Spiral ganglion neurons (SGNs) carry sound information from the cochlea to the hindbrain, and innervate either inner or outer hair cells. Type II SGNs (SGNIIs) extend peripheral afferents towards outer hair cells, which make a characteristic 90° turn towards the cochlear base and innervate multiple outer hair cells. It has been shown that the planar cell polarity (PCP) pathway acts non-autonomously in the cochlear epithelium to guide SGNII peripheral afferent turning. However, the underlying mechanisms are unknown. Here, we show that PCP signaling regulates junctional localization of the small GTPase Rac1 and the cell adhesion molecule Nectin3 in mouse cochlear supporting cells (SCs), which serve as intermediate targets of SGNII peripheral afferents. Loss of Rac1 or Nectin3 partially phenocopied SGNII peripheral afferent turning defects in Vangl2 mutants. We present evidence that Rac1 plays a non-autonomous role in part by regulating the localization of core PCP proteins Vangl2 and Dvl3 at the SC-SC junctions, while Nectin3 likely serves a cell adhesion function to control SGNII afferent turning. Together, these experiments identify Nectin3 and Rac1 as regulators of PCP-directed SGNII axon guidance in the cochlea.

What can we learn about the mechanical processes that shape tissues by simply watching? Several schemes suggest that static cell morphology or junctional connectivity can reveal where chains of cells transmit force or where force asymmetries drive cellular rearrangements. We hypothesize that dynamic cell shape changes from time lapse sequences can be used to distinguish specific mechanisms of tissue morphogenesis. Convergent extension (CE) is a crucial developmental motif wherein a planar tissue narrows in one direction and lengthens in the other. It is tempting to assume that forces driving CE reside within cells of the deforming tissue, as CE may reflect a variety of active processes or passive responses to forces generated by adjacent tissues. In this work, we first construct a simple model of epithelial cells capable of passive CE in response to external forces. We adapt this framework to simulate CE from active anisotropic processes in three different modes: crawling, contraction, and capture. We develop an image analysis pipeline for analysis of morphogenetic changes in both live cells and simulated cells using a panel of mechanical and statistical approaches. Our results allow us to identify how each simulated mechanism uniquely contributes to tissue morphology and provide insight into how force transmission is coordinated. We construct a MEchanism Index (MEI) to quantify how similar live cells are to simulated passive and active cells undergoing CE. Applying these analyses to live cell data of Xenopus neural CE reveals features of both passive motion and active forces. Furthermore, we find spatial variation across the neural plate. We compare the inferred mechanisms in the frog midline to tissues undergoing CE in both the mouse and fly. We find that distinct active modes may have different prevalences depending on the model system. Our modeling framework allows us to gain insight from tissue timelapse images and assess the relative contribution of specific cellular mechanisms to observed tissue phenotypes. This approach can be used to guide further experimental inquiry into how mechanics influences the shaping of tissues and organs during development.

Our sense of hearing is critically dependent on the spiral ganglion neurons (SGNs) that connect the sound receptors in the organ of Corti (OC) to the cochlear nuclei of the hindbrain. Type I SGNs innervate inner hair cells (IHCs) to transmit sound signals, while type II SGNs (SGNIIs) innervate outer hair cells (OHCs) to detect moderate-to-intense sound. During development, SGNII afferents make a characteristic 90-degree turn toward the base of the cochlea and innervate multiple OHCs. It has been shown that the Planar Cell Polarity (PCP) pathway acts non-autonomously to mediate environmental cues in the cochlear epithelium for SGNII afferent turning towards the base. However, the underlying mechanisms are unknown. Here, we present evidence that PCP signaling regulates multiple downstream effectors to influence cell adhesion and the cytoskeleton in cochlear supporting cells (SCs), which serve as intermediate targets of SGNII afferents. We show that the core PCP gene Vangl2 regulates the localization of the small GTPase Rac1 and the cell adhesion molecule Nectin3 at SC-SC junctions through which SGNII afferents travel. Through in vivo genetic analysis, we also show that loss of Rac1 or Nectin3 partially phenocopied SGNII peripheral afferent turning defects in Vangl2 mutants, and that Rac1 plays a non-autonomous role in this process in part by regulating PCP protein localization at the SC-SC junctions. Additionally, epistasis analysis indicates that Nectin3 and Rac1 likely act in the same genetic pathway to control SGNII afferent turning. Together, these experiments identify Nectin3 and Rac1 as novel regulators of PCP-directed SGNII axon guidance in the cochlea.

