Morphogenetic processes often involve the rapid rearrangement of cells held together by mutual adhesion. The dynamic nature of this adhesion endows tissues with liquid-like properties, such that large-scale shape changes appear as tissue flows. Generally, the resistance to flow (tissue viscosity) is expected to depend on the cohesion of a tissue (how strongly its cells adhere to each other), but the exact relationship between these parameters is not known. Here, we analyse the link between cohesion and viscosity to uncover basic mechanical principles of cell rearrangement. We show that for vertebrate and invertebrate tissues, viscosity varies in proportion to cohesion over a 200-fold range of values. We demonstrate that this proportionality is predicted by a cell-based model of tissue viscosity. To do so, we analyse cell adhesion in Xenopus embryonic tissues and determine a number of parameters, including tissue surface tension (as a measure of cohesion), cell contact fluctuation and cortical tension. In the tissues studied, the ratio of surface tension to viscosity, which has the dimension of a velocity, is 1.8 µm/min. This characteristic velocity reflects the rate of cell-cell boundary contraction during rearrangement, and sets a limit to rearrangement rates. Moreover, we propose that, in these tissues, cell movement is maximally efficient. Our approach to cell rearrangement mechanics links adhesion to the resistance of a tissue to plastic deformation, identifies the characteristic velocity of the process, and provides a basis for the comparison of tissues with mechanical properties that may vary by orders of magnitude.
Abstract Objectives Neurofilament light chain (NfL) concentration in blood is a biomarker of neuro-axonal injury in the nervous system and there now exist several assays with high enough sensitivity to measure NfL in serum and plasma. There is a need for harmonization with the goal of creating a certified reference material (CRM) for NfL and an early step in such an effort is to determine the best matrix for the CRM. This is done in a commutability study and here the results of the first one for NfL in blood is presented. Methods Forty paired individual serum and plasma samples were analyzed for NfL on four different analytical platforms. Neat and differently spiked serum and plasma were evaluated for their suitability as a CRM using the difference in bias approach. Results The correlation between the different platforms with regards to measured NfL concentrations were very high (Spearman’s ρ≥0.96). Samples spiked with cerebrospinal fluid (CSF) showed higher commutability compared to samples spiked with recombinant human NfL protein and serum seems to be a better choice than plasma as the matrix for a CRM. Conclusions The results from this first commutability study on NfL in serum/plasma showed that it is feasible to create a CRM for NfL in blood and that spiking should be done using CSF rather than with recombinant human NfL protein.
This article is part two of a two‐part series. In last month's Opflow, the authors discussed how the Southern California Water Co. (SCWC) identifies and records customer complaints related to various operational activities, including flushing, construction use of water, valve maintenance, and fire flow. In this month's conclusion, the authors describe the corrective measures SCWC has taken to reduce the number of complaints and achieve customer satisfaction. Topics covered include: manganese sequestration; controlled flushing programs; and, problematic old pipes.
This article discusses how Southern California Water Company's (SCWC) Central District discovered how to reduce water quality complaints through various operation controls. The district improved its distribution water quality and reduced complaints by up to 75% by implementing five solutions. The second part of this article, published in the November 2001 issue, goes into more detail about each solution. This first part of the article describes how the district came up with these solutions. The use of databases to track what causes the complaints is explained.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract During amphibian gastrulation, presumptive endoderm is internalised as part of vegetal rotation, a large-scale movement that encompasses the whole vegetal half of the embryo. It has been considered a gastrulation process unique to amphibians, but we show that at the cell level, endoderm internalisation exhibits characteristics reminiscent of bottle cell formation and ingression, known mechanisms of germ layer internalisation. During ingression proper, cells leave a single-layered epithelium. In vegetal rotation, the process occurs in a multilayered cell mass; we refer to it as ingression-type cell migration. Endoderm cells move by amoeboid shape changes, but in contrast to other instances of amoeboid migration, trailing edge retraction involves ephrinB1-dependent macropinocytosis and