  2. 
     Nguyen TP
     
  
  
  3. 
     Tokuda S
     
  
  
  4. 
     Sugihara K
     
  
  
  5. 
     Sugawara T
     
  
  
  6. 
     Furuse K
     
  
  
  7. 
     Miura T
     
  
  
  8. 
     Ebnet K
     
  
  
  9. 
     Furuse M
     
  
  
  
  
  2019
  
  
  
  Claudins and JAM-A coordinately regulate tight junction formation and epithelial polarity
  
  
  
  
  J Cell Biol
  
  
  218
  
  :3372–3396
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Peng X
     
  
  
  2. 
     Cuff LE
     
  
  
  3. 
     Lawton CD
     
  
  
  4. 
     DeMali KA
     
  
  
  
  
  2010
  
  
  
  Vinculin regulates cell-surface E-cadherin expression by binding to beta-catenin
  
  
  
  
  J Cell Sci
  
  
  123
  
  :567–577
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Peng X
     
  
  
  2. 
     Maiers JL
     
  
  
  3. 
     Choudhury D
     
  
  
  4. 
     Craig SW
     
  
  
  5. 
     DeMali KA
     
  
  
  
  
  2012
  
  
  
  α-Catenin uses a novel mechanism to activate vinculin
  
  
  
  
  J Biol Chem
  
  
  287
  
  :7728–7737
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Perez AR
     
  
  
  2. 
     Pritykin Y
     
  
  
  3. 
     Vidigal JA
     
  
  
  4. 
     Chhangawala S
     
  
  
  5. 
     Zamparo L
     
  
  
  6. 
     Leslie CS
     
  
  
  7. 
     Ventura A
     
  
  
  
  
  2017
  
  
  
  GuideScan software for improved single and paired CRISPR guide RNA design
  
  
  
  
  Nat Biotechnol
  
  
  35
  
  :347–349
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Perez-Pinera P
     
  
  
  2. 
     Ousterout DG
     
  
  
  3. 
     Brown MT
     
  
  
  4. 
     Gersbach CA
     
  
  
  
  
  2012
  
  
  
  Gene targeting to the ROSA26 locus directed by engineered zinc finger nucleases
  
  
  
  
  Nucleic Acids Res
  
  
  40
  
  :3741–3752
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Perez-Vale KZ
     
  
  
  2. 
     Yow KD
     
  
  
  3. 
     Johnson RI
     
  
  
  4. 
     Byrnes AE
     
  
  
  5. 
     Finegan TM
     
  
  
  6. 
     Slep KC
     
  
  
  7. 
     Peifer M
     
  
  
  
  
  2021
  
  
  
  Multivalent interactions make adherens junction-cytoskeletal linkage robust during morphogenesis
  
  
  
  
  J Cell Biol
  
  
  220
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Poueymirou WT
     
  
  
  2. 
     Auerbach W
     
  
  
  3. 
     Frendewey D
     
  
  
  4. 
     Hickey JF
     
  
  
  5. 
     Escaravage JM
     
  
  
  6. 
     Esau L
     
  
  
  7. 
     Doré AT
     
  
  
  8. 
     Stevens S
     
  
  
  9. 
     Adams NC
     
  
  
  10. 
      Dominguez MG
      
  
  
  11. 
      Gale NW
      
  
  
  12. 
      Yancopoulos GD
      
  
  
  13. 
      DeChiara TM
      
  
  
  14. 
      Valenzuela DM
      
  
  
  
  
  2007
  
  
  
  F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses
  
  
  
  
  Nat Biotechnol
  
  
  25
  
  :91–99
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Rakshit S
     
  
  
  2. 
     Zhang Y
     
  
  
  3. 
     Manibog K
     
  
  
  4. 
     Shafraz O
     
  
  
  5. 
     Sivasankar S
     
  
  
  
  
  2012
  
  
  
  Ideal, catch, and slip bonds in cadherin adhesion
  
  
  