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract Epithelial-to-mesenchymal transition (EMT) is a fundamental process whereby epithelial cells acquire mesenchymal phenotypes and the ability to migrate. EMT is the hallmark of gastrulation, an evolutionarily conserved developmental process. In mammals, epiblast cells ingress at the primitive streak to form mesoderm. Cells ingress and exit the epiblast epithelial layer and the associated EMT is dynamically regulated and involves a stereotypical sequence of cell behaviors. 3D time-lapse imaging of gastrulating mouse embryos combined with cell and tissue scale data analyses revealed the asynchronous ingression of epiblast cells at the primitive streak. Ingressing cells constrict their apical surfaces in a pulsed ratchet-like fashion through asynchronous shrinkage of apical junctions. A quantitative analysis of the distribution of apical proteins revealed the anisotropic and reciprocal enrichment of members of the actomyosin network and Crumbs2 complexes, potential regulators of asynchronous shrinkage of cell junctions. Loss of function analyses demonstrated a requirement for Crumbs2 in myosin II localization and activity at apical junctions, and as a candidate regulator of actomyosin anisotropy. Editor's evaluation This study employs live imaging to investigate the movement of mesodermal cells in early mouse embryos. By examining the dynamics of cell behavior in normal and mutant embryos, the authors propose that apical constriction of cells results from pulsed contraction guided by crumbs2 signals. The paper presents beautiful images and adds to the molecular understanding of cell migration during early development. https://doi.org/10.7554/eLife.84019.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Epithelial-to-mesenchymal transitions (EMTs) are tissue-level morphogenetic programs necessary for normal embryonic development, cancer progression, and metastasis (Francou and Anderson, 2020; Thiery, 2002; Thiery et al., 2009; Ye and Weinberg, 2015). EMT that occurs during embryo gastrulation is an evolutionarily conserved event occurring in amniotes as they generate three definitive germ layers (Lim and Thiery, 2012; Nakaya and Sheng, 2008; Thiery et al., 2009). The onset of gastrulation involves formation of the primitive streak at the posterior of the pluripotent epiblast, at around embryonic (E) 6.5 day in the mouse (Ferrer-Vaquer et al., 2010; Nakaya and Sheng, 2008; Williams et al., 2012; Figure 1A). Primitive streak initiation is regulated by the convergence of WNT, BMP, and Nodal signals, which together with FGF signal trigger the EMT process (Ferrer-Vaquer et al., 2010; Morgani and Hadjantonakis, 2020; Ramkumar and Anderson, 2011). The basement membrane present basally adjacent to epiblast cells is broken down at the primitive streak, thereby facilitating the movement of cells out of the epiblast tissue layer, accompanied by changes in their cell shape, including apical constriction and the basal translocation of cell bodies (Ramkumar et al., 2016; Williams et al., 2012). Basal cell ingression and migration away from the epiblast epithelium is facilitated through the dissolution of apical junctions. As cells at the primitive streak undergo EMT, more laterally positioned epiblast cells converge toward the midline (Williams et al., 2012), replenishing the pool for continued ingression and ensuring epithelial integrity. Figure 1 with 1 supplement see all Download asset Open asset Time-lapse imaging of ZO-1-GFP reporter reveals epithelial-to-mesenchymal transition (EMT) events at the primitive streak of the mouse gastrula and apical constriction associated with cell ingression. (A) Schematic sagittal section view of embryonic day (E)7.5 mouse embryo and 3D time-lapse imaging performed from the posterior side in glass-bottom dishes with the objective positioned adjacent to the primitive streak (PS). In this configuration, the epiblast apical surface situated furthest from the objective (red dashed line shows the microscope light path). (B) Schematic of a view (from the inner cavity) of the apical surface of cells within the epiblast layer. The midline (dotted line) separates the right and left sides of the embryo. (C) High magnification schematic of a sagittal view of an EMT event at the primitive streak depicting a cell constricting its apical surface, elongating basally (dark gray), and ingressing out of the epiblast layer to integrate into the mesoderm (blue). Note the apical surface of epiblast cells is located 40–60 µm away from the imaging objective. (D) Single time points at t=0 min and t=120 min of a time-lapse of a ZO-1-GFP embryo. Movies were analyzed as maximum intensity projections of z-stacks to visualize the apical surfaces of cells. All the cells that can be followed and observed ingressing during 4 hr were tracked. Of 140 cells initially tracked, 95 cells constricted and