trans-endocytosis. Moreover, although cells are separated by wide gaps, they are connected by filiform protrusions, and their migration depends on C-cadherin and the matrix protein fibronectin. Cells move in the same direction but at different velocities, to rearrange by differential migration. https://doi.org/10.7554/eLife.27190.001 eLife digest In most animals, the early embryo consists of a single layer of cells that forms a hollow sphere. This simple structure first gains complexity by organising into multiple layers that are fated to become specialised tissues in the adult, such as muscle or skin. To form the primitive gut, a group of cells known as the endoderm must move from the surface to the interior in a process called gastrulation. Since the early 1900s, the sea urchin and the frog have been the standard species used to study gastrulation. In sea urchin embryos, gastrulation entails bending the sheet of cells that form the surface of the embryo inward at a predetermined site to generate a pocket that will become the digestive system. By contrast, frog embryos begin gastrulation as multilayered structures, and the embryo's surface does not bend. Furthermore, classic studies of frog gastrulation have found that cells do not leave the surface of the embryo to enter its interior. So despite generations of students having learned about how gastrulation occurs in backboned animals from studying frogs, the cell behaviours that internalise the endoderm are still not understood. Wen and Winklbauer now show that endoderm cells in the frog move using the same set of behaviours that cells in other organisms use to break loose from or bend sheets of cells. Individual cells move by simultaneously pushing their front end forward while retracting their rear in a peculiar manner, by engulfing their own cell surface at a large scale. In the frog embryo, this movement is coordinated into an organised pattern where cells use the surfaces of their slower or stationary neighbours to propel past each other, and then slow down to return the favour. This constitutes a newly defined type of movement referred to as ingression-type migration. Frog embryos are remarkably large because each of their cells is packed with yolk to support development until the animals are able to feed. As an adaptation to this large size, some frog gastrulation movements appear unusual. However, Wen and Winklbauer show that the cell behaviours that drive these movements are similar to the behaviours of cells in single-layered embryos, and indeed the behaviour of single-celled organisms such as amoebae. Further research is now needed to investigate how these cells find their way straight to the interior of the embryo. https://doi.org/10.7554/eLife.27190.002 Introduction The basic body plan of metazoans is established by gastrulation, and at its core is the movement of endoderm and mesoderm from the surface to the interior of the embryo. Among invertebrates, the pre-gastrulation embryo typically consists of a single-layered epithelium, and a common mechanism of germ layer internalisation is invagination, the inward bending of an epithelium at a pre-localised site. A classic example of gastrulation by invagination is the sea urchin embryo (Kominami and Takata, 2004), and more recently, the invagination of the mesoderm during gastrulation in the fruit fly Drosophila melanogaster has been thoroughly studied (Rauzi et al., 2013). Another major internalisation mechanism is ingression, where individual cells leave the epithelial layer to move interiorly. Both modes of internalisation can occur in the same organism. For example, primary mesenchyme ingression precedes invagination in the sea urchin embryo (Katow and Solursh, 1980; Kominami and Takata, 2004). Within chordates, cephalochordates and tunicates develop from a single-layered blastula. Ingression is not observed in these groups, and internalisation of germ layers occurs by invagination (Shook and Keller, 2008). Although the blastula wall is single-layered in ascidian tunicates, it is thick relative to the size of the embryo, and the vegetal cells in particular are comparatively large, which gives ascidian invagination a distinctive appearance (Satoh, 1978; Sherrard et al., 2010). The transition to the third chordate group, vertebrates, is characterised by a sharp increase in egg size along with the formation of a thick multilayered epithelium that surrounds a blastocoel cavity. Whereas the animal side of the embryo can secondarily become single-layered, the vegetal half always remains as a multilayered cell mass. The corresponding ancestral mode of vertebrate gastrulation, conserved in lampreys, lungfish, and amphibians (Collazo et al., 1994; Shook and Keller, 2008), must adapt to this condition. In a second wave of further egg size increase, meroblastic cleavage again requires adaptation of gastrulation movements in various vertebrate groups. For example, germ layer internalisation occurs by ingression at a novel