  
  Proc Natl Acad Sci U S A
  
  
  109
  
  :18815–18820
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Rauskolb C
     
  
  
  2. 
     Cervantes E
     
  
  
  3. 
     Madere F
     
  
  
  4. 
     Irvine KD
     
  
  
  
  
  2019
  
  
  
  Organization and function of tension-dependent complexes at adherens junctions
  
  
  
  
  J Cell Sci
  
  
  132
  
  :jcs224063
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Razzell W
     
  
  
  2. 
     Bustillo ME
     
  
  
  3. 
     Zallen JA
     
  
  
  
  
  2018
  
  
  
  The force-sensitive protein Ajuba regulates cell adhesion during epithelial morphogenesis
  
  
  
  
  J Cell Biol
  
  
  217
  
  :3715–3730
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Sawyer JK
     
  
  
  2. 
     Choi W
     
  
  
  3. 
     Jung K-C
     
  
  
  4. 
     He L
     
  
  
  5. 
     Harris NJ
     
  
  
  6. 
     Peifer M
     
  
  
  
  
  2011
  
  
  
  A contractile actomyosin network linked to adherens junctions by Canoe/afadin helps drive convergent extension
  
  
  
  
  Mol Biol Cell
  
  
  22
  
  :2491–2508
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Schindelin J
     
  
  
  2. 
     Arganda-Carreras I
     
  
  
  3. 
     Frise E
     
  
  
  4. 
     Kaynig V
     
  
  
  5. 
     Longair M
     
  
  
  6. 
     Pietzsch T
     
  
  
  7. 
     Preibisch S
     
  
  
  8. 
     Rueden C
     
  
  
  9. 
     Saalfeld S
     
  
  
  10. 
      Schmid B
      
  
  
  11. 
      Tinevez J-Y
      
  
  
  12. 
      White DJ
      
  
  
  13. 
      Hartenstein V
      
  
  
  14. 
      Eliceiri K
      
  
  
  15. 
      Tomancak P
      
  
  
  16. 
      Cardona A
      
  
  
  
  
  2012
  
  
  
  Fiji: an open-source platform for biological-image analysis
  
  
  
  
  Nat Methods
  
  
  9
  
  :676–682
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Schmidt H
     
  
  
  2. 
     Zhang M
     
  
  
  3. 
     Chakarov D
     
  
  
  4. 
     Bansal V
     
  
  
  5. 
     Mourelatos H
     
  
  
  6. 
     Sánchez-Rivera FJ
     
  
  
  7. 
     Lowe SW
     
  
  
  8. 
     Ventura A
     
  
  
  9. 
     Leslie CS
     
  
  
  10. 
      Pritykin Y
      
  
  
  
  
  2025
  
  
  
  Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2
  
  
  
  
  Genome Biol
  
  
  26
  
  :41
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Schoenwolf GC
     
  
  
  2. 
     Smith JL
     
  
  
  
  
  1990
  
  
  
  Mechanisms of neurulation: traditional viewpoint and recent advances
  
  
  
  
  Development
  
  
  109
  
  :243–270
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Sheppard L
     
  
  
  2. 
     Green DG
     
  
  
  3. 
     Lerchbaumer G
     
  
  
  4. 
     Rothenberg KE
     
  
  
  5. 
     Fernandez-Gonzalez R
     
  
  
  6. 
     Tepass U
     
  
  
  
  
  2023
  
  
  
  The α-Catenin mechanosensing M region is required for cell adhesion during tissue morphogenesis
  
  
  
  
  J Cell Biol
  
  
  222
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Shioi G
     
  
  
  2. 
     Hoshino H
     
  
  
  3. 
     Abe T
     
  
  
  4. 
     Kiyonari H
     
  
  
  5. 
     Nakao K
     
  
  
  6. 
     Meng W
     
  
  
  7. 
     Furuta Y
     
  
  
