Figure 1.
Cell organization, polarization and matrix organization in rostral PSM and epithelial somites (see also Videos S1 and S2).
A) Coronal confocal section of chick embryo PSM immunostained for N-cadherin showing s-II, s-I and s0. Different cell shapes can be recognized: mesenchymal polygonal cells (blue), medial cuboidal cells (red) which are aligned forming a cuboidal epitheloid layer (between arrowheads) and elongated spindle-shaped cells (yellow). Red cells in s-I have started elongating and yellow cells in s0 are spindle-shaped. B and C) Coronal confocal sections of a chick embryo PSM stained for DNA showing a detail of the medial epithelium at the level of s-I (B) where cell nuclei are round and basally aligned (red dots), and at level sI (C), where they are oval-shaped and non-aligned basally as in a pseudostratified epithelium (green dots). D) Coronal confocal section of a chick embryo PSM stained for N-cadherin, showing in detail the extension of pseudopodia that connect the PSM cells to their surroundings. E) 3D surface reconstruction of the rostral PSM and sI showing a dorsal (E) and medial view (E′). Transparent surface represents the epithelium and in dark gray the mesenchymal core. The s0 somite is still inserted in the “socket-like” PSM [1], but indentations (arrowheads) reveal where the inter-somitic cleft will soon appear. The rostral and lateral sides of s0 are still mesenchymal. As a result of cell rearrangements between s-II and sI, the PSM narrows medio-laterally and thickens dorso-ventrally (dotted lines in E and E′). F) 3D surface reconstruction (white transparent surface) of s0 somite viewed medially (F), rostrally (F′) and dorsally (F″), showing a volume reconstruction of N-cadherin immunostaining (green) inside. N-cadherin is enriched in the medial, dorsal, ventral and caudal sides, and less so in rostral and lateral sides. Thus the N-cadherin-staining forms a 3D “adhesion basket” in s0. G) 3D surface reconstruction (white transparent surface) of s0 somite viewed dorsally (G) and medially (G′) showing representative cells in the epithelial layer inside, also surface reconstructed (multiple colors). Rostral and lateral cells are elongated but not yet oriented centripetally (dotted circles), while cells in other sides are already aligned. See also Video S1. H) Projection of tangential confocal coronal sections FN-positive fibrils (green) extending away from somite surface, and demonstrating a patchy pattern of laminin immunoreactivity (red). I) 3D volume reconstruction of FN matrix organization (green) surrounding the rostral PSM including s-I and s0 (brown surface) and sI (blue surface). Rostral is to the right and medial to the top. Cables of FN connect the PSM and sI to surrounding tissues (arrowheads). J) Rostral view of FN matrix in a 3D volume reconstructed transversal slab of PSM as shown in I (black plane) at the level of the forming cleft between s-I and s0. Dorsal is upwards, medial is to the right. Cables of FN penetrate inwards into the interior of the somite (yellow arrows), along the lateral surface of epitheloid cells, and between somites, into the nascent cleft. Tissues are represented in light blue. J' is a detail of panel J, showing the cables of FN penetrating the intersomitic cleft. The tissues have been digitally removed to show only the FN matrix (green).
Figure 2.
Morphogenetic movements during somite formation in chick embryos (see also Video S3).
A) Panels represent seven time-points (spaced 1 hour) of a 4D two-photon imaging sequence (each is a 3D reconstructed 30 µm coronal “slab” through the somite's equator (rostral is to the top, medial to the left) of embryos expressing a mosaic of GFP-positive and negative cells. Thin horizontal arrows point to areas where intersomitic boundaries form. Five cells were traced (outlined in color) to depict different morphogenetic movements: i) Cuboidal medial cells elongate and become spindle-shaped (blue). ii) Some cells are recruited to the medial epithelium via accretion (red) and undergo a change in shape and orientation to conform to the orientation of medial elongated cells. iii) Lateral PSM cells and core mesenchymal cells (both yellow) converge and elongate. iv) Core cells egress (green) into the epithelium, becoming spindle-shaped and centripetally aligned. In the last panel (A+6h) the initial positions of the cells (A+0h) are drawn with dotted lines, and colored arrows represent the overall morphogenetic movements. The size of the mesenchymal region, i.e. the somitocoel (black dotted lines) becomes progressively smaller during these 6 hours. B) Detailed view of time-points 3:50 (B) and 4:00 (B′) showing pseudopodia retracting (orange arrowheads) and new ones forming (green arrowheads), similar to the tapering extension observed in fixed embryos (see Figure 1D and last segment of Video S3). Furthermore, massive cytoplasm movements inside cells are also visible (asterisks; where green marks cytoplasm translocations; see also Video S3). These movements occurred throughout the image recording period indicating that the dynamic behavior was retained even after cells became epitheloid.
Figure 3.
Inhibiting FN matrix assembly impairs somite formation by affecting cell elongation and alignment.