structure, the primitive streak, in birds and mammals (Arendt and Nübler-Jung, 1999). In the ancestral mode of vertebrate gastrulation, mesoderm is internalised by involution or ingression at the blastopore lip, and the supra-blastoporal endoderm by involution (Shook and Keller, 2008). The multilayered structure of the sub-blastoporal endoderm of the vegetal cell mass precludes invagination, and ingression of the vegetal surface is also absent. Thus, the question arises of how the vegetal endoderm is internalised. Surprisingly, despite endoderm internalisation being a defining feature of gastrulation, it has scarcely been studied in lower vertebrates. Even in the African clawed frog, Xenopus laevis, the most extensively studied model of vertebrate gastrulation, the inward movement of the sub-blastoporal vegetal endoderm has only been analysed at the tissue level (Winklbauer and Schürfeld, 1999; Ibrahim and Winklbauer, 2001; Papan et al., 2007). In X. laevis, the cone-shaped vegetal endoderm is initially narrow inside at the blastocoel floor (BCF), and wide at its outer, epithelial surface. At the equator, it is surrounded by an annulus of mesoderm (Figure 1A) (Keller, 1975; Keller, 1976; Bauer et al., 1994). At the onset of gastrulation, the vegetal cell mass surges animally into the embryo. It narrows at its vegetal-most part, expands at the BCF, rolls the anterior mesoderm against the ectoderm and displaces the posterior mesoderm in the vegetal direction (Figure 1A) (Winklbauer and Schürfeld, 1999; Ibrahim and Winklbauer, 2001; Papan et al., 2007; Winklbauer and Damm, 2012). When a mid-sagittal slice of the vegetal half of the gastrula is explanted (Figure 1B), the entire process continues in isolation and appears as rotational movements on the dorsal and the ventral sides, which gave rise to the term vegetal rotation (Video 1). Further dissection of explants revealed that vegetal rotation is based on active, region-specific tissue deformations within the vegetal cell mass (Winklbauer and Schürfeld, 1999; Ibrahim and Winklbauer, 2001). However, the cellular mechanisms that drive vegetal rotation are not known, and it is not understood how these processes are related to other modes of gastrulation in chordates. Figure 1 Download asset Open asset Tissue movements during Xenopus laevis gastrulation. (A) Fate map and tissue deformation of X. laevis germ layers for stages 10–13. Movements of the ectoderm (white), mesoderm (blue), and endoderm (yellow) are indicated (top row). Blastocoel floor expansion throughout developmental stages is shown (red line). Mid-sagittally fractured gastrulae at stages 10–13 (mid row). Animal is to the top, vegetal to the bottom, ventral to the left, and dorsal to the right. Early, mid, and late stage gastrulae are shown together with the corresponding developmental stage and timeline (bottom row). The onset of gastrulation is set as 0:00 in hours and minutes. Blastocoel (bc) and archenteron (arc) are indicated. (B) Schematic of vegetal explant. The ectodermal BCR was removed with incisions shown (red lines). A mid-sagittal slice of about 5 cell layers thick was removed from the vegetal half of stage 10 embryos and placed under a coverslip for observation. Discarded regions are indicated (X's). Arrows indicate that the explant was tilted 90° toward the viewer to provide an overhead view, and then flipped back to the sagittal view. https://doi.org/10.7554/eLife.27190.003 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 Vegetal 'slice' explant. Explant was excised from a stage 10 gastrula. Movie shows tissue autonomous vegetal rotation movement over the course of 60 min. Cells are labelled with membrane-GFP. Animal is to the top, vegetal to the bottom. https://doi.org/10.7554/eLife.27190.004 In the present work, we have analysed the cellular mechanism of vegetal rotation. We show that endoderm cells undergo elongation and region-specific re-orientation at the onset of gastrulation and move by amoeboid migration without involving lamellipodial, filopodial or bleb-like protrusions. Spatially graded differences in movement velocity lead to orderly cell rearrangements as cells move over and between each other. Rearrangement by such differential migration narrows the vegetal-most part of the tissue and expands the animal part, which leads to the inward surge of the vegetal mass. During migration, endoderm cells are separated by wide interstitial gaps, which are bridged by dynamic filiform protrusions. Despite these gaps, C-cadherin is required to maintain cell migration, and interaction with the extracellular matrix (ECM) protein fibronectin (FN) is also necessary. A peculiar mode of ephrinB1-dependent trailing edge retraction by macropinocytosis and trans-endocytosis, combined with the amoeboid characteristics of endoderm cell translocation, suggests that vegetal rotation is a modification of invagination or ingression adapted to the multilayered structure of the vegetal mass, and is based on