  8. 
     Fujimori T
     
  
  
  9. 
     Aizawa S
     
  
  
  
  
  2017
  
  
  
  Apical constriction in distal visceral endoderm cells initiates global, collective cell rearrangement in embryonic visceral endoderm to form anterior visceral endoderm
  
  
  
  
  Dev Biol
  
  
  429
  
  :20–30
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Spadaro D
     
  
  
  2. 
     Le S
     
  
  
  3. 
     Laroche T
     
  
  
  4. 
     Mean I
     
  
  
  5. 
     Jond L
     
  
  
  6. 
     Yan J
     
  
  
  7. 
     Citi S
     
  
  
  
  
  2017
  
  
  
  Tension-dependent stretching activates ZO-1 to control the junctional localization of its interactors
  
  
  
  
  Curr Biol
  
  
  27
  
  :3783–3795
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Stephenson RE
     
  
  
  2. 
     Higashi T
     
  
  
  3. 
     Erofeev IS
     
  
  
  4. 
     Arnold TR
     
  
  
  5. 
     Leda M
     
  
  
  6. 
     Goryachev AB
     
  
  
  7. 
     Miller AL
     
  
  
  
  
  2019
  
  
  
  Rho flares repair local tight junction leaks
  
  
  
  
  Dev Cell
  
  
  48
  
  :445–459
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Sun X
     
  
  
  2. 
     Phua DYZ
     
  
  
  3. 
     Axiotakis L
     
  
  
  4. 
     Smith MA
     
  
  
  5. 
     Blankman E
     
  
  
  6. 
     Gong R
     
  
  
  7. 
     Cail RC
     
  
  
  8. 
     de Los Reyes S Espinosa
     
  
  
  9. 
     Beckerle MC
     
  
  
  10. 
      Waterman CM
      
  
  
  11. 
      Alushin GM
      
  
  
  
  
  2020
  
  
  
  Mechanosensing through direct binding of tensed F-actin by LIM domains
  
  
  
  
  Dev Cell
  
  
  55
  
  :468–482
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Taguchi K
     
  
  
  2. 
     Ishiuchi T
     
  
  
  3. 
     Takeichi M
     
  
  
  
  
  2011
  
  
  
  Mechanosensitive EPLIN-dependent remodeling of adherens junctions regulates epithelial reshaping
  
  
  
  
  J Cell Biol
  
  
  194
  
  :643–656
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Tam PPL
     
  
  
  2. 
     Rossant J
     
  
  
  
  
  2003
  
  
  
  Mouse embryonic chimeras: tools for studying mammalian development
  
  
  
  
  Development
  
  
  130
  
  :6155–6163
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Tarazi S
     
  
  
  2. 
     Aguilera-Castrejon A
     
  
  
  3. 
     Joubran C
     
  
  
  4. 
     Ghanem N
     
  
  
  5. 
     Ashouokhi S
     
  
  
  6. 
     Roncato F
     
  
  
  7. 
     Wildschutz E
     
  
  
  8. 
     Haddad M
     
  
  
  9. 
     Oldak B
     
  
  
  10. 
      Gomez-Cesar E
      
  
  
  11. 
      Livnat N
      
  
  
  12. 
      Viukov S
      
  
  
  13. 
      Lukshtanov D
      
  
  
  14. 
      Naveh-Tassa S
      
  
  
  15. 
      Rose M
      
  
  
  16. 
      Hanna S
      
  
  
  17. 
      Raanan C
      
  
  
  18. 
      Brenner O
      
  
  
  19. 
      Kedmi M
      
  
  
  20. 
      Keren-Shaul H
      
  
  
  21. 
      Lapidot T
      
  
  
  22. 
      Maza I
      
  
  
  23. 
      Novershtern N
      
  
  
  24. 
      Hanna JH
      
  
  
  
  
  2022
  
  
  