ingressed over the course of 120 min (45 tracked cells remaining). The time-lapse was acquired with a 5 min time interval, 290 cells were in the field of view, with 140 ingressing cells tracked. (E) Cumulative tracking of the 140 cells showing cell tracks over time (lines) and cell position at the time of ingression (dots). Epiblast cells converge toward the primitive streak (~40 µm, dotted lines) where the majority of ingression events occur. (F) Graphs showing 44 ± 2.1% of cells in the primitive streak ingress each hour, with 48 ± 1.1% ingressing as single cells, 37 ± 1.7% as pairs of cells, 11 ± 1.5% as triplets (groups of three cells), and 4% as groups of four cells (of a total of 378 ingressing cells analyzed in three embryos). (G) High magnification view showing the apical constriction of three cells at the primitive streak. Pr: proximal, D: distal, A: anterior, P: posterior, R: right, L: left, Ext Emb: extra embryonic region, PS: primitive streak. Error bars represent s.e.m. See Supplementary file 1 for n. Scale bars, D, 40µm; G, 10µm. Figure 1—source data 1 Ingression events per hour and percentage of different types of events. https://cdn.elifesciences.org/articles/84019/elife-84019-fig1-data1-v1.xlsx Download elife-84019-fig1-data1-v1.xlsx Apical constriction is an epithelial cell shape change associated with morphogenetic processes such as tissue bending, cell delamination, and internalization (An et al., 2017; Chung et al., 2017; Lecuit and Lenne, 2007; Martin and Goldstein, 2014; Nishimura et al., 2012; Simões et al., 2017). The dynamics of apical constriction at gastrulation have been studied in invertebrates, particularly in Drosophila, and shown to be controlled by apical actomyosin contractility (Marston et al., 2016; Martin and Goldstein, 2014; Martin et al., 2009; Mason et al., 2013; Roh-Johnson et al., 2012). Apical constriction has been associated with cell ingression during the gastrulation EMT in chick and mouse embryos (Rozbicki et al., 2015; Serrano Nájera and Weijer, 2020; Williams et al., 2012), but how cells dynamically constrict their apical surfaces during EMT, and what regulates this process remain open questions. The process of gastrulation occurs along divergent time-scales (~1 hr vs. >24 hr) in Drosophila and mouse, and involves distinct mechanisms and spatial parameters (tissue invagination vs. cell ingression). In the chick embryo, Myosin II plays a role in the formation of the primitive streak, and cell ingression from the epiblast layer (Chuai et al., 2006; Rozbicki et al., 2015; Serrano Nájera and Weijer, 2020; Voiculescu et al., 2007; Voiculescu et al., 2014). Crumbs2, a protein shown to regulate apical polarity in epithelia, is critical for cell ingression during gastrulation EMT in mouse embryos (Ramkumar et al., 2016). Apical Crumbs2 and myosin heavy chain IIB show an anisotropic accumulation in mouse epiblast cells in the vicinity of the primitive streak (Ramkumar et al., 2016), reminiscent of patterns controlling apical constriction during Drosophila salivary gland development (Röper, 2012), suggesting these proteins are potential regulators of apical constriction during mouse EMT. Visualizing the cellular dynamics of the epiblast during mouse gastrulation has been a longstanding challenge due to the internal location of the tissue which limits optical access and visualization at high resolution. Using live imaging of ex utero cultured mouse embryos, and high-resolution visualization and segmentation of epiblast cell membranes, we performed a dynamic quantitative analysis of the apical constriction associated with ingression during the mouse gastrulation EMT. We observed cells undergoing EMT at the mouse primitive streak in a scattered and apparently stochastic manner. Epiblast cells constricted their apical surfaces in a ratchet-like pulsed fashion through asynchronous shrinkage of their apical cell-cell junctions. By analyzing and quantifying the distribution of apical proteins, we uncovered an anisotropic and reciprocal enrichment of actomyosin network and Crumbs complex proteins. Such reciprocal distribution is consistent with actomyosin and Crumbs complex playing a role in regulating junctional shrinkage and apical constriction. The localization of the apical actomyosin network, as well as two kinases aPKC and Rock1, was perturbed in Crb2 mutants, thereby identifying them as key components of a putative regulatory network driving apical constriction. Results Epiblast cells undergo apical constriction and isolated ingression at the mouse primitive streak The mouse gastrulation EMT occurs at the primitive streak, which, by contrast to the chick, does not form a morphologically distinct domain (Figure 1A and B). Columnar epithelial epiblast cells with their apical surfaces facing the inner (amniotic) cavity of the embryo apically constrict and elongate basally as they ingress out of the epiblast