ingression-type cell migration. Results The BCF expands by endoderm cell rearrangement and oriented cell shape changes To analyse vegetal rotation, we first quantified tissue shape changes performed by vegetal slice explants. Beginning at gastrulation, the upper region of explants expanded, and the BCF widened by 1.8-fold in two hours before reaching a plateau. Simultaneously, the equatorial waist of explants narrowed by 0.75-fold (Figure 2A). At the cell level, cell rearrangement within the exposed-surface plane of the explant, rearrangement by cells moving in and out of this plane, and cell elongation in region-specific patterns were apparent (Figure 2B). In the absence of strong cell division or cell growth during gastrulation (Saka and Smith, 2001; Kurth, 2005); four cells divided in explant shown in Figure 2B), changes in apparent cell size must be due to cell shape changes and incomplete intercalation in and out of the plane of view (Figure 2B). Together, the changes in cell shape and position expanded the upper part of the explant at the expense of the lower, narrowing part. Figure 2 with 1 supplement see all Download asset Open asset Cellular basis of vegetal rotation. (A) Tissue autonomous movement in live explants. Blastocoel floor (BCF) expansion was followed in explants (red line). BCF length was determined by tracking positions of peripheral endoderm cells (red dots). The equatorial waist (white dashed line) at the explant mid-point runs at the level of the dorsal blastopore (red arrowhead). Animal (An) is to the top, vegetal (Vg) to the bottom, ventral (V) to the left, and dorsal (D) to the right. (B) Cell behaviours in explants. Cells are outlined for the explant shown in Figure 2A, and morphogenetic cell behaviours from 30 to 90 min are indicated (coloured cells). Elongated marginal cells (purple zones) are shown with respect to the cells of the vegetal cell mass (white zone) of the equatorial waist (white and purple solid line). Cells that disappeared from the surface (Brown), cells that emerged at the surface from deep layers (Yellow), cells undergoing division (Blue), cells that reduced their area at the surface as they migrated into deep layers (Red), and cells that increased their surface as they spread out during migration (Green) are shown. (C) Quantification of cell numbers at the BCF and equatorial waist. Error bars indicate S.D. (D) Quantification of cell orientation. Rose diagrams indicate the number of cells in explants (left) or in embryos (right) oriented at given angles relative to the A–V axis in the top (yellow), mid (orange), and bottom (red) cell layers. At stage 10, the endoderm has an average of twelve cell layers, which were evenly divided into three regions. The lengths of bars indicate the number of occurrences in 5° bins. (E) Endoderm cells depicted in scanning electron micrographs of mid-sagittally fractured gastrulae. An apically constricted epithelial surface cell is indicated (asterisk). Corresponding stages are indicated on the left. A schematic of the region of interest (red box) is indicated in the top right corner of select panels. A ruler corresponding to the approximate position of top (yellow), mid (orange), and bottom (red) cell layers is shown in each panel. Panels show data from 14 embryos collected from different egg batches. https://doi.org/10.7554/eLife.27190.005 Figure 2—source data 1 Quantification of cellular changes during vegetal rotation. https://doi.org/10.7554/eLife.27190.007 Download elife-27190-fig2-data1-v2.xlsx Quantitatively, overall cell rearrangement was indicated by changes in cell numbers along landmark lines over time. Average cell number increased from 11.7 to 17.3 at the BCF, remained unchanged along the animal–vegetal (A–V) axis, and decreased from 21.3 to 14.3 along the waists of explants (Figure 2C; Figure 2—source data 1). Cells also elongated slightly with the onset of rearrangement in explants (Figure 2B). In embryos, cell elongation was less pronounced, as seen by scanning electron microscopy (SEM) (Figure 2E). Cell elongation was accompanied by cell re-orientation and alignment, and elongation was predominantly in the direction of movement (Figure 2D; Figure 2—figure supplement 1A; Figure 2—figure supplement 1A—source data 1). After explant excision, cells were on average obliquely oriented in all regions. During the next half hour, cells in the top layers turned parallel to the BCF, while those in the middle layers aligned with the A–V axis. Thus, cells near the BCF became perpendicularly oriented relative to cells in the center. Orientation changed similarly in the embryo (Figure 2D; Figure 2 Data; Figure 2—figure supplement 1B—source data 1). In particular, cells of the expanding BCF flattened in parallel to the tissue surface, perpendicular to cells located farther vegetally (Figure 2E). Cells of the vegetal epithelial layer are known to remain at the surface (Keller, 1978), but occasionally they showed apical constriction and