  ¬Post-Gastrulation Synthetic Embryos Generated Ex Utero from Mouse Naïve ESCs
  
  
  
  
  Cell
  
  
  0
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Thévenaz P
     
  
  
  2. 
     Ruttimann UE
     
  
  
  3. 
     Unser M
     
  
  
  
  
  1998
  
  
  
  A pyramid approach to subpixel registration based on intensity
  
  
  
  
  IEEE Trans Image Process
  
  
  7
  
  :27–41
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Thomas WA
     
  
  
  2. 
     Boscher C
     
  
  
  3. 
     Chu Y-S
     
  
  
  4. 
     Cuvelier D
     
  
  
  5. 
     Martinez-Rico C
     
  
  
  6. 
     Seddiki R
     
  
  
  7. 
     Heysch J
     
  
  
  8. 
     Ladoux B
     
  
  
  9. 
     Thiery JP
     
  
  
  10. 
      Mege R-M
      
  
  
  11. 
      Dufour S
      
  
  
  
  
  2013
  
  
  
  α-Catenin and vinculin cooperate to promote high E-cadherin-based adhesion strength
  
  
  
  
  J Biol Chem
  
  
  288
  
  :4957–4969
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Tucker KL
     
  
  
  2. 
     Wang Y
     
  
  
  3. 
     Dausman J
     
  
  
  4. 
     Jaenisch R
     
  
  
  
  
  1997
  
  
  
  A transgenic mouse strain expressing four drug-selectable marker genes
  
  
  
  
  Nucleic Acids Res
  
  
  25
  
  :3745–3746
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Ukai H
     
  
  
  2. 
     Kiyonari H
     
  
  
  3. 
     Ueda HR
     
  
  
  
  
  2017
  
  
  
  Production of knock-in mice in a single generation from embryonic stem cells
  
  
  
  
  Nat Protoc
  
  
  12
  
  :2513–2530
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     van den Goor L
     
  
  
  2. 
     Iseler J
     
  
  
  3. 
     Koning KM
     
  
  
  4. 
     Miller AL
     
  
  
  
  
  2024
  
  
  
  Mechanosensitive recruitment of Vinculin maintains junction integrity and barrier function at epithelial tricellular junctions
  
  
  
  
  Curr Biol
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     van der Stoel MM
     
  
  
  2. 
     Kotini MP
     
  
  
  3. 
     Schoon RM
     
  
  
  4. 
     Affolter M
     
  
  
  5. 
     Belting H-G
     
  
  
  6. 
     Huveneers S
     
  
  
  
  
  2023
  
  
  
  Vinculin strengthens the endothelial barrier during vascular development
  
  
  
  
  Vasc Biol
  
  
  5
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Vijayraghavan DS
     
  
  
  2. 
     Davidson LA
     
  
  
  
  
  2017
  
  
  
  Mechanics of neurulation: From classical to current perspectives on the physical mechanics that shape, fold, and form the neural tube
  
  
  
  
  Birth Defects Res
  
  
  109
  
  :153–168
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Wallingford JB
     
  
  
  2. 
     Niswander LA
     
  
  
  3. 
     Shaw GM
     
  
  
  4. 
     Finnell RH
     
  
  
  
  
  2013
  
  
  
  The continuing challenge of understanding, preventing, and treating neural tube defects
  
  
  
  
  Science
  
  
  339
  
  :1222002
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Wang H
     
  
  
  2. 
     Yang H
     
  
  
  3. 
     Shivalila CS
     
  
  
  4. 
     Dawlaty MM
     
  
  
  5. 
     Cheng AW
     
  
  
  6. 
     Zhang F
     
  
  
  7. 
     Jaenisch R
     
  
  
  
  
  2013
  
  
  
  One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering
  
  
  
  
  Cell
  
  
  153
  
  :910–918
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Wang Y
     
  
  
  2. 
     Li J
     
  
  
  3. 
     Xiang J
     
  
  
  4. 
     Wen B
     
  
  