tissue layer (Figure 1A and C; Williams et al., 2012). To visualize the dynamic changes in the shapes of all cells in tissue context at single-cell resolution during the gastrulation EMT, we sought to label the membranes and junctions of all epiblast cells and perform time-lapse imaging. Time-lapse imaging of epiblast cells in the vicinity of the primitive streak of developing mouse embryos is challenging for several reasons. Embryos need to be kept intact and tissues cannot be microdissected as epiblast integrity would be lost. The epiblast is cup-shaped with inherent curvature, and it is the tissue located within the deepest extremity of the embryo (up to 60 µm away from the objective), needing to be imaged through the adjacent visceral endoderm and mesoderm tissue layers (Figure 1C). Even light-sheet systems, which can provide in toto visualization of embryos and have predominantly been used to visualize and segment nuclear-localized reporters (McDole et al., 2018), present limitations in imaging membrane and junctional reporters. We used a ZO-1-GFP protein fusion reporter (Foote et al., 2013) to visualize tight junctions, and by extension, the apical surface of epiblast cells. This allowed us to quantify apical surface dynamics (Figure 1D). We also used a membrane-localized Rosa26mT/mG reporter to visualize their entire plasma membrane, and to identify completion of the apical constriction associated with cell ingression out of the epithelial epiblast layer (Figure 1—figure supplement 1A and Video 1). 3D time-lapse imaging of embryos at mid/late-streak stage (E7.5) was performed to image the primitive streak (Figure 1A and B). Time-lapse imaging of ZO-1-GFP embryos revealed that epiblast cells undergo extensive rearrangements as cells on the left and right sides of the embryo converge toward the primitive streak (Figure 1D, Figure 1—figure supplement 1C, Videos 2 and 3; Williams et al., 2012). The majority of epiblast cells underwent apical constriction and ingression within an ~40 µm region at the posterior midline (Figure 1D and E, Video 2). This region corresponded to the domain of Snail expression and basement membrane breakdown within the epiblast (Francou and Anderson, 2020; Ramkumar et al., 2016; Williams et al., 2012), which by convention defines the primitive streak (Figure 1E, dotted lines). Rare apical constriction and ingression events were also observed outside the domain of the primitive streak (Figure 1—figure supplement 1B and Video 3, bottom). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse imaging of a membrane-GFP reporter (recombined mT/mG) expressing embryonic day (E)7.5 embryo. Apical constriction (right panel) associated with ingression, as cells move out of the epiblast epithelial layer basally to join the underlying mesoderm (left panel). Images representing single planes of optical sections and apical cell surfaces were denoised and deconvolved. The time-lapse spans 70 min with 10 min time interval between frames. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse imaging of the posterior side of a ZO-1-GFP reporter expressing embryonic day (E)7.5 embryo in the vicinity of the primitive streak. Tracking of individual cells reveals lateral epiblast cells converging toward the midline prior to their ingression. Cells around the midline (middle row) do not exhibit extensive movement in the plane of the epithelium. Movie of 240 min with 5 min interval between frames. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse of ZO-1-GFP reveals dynamic cell behaviors at the primitive streak. Rearrangement of cells in the epiblast near the primitive streak (top panel, 125 min), and cell apical constriction and ingression away from the primitive streak, as the epiblast flows (to the left side) toward the midline (bottom panel, 55 min). Five min time interval between frames. Within a 1 hr period 44 ± 2% of cells within the primitive streak domain, constricted and ingressed (mean ± s.e.m., n=378 cells, three embryos) (Figure 1D, E and F, Video 2), with ingression events scattered through the streak appearing to occur stochastically. Among the ingressing cells tracked, 48% constricted and ingressed as isolated events (more than 30 min apart from adjacent neighboring cells), while the remaining 52% ingressed as pairs, and occasionally as groups of three to four cells (less than 30 min apart from adjacent neighboring cells) (Figure 1F). At the tissue level the epiblast underwent extensive rearrangements, with some cells moving and acquiring new neighbors prior to their coordinate ingression (Figure 1—figure supplement 1D, E), while others, which were initially neighbors, ingressed at different times (more than 30 min apart) (Figure 1—figure supplement 1D, F). Epiblast cells undergo a ratchet-like pulsed apical constriction during ingression We next focused on changes associated with the apical surface of cells during