became wedge-shaped (Figure 2D). Our data support the notion that a combination of cell rearrangement and oriented cell elongation underlies the distinct shape change of vegetal explants. To estimate the relative importance of these processes, we considered in detail a representative explant (Figure 2B). Here, the narrowing of the equator was due to a decrease in cell number, as in other explants (Figure 2C). In part, this was because of a disappearance of cells at the lateral explant margins, which was to some extent offset by the elongation of former sub-marginal cells in parallel to the equator (Figure 2B). We suggest that this 'edge effect' was an explant artifact. In the remaining central section of the equator, cell numbers decreased by 0.64 (from 14 to 9 cells), matching the 0.64-fold decrease in equator length. Apparently, the slight elongation of cells perpendicular to the equator in the center of the explant was offset by a similarly slight net increase in apparent cell size. Cell disappearance was rare in the center of the explant (Figure 2B); thus, in-plane cell rearrangement constituted the major morphogenetic process to narrow the lower part of the explant. Cells disappeared at the lateral margins both above and below the equator, and to a lesser extent sub-marginally below the equator. However, cells appeared at the explant surface only above the equator, which contributed to expansion of the explant upper region (Figure 2B). As shown below, this type of intercalation was mostly due to superficial cells moving laterally to expose deeper cells. Importantly, cell lengthening parallel to the BCF also contributed to the region's lateral expansion. Directly at the BCF, net cell number increased 1.3-fold (from 13 to 17 cells), and the remainder of the total 1.8-fold length increase (i.e. a 1.4-fold contribution) was due to oriented cell lengthening and some increase in apparent cell size (Figure 2B). As described in the following paragraph, cell lengthening is an integral part of endoderm cell movement; thus, cell rearrangement and its associated cell movements seemed to be the main mechanisms driving vegetal rotation. Amoeboid migration of endoderm cells We next determined the mechanism by which vegetal cells rearrange. Generally, two basic processes of cell neighbour exchange have been identified. An intensively studied paradigm is epithelial cell intercalation by junction remodelling (Bertet et al., 2004). For example, in D. melanogaster gastrula ectoderm, a cell–cell boundary constricts and resolves to separate two neighbouring cells while a new, perpendicularly oriented contact is formed between previously non-attached cells. An analogous mechanism was proposed for mesenchymal cell rearrangement in X. laevis mesoderm (Shindo and Wallingford, 2014). However, mesenchymal rearrangement can also be driven by the migration of cells over each other. A defining feature of migration is that a cell establishes new contacts on a substratum and detaches from previous contacts, thus changing its position. When two cells migrate over each other, one cell serves as substratum at a given instance for the other to translocate across it. For rearrangement by junction remodelling, no such distinction can be made as the common contact areas between two cells shrink or expand together. During vegetal rotation, the endoderm cells rearranged by amoeboid migration (Figure 3A). While cells wedged themselves between neighbours, they underwent cycles of cell body elongation in the direction of movement, expansion of the cell front, narrowing of the cell rear, and retraction of the trailing edge. Cell shapes reminiscent of the amoeboid motility cycle were also seen in the embryo using SEM (Figure 3B–C). Whereas cell tails were often flattened against other cells, leading edges were blunt and locomotory protrusions such as lamellipodia or filopodia were notably absent. Figure 3 Download asset Open asset Amoeboid cell behaviours. (A) Amoeboid migration of vegetal endoderm cells. mGFP-labelled cells (top row) are shown. Major cell shape changes (dashed arrows) are indicated. Yolk platelets in the same cells are shown in bottom row. A select cell (pink dashed outline) moves with respect to neighbouring cells (grey dashed outlines). Select yolk platelets (pink and yellow outlines) within the moving cell and platelets in a neighbour cell (blue and orange platelets) are indicated. Platelets in different cells move relative to each other, indicating cell migration. Degree of platelet displacement is indicated (Δd, white double arrow). (B, C) Endoderm cell morphology in the embryo, as seen in SEM. (B) Morphology is consistent with amoeboid movement, cells are numbered as showing (1) cell elongation, (2) leading edge expansion, (3) trailing edge recession, and (4) retraction. (C) Higher magnification of cells undergoing leading edge expansion (left), trailing edge recession (center), and trailing edge retraction (right). Cell front (yellow