  5. 
     Mu H
     
  
  
  6. 
     Zhang W
     
  
  
  7. 
     Han J
     
  
  
  
  
  2016
  
  
  
  Highly efficient generation of biallelic reporter gene knock-in mice via CRISPR-mediated genome editing of ESCs
  
  
  
  
  Protein Cell
  
  
  7
  
  :152–156
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Wang Y
     
  
  
  2. 
     Qin Y
     
  
  
  3. 
     Peng R
     
  
  
  4. 
     Wang H
     
  
  
  
  
  2021
  
  
  
  Loss-of-function or gain-of-function variations in VINCULIN (VCL) are risk factors of human neural tube defects
  
  
  
  
  Mol Genet Genomic Med e
  
  
  1563
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Wen K-K
     
  
  
  2. 
     Rubenstein PA
     
  
  
  3. 
     DeMali KA
     
  
  
  
  
  2009
  
  
  
  Vinculin nucleates actin polymerization and modifies actin filament structure
  
  
  
  
  J Biol Chem
  
  
  284
  
  :30463–30473
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Wilde JJ
     
  
  
  2. 
     Petersen JR
     
  
  
  3. 
     Niswander L
     
  
  
  
  
  2014
  
  
  
  Genetic, epigenetic, and environmental contributions to neural tube closure
  
  
  
  
  Annu Rev Genet
  
  
  48
  
  :583–611
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Williams M
     
  
  
  2. 
     Yen W
     
  
  
  3. 
     Lu X
     
  
  
  4. 
     Sutherland A
     
  
  
  
  
  2014
  
  
  
  Distinct apical and basolateral mechanisms drive planar cell polarity-dependent convergent extension of the mouse neural plate
  
  
  
  
  Dev Cell
  
  
  29
  
  :34–46
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Wu X
     
  
  
  2. 
     Hammer JA
     
  
  
  
  
  2021
  
  
  
  ZEISS Airyscan: Optimizing usage for fast, gentle, super-resolution imaging
  
  
  
  
  Methods Mol Biol
  
  
  2304
  
  :111–130
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Xu W
     
  
  
  2. 
     Baribault H
     
  
  
  3. 
     Adamson ED
     
  
  
  
  
  1998
  
  
  
  Vinculin knockout results in heart and brain defects during embryonic development
  
  
  
  
  Development
  
  
  125
  
  :327–337
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Yagi T
     
  
  
  2. 
     Tokunaga T
     
  
  
  3. 
     Furuta Y
     
  
  
  4. 
     Nada S
     
  
  
  5. 
     Yoshida M
     
  
  
  6. 
     Tsukada T
     
  
  
  7. 
     Saga Y
     
  
  
  8. 
     Takeda N
     
  
  
  9. 
     Ikawa Y
     
  
  
  10. 
      Aizawa S
      
  
  
  
  
  1993
  
  
  
  A novel ES cell line, TT2, with high germline-differentiating potency
  
  
  
  
  Anal Biochem
  
  
  214
  
  :70–76
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Yilmaz A
     
  
  
  2. 
     Gurhan G
     
  
  
  3. 
     Comar M-Y
     
  
  
  4. 
     Viukov S
     
  
  
  5. 
     Serfaty I
     
  
  
  6. 
     Gayretli M
     
  
  
  7. 
     Golenchenko S
     
  
  
  8. 
     Lokshtanov D
     
  
  
  9. 
     Ashouokhi S
     
  
  
  10. 
      Polanco A
      
  
  
  11. 
      Berlad I
      
  
  
  12. 
      Ha T-W
      
  
  
  13. 
      Aguilera-Castrejon A
      
  
  
  14. 
      Tarazi S
      
  
  
  15. 
      Cohen M
      
  
  
  16. 
      Livnat N
      
  
  
  17. 
      Kumar K
      
  
  
  18. 
      Cholakkal H
      
  
  