their ingression. Apical constriction has been extensively studied in Drosophila, including during mesoderm formation and neuroblast delamination (An et al., 2017; Martin et al., 2009; Mason et al., 2013; Simões et al., 2017). Very few studies have investigated the gastrulation EMT in an amniote, and it is unknown how cells constrict their apical surfaces during single-cell ingression in the mouse embryo versus the more global tissue-level constriction and invagination during Drosophila gastrulation. To follow changes in apical surfaces during cell ingression, cell membranes identified on projections of Z-stacks from time-lapse data were segmented using Tissue Analyzer software (Aigouy et al., 2010; Aigouy et al., 2016). By visualizing cells at the primitive streak, we identified apical constriction events associated with cell ingression and quantified several parameters, including apical surface area, apical cell surface elongation and orientation, and number of apical junctions (Figure 1G, Figure 2—figure supplement 1, Videos 4 and 5). Epiblast cells exhibited a variety of apical surface sizes and shapes that fluctuated over time as they flowed toward the primitive streak. As they initiated apical constriction at the primitive streak, cells exhibited 20–80 µm2 apical surface area, and completed ingression within 25–90 min (Figure 2A and Figure 2—figure supplement 1D). With a 5 min image data acquisition time interval, the majority of ingressing cells exhibited pulsed constrictions, generally consisting of two to four major phases of contraction (Figure 2B and C and Figure 2—figure supplement 1G). As cells progressed toward ingression, the number of apical junctions decreased (Figure 2—figure supplement 1J, Video 5). We observed a similar behavior of pulsed apical constrictions with both the ZO-1-GFP and a myosin heavy chain IIB-GFP reporter (Figure 2—figure supplement 1A–C). Figure 2 with 2 supplements see all Download asset Open asset Pulsed racket-like apical constriction during epiblast cell ingression and asynchronous constriction at the multi-cellular level. (A) Graph showing the apical surface area of a few cells during constriction preceding ingression. Data are aligned to the start of constriction. (B) Heat map showing the rate of change of apical surface area of cells during the final constriction period, revealing minimal expansion and pulsed contractions. Each row represents data for an individual cell, with all data aligned to the end of the constriction period. (C) Graph of apical surface area and rate of constriction of a single cell exhibiting a slight oscillation in its apical surface before undergoing a final constriction period with (three – beige regions) pulses of constriction. (D) Membrane segmentation and color-coded time-series of apical surface area and constriction rate of a single cell exhibiting three pulses of constriction separated by periods of stability. The highlighted cell corresponds to the plot shown in C. (E) Plot showing the average rate of changes in apical area. Epiblast cells located at a distance from the primitive streak before ingression show low and equivalent levels of contraction and expansion. Cells at the primitive streak show no significant differences between contraction and expansion prior to their period of constriction, whereas rate of contraction is significantly higher, and expansion reduces during the subsequent constriction period. (F) Color-coded time-series and heat map of rate of change of apical surface area showing asynchronous oscillation in area and asynchronous constriction and ingression among neighbors. Asterisks show ingression events. Forty-one cells quantified from four embryos. Error bars represent s.e.m. See Supplementary file 1 for n and p values. Scale bars: 10µm. *p<0.05, ***p<0.001 (unpaired bilateral Mann-Whitney test). Figure 2—source data 1 Apical constriction and constriction rate. https://cdn.elifesciences.org/articles/84019/elife-84019-fig2-data1-v1.xlsx Download elife-84019-fig2-data1-v1.xlsx Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse of ZO-1-GFP highlighting three cells apically constricting and ingressing in the primitive streak region. Sixty min total with 5 min time interval between frames. Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse of ZO-1-GFP highlighting a single cell at the primitive streak apically constricting. Membrane segmentation is performed over time such that different parameters can be followed and quantified, including the apical surface area, cell elongation and orientation, and the number of junctions: 95 min total with 5 min interval between frames. A closer examination of individual cells allowed us to quantify several features of apical surface area throughout the constriction period. The example in Figure 2D depicts the apical surface area of a cell exhibiting three main pulses of constriction each separated by more stable intermediate steps (Figure 2C and D, Video 6). While cells showed an average rate of constriction of 1.1±0.1 µm2/min (mean ± s.e.m., n=51 cells, four embryos), they exhibited constriction pulses with higher rates of contraction (up to 4.6 µm2/min), and an average pulse magnitude of 1.4±0.1 µm2/min (Figure 2B, C and E and Figure 2—figure supplement 1H). Cells at the primitive streak fluctuated between expansion and contraction before ingression, as did epiblast cells located some distance from the streak (Figure 2C and Figure 2—figure supplement 1D, I). During ingression, cells spent 90% of the time contracting apical surfaces (compare with 48% and 55% for epiblast cells away from the streak and before ingression) with an average rate of contraction of 1.1 µm2/min, higher than the average expansion rate of 0.1 µm2/min (Figure 2E and Figure 2—figure supplement 1F). To conclude, epiblast cells exhibited fluctuations in their apical surface areas before ingression, and pulsed constrictions during ingression at the primitive streak. We quantified the elongation of the apical surface, its orientation and the number of edges per cell at the onset and throughout the constriction in order to identify any parameters that could predict and identify cells that will initiate constriction and ingression, but no significant trend of hallmark were observed (Figure 2—figure supplement 1J–L). Video 6 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Segmentation of time-lapse showing color code of cell apical surface (left panel), constriction rate (middle panel), and junction length (right panel) of a cell shown in Figure 2: 40 min total with 5 min interval between frames. Asynchronous apical constriction at multi-cellular level To uncover details of the apical constriction behavior at the multi-cellular level, we analyzed clusters of cells at the primitive streak. Segmenting cell clusters allowed tracking of cells and documentation of changes in their apical surfaces through time, as cells ingressed (Figure 2F, Figure 2—figure supplement 2, Video 7). Cells underwent fluctuations in their apical surface area, as some cells constricted and ingressed (Figure 2—figure supplement 2B, Video 7). By color-coding and plotting the rate of apical surface area change, we observed asynchronous oscillation of expansion and contraction phases among neighboring cells. As cells constricted and ingressed, their neighbors did not always exhibit the same behavior; some contracted at the same time, while others slightly expanded or maintained a constant surface area (Figure 2F, asterisk shows ingression; Figure 2—figure supplement 2C and Video 7). Thus, neighboring epiblast cells undergoing EMT and ingressing at the primitive streak displayed asynchronous apical behaviors and did not simultaneously ingress, with some neighboring cells ingressing 2 hr apart, in comparison to Drosophila gastrulation where all ventral furrow cells coordinately constrict in a time frame of less than 10 min. Video 7 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse of ZO-1-GFP showing a cell cluster at the primitive streak. Most of the cells gradually constrict and ingress, and cells tracked after segmentation and color-coded for apical surface area and changes in surface area. Images were denoised and deconvolved: 115 min with 5 min interval between frames. Asynchronous shrinkage of junctions correlates with apical constriction during cell ingression Most apical junctions of any given cell decreased in length in a pulsed manner, with one to three pulses of shrinkage (Figure 3A and B), with junctions appearing to shrink asynchronously (Figure 3A, Video 6). By analyzing and quantifying the behavior of each junction of any one cell during constriction, we noted that the average rate of shrinkage was significantly higher during cell constriction pulses (Figure 3C). Junctions of individual cells shrunk asynchronously (Figure 3D–F and H and Figure 3—figure supplement 1A–C, E, F), differentially and with different magnitudes between one constriction pulse and the next (Figure 3G and Figure 2—figure supplement 1D), suggesting that asynchronous differential junctional shrinkage may trigger constriction of the cell surface. In sum, epiblast cells at the mouse primitive streak constrict their apical surfaces in a ratchet-like manner, with associated asynchronous shrinkage of junctions (Figure 3F). Figure 3 with 1 supplement see all Download asset Open asset Asynchronous junctional shrinkage during apical constriction. (A) Membrane segmentation and color-coded time-series of a single junction showing a reduction of the junction's length during apical constriction. Here, the six initial junctions are numbered, and two are highlighted (arrowheads) and shrink at different time. (B) Graph showing the length and rate of shrinkage of the junction identified with the green arrowhead in A, indicating the