arrows) and rear (blue arrows) are indicated. (D) Coordinate behaviors during cell locomotion. An elongated cell maintains stable lateral borders (yellow arrowed line) throughout time interval (parallel dashed white lines). To advance, the leading edge is extended (green arrow) relative to its initial length (top grey line), while the trailing edge is retracted (red arrow) relative to its initial length (bottom grey line). Extracellular debris attached to the leading cell (blue arrow) and lagging cell (white arrow) are indicated to show displacement. Enlargement of the trailing edge shows contact reduction (mRFP panels, dashed line flanked by arrows) between cells. AvidinFITC puncta are visible (white arrows) at sites of membrane undulation. Interpretation of trailing edge retraction is depicted in bottom rows. AvidinFITC puncta are indicated (black arrows). Area in green corresponds to interstitial space. In all panels, animal (An) is to the top, and vegetal (Vg) to the bottom. Schematic showing the region of interest (red box) is indicated in the top right corner of select panels. https://doi.org/10.7554/eLife.27190.009 To directly show that cells translocated relative to their neighbours, yolk platelets were used as markers of cytoplasmic positions (Kubota, 1981; Selchow and Winklbauer, 1997). Platelets within an advancing cell maintained their relative positions during migration (Figure 3A), which indicated that the cytoplasm of the cell body advanced as a whole. Cell displacement occurred relative to the yolk platelets of stationary cells on the sides, and contact with these cells was reduced at the rear while new contacts were formed at the front (Figure 3A). The leading edge of the cell remained in contact with the cell ahead of it, and both cells moved in tandem. On other occasions, cells invaded the space evacuated by the retraction of a cell (Figure 4A). At the rear, a lagging cell followed closely, although contact with that cell was gradually reduced (Figure 3A). Figure 4 with 1 supplement see all Download asset Open asset Differential cell migration. (A) Cell rearrangement in explants. A leading cell (blue) moves away from the lagging cell (red), neighbouring cells (orange, green) converge to fill the gap. Corresponding trajectories are shown (coloured arrows). (B) Cell displacement in explants. Displacements were recorded starting 15 min after explantation. Panels indicate changes in cell positions between 15–30, 30–45, and 45–60 min. Direction and magnitude of displacements (white arrows) are indicated. (C) Migration velocity variability in explants. Velocities correspond to displacements shown in (B). Cells 1–4 move as described in (A). Colours refer to velocity scale (right). Grey cells were not tracked due to poor visibility. (D) Migration velocity in cell columns (left) and rows (right). Plots show average instantaneous velocities at different time intervals after explantation (colours). Bars indicate S.E. Schematic marks the explant center (Cn), periphery (Pr), animal (An), and vegetal (Vg) boundaries. Panels show data from three embryos from different egg batches. (E) Total velocity is maintained while its vertical component is reduced at the BCF as cells change orientation. Cell movement (top row). Cohort (top-mid row) shows a leading cell (green) advancing laterally relative to the lagging cell (orange), the gap that opens is filled by an inserting deep cell (purple). Cell near the surface (red) remained parallel to the BCF. A deep cell (blue) initially oriented perpendicular to the surface re-oriented to a parallel alignment with the BCF. Cell re-orientation (bottom-mid row) of deep cell (blue dashed outline) relative to its previous position (grey dashed outline) is indicated (arrow). Re-orienting cell velocity vectors (bottom) showing the vertical component (black arrow) relative to the total velocity vector (grey arrow). (F) Cell re-orientation. Optical section shows that the entire cell body is rotated during re-orientation. Movement is indicated (orange arrow). (G, H) Cell morphology in the embryo is consistent with cell reorientation and insertion at the BCF. SEM of cells at the BCF of stage 10.5 gastrulae (left), elongated cell is highlighted (orange, black arrow), blastocoel (bc) is indicated. endo, vegetal endoderm cells. (I, J) Interstitial gaps between cells in the embryo. (I) TEM section through the endoderm (left), negative of TEM (right). Gap width varies between the top (yellow arrow), mid (orange arrow), and bottom cell layers (red arrow). (J) A vertical series of gaps shows gap-width increase in a vegetal to animal direction. (K) Gap width in cell columns (left) and rows (right) in gastrulae. Error bars indicate S.E. Panels show data from six embryos from different egg batches. Schematic of the region of interest (red box) is indicated in the top right corner of select panels. https://doi.org/10.7554/eLife.27190.010 Figure 4—source data 1 Quantification of differential cell