  19. 
      Levy N
      
  
  
  20. 
      Yosef N
      
  
  
  21. 
      Khatib N
      
  
  
  22. 
      Kakun RR
      
  
  
  23. 
      Kedmi M
      
  
  
  24. 
      Nachman IB
      
  
  
  25. 
      Keren-Shaul H
      
  
  
  26. 
      Addadi Y
      
  
  
  27. 
      Orenbuch A-H
      
  
  
  28. 
      Korovin K
      
  
  
  29. 
      Molchadsky A
      
  
  
  30. 
      Hochedlinger K
      
  
  
  31. 
      Gafni O
      
  
  
  32. 
      Maza I
      
  
  
  33. 
      Novershtern N
      
  
  
  34. 
      Oldak B
      
  
  
  35. 
      Hanna JH
      
  
  
  
  
  2025
  
  
  
  Transgene-free generation of mouse post-gastrulation whole embryo models solely from naive ESCs and iPSCs
  
  
  
  
  Cell Stem Cell
  
  
  32
  
  :1545–1562
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Yonemura S
     
  
  
  2. 
     Wada Y
     
  
  
  3. 
     Watanabe T
     
  
  
  4. 
     Nagafuchi A
     
  
  
  5. 
     Shibata M
     
  
  
  
  
  2010
  
  
  
  . alpha-Catenin as a tension transducer that induces adherens junction development
  
  
  
  
  Nat Cell Biol
  
  
  12
  
  :533–542
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Yu HH
     
  
  
  2. 
     Zallen JA
     
  
  
  
  
  2020
  
  
  
  Abl and Canoe/Afadin mediate mechanotransduction at tricellular junctions
  
  
  
  
  Science
  
  
  370
  
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Zaganjor I
     
  
  
  2. 
     Sekkarie A
     
  
  
  3. 
     Tsang BL
     
  
  
  4. 
     Williams J
     
  
  
  5. 
     Razzaghi H
     
  
  
  6. 
     Mulinare J
     
  
  
  7. 
     Sniezek JE
     
  
  
  8. 
     Cannon MJ
     
  
  
  9. 
     Rosenthal J
     
  
  
  
  
  2016
  
  
  
  Describing the Prevalence of Neural Tube Defects Worldwide: A Systematic Literature Review
  
  
  
  
  PLoS One
  
  
  11
  
  :e0151586
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Zemljic-Harpf AE
     
  
  
  2. 
     Godoy JC
     
  
  
  3. 
     Platoshyn O
     
  
  
  4. 
     Asfaw EK
     
  
  
  5. 
     Busija AR
     
  
  
  6. 
     Domenighetti AA
     
  
  
  7. 
     Ross RS
     
  
  
  
  
  2014
  
  
  
  Vinculin directly binds zonula occludens-1 and is essential for stabilizing connexin-43-containing gap junctions in cardiac myocytes
  
  
  
  
  Development
  
  
  141
  
  :e708–e708
  
  
  
  
  PubMed
  
  
  Google Scholar
  
  
  


• 
  
  
  
  1. 
     Zemljic-Harpf AE
     
  
  
  2. 
     Miller JC
     
  
  
  3. 
     Henderson SA
     
  
  
  4. 
     Wright AT
     
  
  
  5. 
     Manso AM
     
  
  
  6. 
     Elsherif L
     
  
  
  7. 
     Dalton ND
     
  
  
  8. 
     Thor AK
     
  
  
  9. 
     Perkins GA
     
  
  
  10. 
      McCulloch AD
      
  
  
  11. 
      Ross RS
      
  
  
  
  
  2007
  
  
  
  Cardiac-myocyte-specific excision of the vinculin gene disrupts cellular junctions, causing sudden death or dilated cardiomyopathy
  
  
  
  
  Mol Cell Biol
  
  
  27
  
  :7522–7537
  
  
  
  
  PubMed
  
  
  Google Scholar