three main pulses of shrinkage (hatched regions). (C) Graph showing increased rate of junctional shrinkage during the pulses of cell constriction compared to stable phases between constriction pulses from multiple cells (three embryos, 51 junctions). (D) Graph of apical surface area and rate of constriction of a single cell showing pulses of constriction, and associated graph showing the length of its individual junctions over time (E). Junctions reduce their length differentially during the constriction pulses (beige regions). (F) Heat map of the shrinkage rate of junctions, showing junctions shrinking asynchronously during the constriction pulses (beige time points). Each row represents data for an individual junction, junctions are color-coded as corresponding in E, and ordered as neighboring junctions. (G) Plot of the average shrinkage rate of each junction over time during each constriction pulse and intervening stable phases, illustrating the asynchronous shrinkage of different sets of junctions with variable magnitude during the different cell constriction pulses. Junctions are color-coded as neighbors, as in E. (H) Matrix heat map showing the correlation of junction length changes over time. Some junctions show correlation and change their length, as others show anti-correlation. Each row and column represent a single junction and are color-coded as in E. Numbers represent the correlation coefficients for each pair of junctions. (I) Model of ratchet-like pulsed constriction associated with asynchronous junctional shrinkage. Error bars represent s.e.m. *p<0.05 (unpaired bilateral Mann-Whitney test). See Supplementary file 1 for n and p values. Scale bars, 5µm. Figure 3—source data 1 Junction length and shrinkage rate. https://cdn.elifesciences.org/articles/84019/elife-84019-fig3-data1-v1.xlsx Download elife-84019-fig3-data1-v1.xlsx Anisotropic accumulation of components at apical junctions of epiblast cells We sought to analyze the localization of apical proteins implicated in epithelial apical constriction in different tissues and species, that could potentially be involved in the apical constriction associated with the mouse gastrulation EMT. E7.5 embryos were immunostained, microdissected and the posterior region mounted so that the apical surface of epiblast cells could be positioned adjacent to the objective, and imaged in the vicinity of the middle of the primitive streak, which has the least curvature and is preferable for such an analysis (Figure 4A and Figure 4—figure supplement 1A). Crumbs2 and myosin heavy chain IIB have been described as exhibiting heterogenous and reciprocal enrichment at the apical junctions of epiblast cells (Ramkumar et al., 2016), and our data showed myosin heavy chain IIB and Crumbs2 to be predominantly anisotropically distributed, with Crumbs2 exhibiting a stronger anisotropy, being almost absent from some junctions, while myosin heavy chain IIB was observed on all junctions but at varying levels (Figure 4D and Figure 4—figure supplement 1B). Figure 4 with 2 supplements see all Download asset Open asset Anisotropic distribution of apical components in epiblast cells at the primitive streak. (A) Schematic of embryo processed for fixed tissue imaging after immunostaining. Embryos are microdissected and their posterior side is mounted between a slide and coverslip so that the apical surface of the epiblast is directly adjacent to the objective for imaging. (B) ZO-1 apical membrane localization, and corresponding skeleton of membrane segmentation used to quantify junctional intensi

Morphogenesis of the vertebrate neural tube occurs by elongation and bending of the neural plate, tissue shape changes that are driven at the cellular level by polarized cell intercalation and cell shape changes, notably apical constriction and cell wedging. Coordinated cell intercalation, apical constriction, and wedging undoubtedly require complex underlying cytoskeletal dynamics and remodeling of adhesions. Mutations of the gene encoding Scribble result in neural tube defects in mice, however the cellular and molecular mechanisms by which Scrib regulates neural cell behavior remain unknown. Analysis of Scribble mutants revealed defects in neural tissue shape changes, and live cell imaging of mouse embryos showed that the Scrib mutation results in defects in polarized cell intercalation, particularly in rosette resolution, and failure of both cell apical constriction and cell wedging. Scrib mutant embryos displayed aberrant expression of the junctional proteins ZO-1, Par3, Par6, E- and N-cadherins, and the cytoskeletal proteins actin and myosin. These findings show that Scribble has a central role in organizing the molecular complexes regulating the morphomechanical neural cell behaviors underlying vertebrate neurulation, and they advance our understanding of the molecular mechanisms involved in mammalian neural tube closure.