migration. https://doi.org/10.7554/eLife.27190.012 Download elife-27190-fig4-data1-v2.xlsx To confirm this mode of translocation, we followed the lengths of lateral contacts, and the distances of these from leading and trailing edges over a time interval (Figure 3D). While lateral contacts remained stationary, the leading edge advanced and the trailing edge retracted, which led to a net translocation of the cell relative to its neighbours. Again, the trailing edge reduced its contact with the cell behind, but membrane undulations suggesting cell detachment also occurred laterally. Occasionally, vesicles appeared inside the cell. Taken together, endoderm cell displacement shows the hallmarks of migration; that is, contact formation at the cell front and contact resolution at the rear. While lateral contacts remain stationary, the cytoplasmic content of a cell moves forward into the advancing front region. Endoderm cells rearrange by differential migration During cell-on-cell migration, cells necessarily move past one another and therefore rearrange locally. To achieve tissue remodelling, rearrangements must be patterned globally such that small local cell displacements translate into large-scale shape change at the tissue level. In the endoderm, the more vegetal part narrows laterally, whereas the animal part expands. In the narrowing region, the elementary rearrangement event consisted of a merging of cell columns. In groups of contiguous cells, animal–vegetal neighbours separated because of the higher velocities of more animally positioned cells, whereas their lateral neighbours converged to fill the spaces left after separation (Figure 4A; Video 2). Cells also partially or completely disappeared into the deeper layers (Figures 2B and 4A). To a lesser extent, this type of rearrangement also contributed to the narrowing of explants below the equator (Figure 2B). 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 Endoderm cell rearrangement. Neighbour exchange of central endoderm cells during differential migration viewed from a magnification of Video 1. Cells are labelled with membrane-GFP. Animal is to the top, vegetal to the bottom. https://doi.org/10.7554/eLife.27190.014 For oriented rearrangements to occur across the whole expanse of the narrowing zone, cell velocity must increase continuously from vegetal to animal. Such a velocity gradient was observed (Figure 4B). Relative to the vegetal surface, cells moved faster when they were located farther animally, up to a zone near the BCF (Figure 4D; Figure 4—source data 1). Movement was also slightly faster in the center of the explant compared to the periphery (Figure 4D); that is, cell movements were not restricted to the endoderm periphery as previously suggested (Winklbauer and Schürfeld, 1999). The timing of explant excision could account for this discrepancy, as vegetal rotation initially spreads from the periphery to the center of the vegetal mass. Rearrangement by the merging of cell columns
SUMMARY We characterize the morphogenic process of convergent thickening (CT), which occurs in the involuting marginal zone (IMZ) during gastrulation of Xenopus , the African clawed frog. CT was described previously as the tendency of explants of the ventral IMZ of Xenopus to converge their circumblastoporal dimension and thicken their radial dimension (Keller and Danilchik 1988). Here we show that CT occurs from the onset of gastrulation, initially throughout the pre-involution IMZ. We suggest that CT is driven by an increase in the interfacial tension between the deep IMZ and its epithelium, resulting in cells of the deep IMZ tending to minimize their surface area. In explants, this results in a progressive shortening (convergence) of the IMZ along its longer mediolateral axis and thickening in the orthogonal planes, and can generate tensile force (Shook et al. 2018). In vivo, convergence of the annular IMZ generates circumferential tension, closing the blastopore. These results provide the first clear example of a tensile morphogenic force from a Holtfreterian/Steinbergian change in tissue affinity.
Xenopus provides a well-studied model of vertebrate gastrulation, but a central feature, the movement of the mesoderm to the interior of the embryo, has received little attention. Here, we analyze mesoderm involution at the Xenopus dorsal blastopore lip. We show that a phase of rapid involution – peak involution – is intimately linked to an early stage of convergent extension, which involves differential cell migration in the prechordal mesoderm and a new movement of the chordamesoderm, radial convergence. The latter process depends on Xenopus Brachyury, the expression of which at the time of peak involution is controlled by signaling through the ephrin receptor, EphA4, its ligand ephrinB2 and its downstream effector p21-activated kinase. Our findings support a conserved role for Brachyury in blastopore morphogenesis.
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