The primitive streak, a transient embryonic structure, marks bilateral symmetry in mammalian and avian embryos and helps confer anterior-posterior and dorsal-ventral spatial information to early differentiating cells during gastrulation. Its recapitulation in vitro may facilitate derivation of tissues and organs with in vivo–like complexity. Proper understanding of the primitive streak and what it entails in human development is key to achieving such research objectives. Here we provide an overview of the primitive streak and conclude that this structure is neither conserved nor necessary for gastrulation or early lineage diversification. We offer a model in which the primitive streak is viewed as part of a morphologically diverse yet molecularly conserved process of spatial coordinate acquisition. We predict that recapitulation of the primitive streak is dispensable for development in vitro.

The Drosophila Afadin and ZO-1 homologues Canoe and Polychaetoid act in parallel to maintain epithelial

Cell and tissue shape changes are the fundamental elements of morphogenesis that drive normal development of embryos into fully functional organisms. This requires a variety of cellular processes including establishment and maintenance of polarity, tissue growth and apoptosis, and cell differentiation, rearrangement, and migration. It is widely appreciated that the cytoskeletal networks play an important role in regulating many of these processes and, in particular, that pulsed actomyosin contractions are a core cellular mechanism driving cell shape changes and cell rearrangement. In this review, we discuss the role of pulsed actomyosin contractions during developmental morphogenesis, advances in our understanding of the mechanisms regulating actomyosin pulsing, and novel techniques to probe the role of pulsed actomyosin processes in model systems.

Scribble (Scrib) is a scaffold protein with multifunctional roles in PCP, tight junction and Hippo signaling. This study shows that Scrib is expressed in stromal cells around the implantation chamber following implantation. Stromal cells transform into epithelial-like cells to form the avascular primary decidual zone (PDZ) around the implantation chamber (crypt). The PDZ creates a permeability barrier around the crypt restricting immune cells and harmful agents from maternal circulation to protect embryonic health. The mechanism underlying PDZ formation is not yet known. We found that uterine deletion of Scrib by a Pgr-Cre driver leads to defective PDZ formation and implantation chamber (crypt) formation, compromising pregnancy success. Interestingly, epithelial-specific Scrib deletion by a lactoferrin-Cre (Ltf-Cre) driver does not adversely affect PDZ formation and pregnancy success. These findings provide evidence for a previously unknown function of stromal Scrib in PDZ formation, potentially involving ZO-1 and Hippo signaling. PMID: 31780662

The actomyosin cytoskeleton is responsible for many changes in cell and tissue shape.For a long time, the actomyosin cytoskeleton has been known to exhibit dynamic contractile behavior.Recently, discrete actomyosin assembly/disassembly cycles have also been observed in cells.These so-called actomyosin pulses have been observed in a variety of contexts, including cell polarization and division, and in epithelia, where they occur during tissue contraction, folding, and extension.In epithelia, evidence suggests that actomyosin pulsing, and more generally, actomyosin turnover, is required to maintain tissue integrity during contractile processes.This review explores possible functions for pulsing in the many instances during which pulsing has been observed, and also highlights proposed molecular mechanisms that drive pulsing.

This review is a comprehensive analysis of the cell biology and biomechanics of Convergent Extension in Xenopus.