A and B) Bright-field images showing equivalent halves of embryos cultured for 6 hours either under control (BSA) conditions (A) or with the 70 kDa FN fragment (B). The control embryo formed three complete somites (sI-sIII) and an advanced s0 (A), while the 70 kDa fragment-treated embryo formed only one somite (marked “sI”) and an incipient “s0” (B). B′ is a two-photon section showing an “sI” of a GFP-electroporated and 70 kDa fragment-treated embryo showing impaired cell elongation and alignment (see also Video S4), particularly in the caudal side (B″; compare with Figure 2A+6h). C and D) FN matrix covering the dorsal surface of the PSM at equivalent axial positions of control explants (s0, C) and explants treated with the 70kDa fragment (“s-II”, D). Ectoderm-associated FN was digitally removed to show only the PSM FN matrix. Explants treated with the 70 kDa fragment (D) show numerous large holes in the FN matrix (red arrows) interspersed with dense agglomerates of FN (green arrows), contrasting with the more uniform fibrillar matrix of control explants (C). E) Timing of intersomitic cleft formation as determined from analysis of bright-field time-lapse image sequences. Control embryos (n = 3) formed three new somites in approximately 4 hours, while experimental embryos (n = 4) took almost 5 hours to form either one (n = 2) or two (n = 2) new somites. F) Graphical representation of cell lengths and centripetal angles of rostral (n = 10 cells/embryo) versus caudal (n = 10 cells/embryo) somitic cells in control embryos at mid-culture (s0, n = 3), experimental embryos at 6 hours (“s0”, n = 3) and control embryos at 6 hours (sII, n = 3). Comparing cells in “s0” with the equivalent cells of control embryos, which had matured to sII stage, showed that caudal cells were significantly less elongated and aligned (P<0.001) in the “s0”, and so were rostral cells (P = 0.004 and P<0.001, respectively). Comparing cells in “s0” to cells of control s0 revealed significant differences in caudal cell elongation (P<0.001) and alignment (P = 0.007) whereas no differences in those parameters were detected rostrally (P = 0.643 and P = 0.368, respectively). Error bars represent 95% confidence intervals. Asterisks represent significant differences of “s0” when compared to sII (bottom asterisk) or s0 (upper asterisk).
Figure 4.
N-cadherin fails to polarize apically when FN fibrillogenesis is inhibited.
Bright field (A, D), coronal confocal sections (A′, A″, A″′, D′, D″, D′″) of control (A) and 70 kDa FN fragment-treated (D) explants cultured for 6 hours, immunostained for N-cadherin (green) and labeled for DNA (red) and 3D surface reconstruction with N-cadherin labeling (green in B,C,E,F) of selected volumes. A) Control explants form four new somites. In s0 (A′), medial cells are starting to elongate and to polarize their N-cadherin apically. In sIII (A″) and sV (A″′), cells have become centripetally aligned, with oval-shaped nuclei and apically restricted N-cadherin immunoreactivity. B–C) Lateral view of N-cadherin labeling shows a “3D adhesion basket” in s0 (B). By sIII (C) the N-cadherin labeling has become more apically restricted and has closed rostrally and laterally thus forming a ball. D) 70 kDa fragment-treated explants form one or two somites; the depicted embryo formed one (“sI”) and has an advanced “s0”. The tissue at the same axial level as A′ (“s-III”, D′) shows no sign of epithelialization or cell elongation. Medial cells in the forming somites of 70kDa fragment-treated explants (“s0”, D″; axially equivalent to A″) show incipient elongation and N-cadherin polarization. Cells of somites that had formed before culture (“sII”, D″′) are less polarized and less elongated than cells at the equivalent axial level in control explants (sV). E–F) Lateral view of N-cadherin labeling in 70kDa fragment-treated explants show dispersed N-cadherin localization in the “s-III” (axial equivalent to B). The “s0” (axial equivalent to C) depicts a more intense labeling in the rostral portion, resulting in an “adhesion basket” that is opened caudally.
Figure 5.
Inhibition of FN fibrillogenesis impairs egression of cells from the somitocoel to the somite epithelium.
A–B) Representative examples of 3D surface reconstructions of somites (blue; dorsal cap digitally removed) and their somitocoels (purple) formed in embryo explants at the end of 6 hours of culture with BSA (n = 4; A) or with 70 kDa fragment (n = 6; B). Three of the six explants treated with the fragment formed two somites as depicted in B; the remaining ones formed only one somite. C) Quantification of somite and somitocoel volumes and cell densities (fluorescence intensity of DNA labeling). Only somites fully separated from the PSM (≥ sI) were measured. Since an ANOVA revealed no significant differences between somites formed during the 6 hour culture period within each treatment, we pooled the measurements of the different somites to an average per explant (C). There is no significant difference (P = 0.129) in somite volume between control and fragment-treated explants, but somitocoels of fragment-treated explants are significantly larger (P<0.0001). D) Quantification of cell density in the whole somites shows no difference (P = 0.990) between control and 70 kDa fragment-treated explants. However, the cell density ratio (somitocoel/epithelial portion) is significantly higher (P = 0.043) in somites of 70 kDa fragment-treated explants. Bars represent 95% confidence intervals.
Figure 6.
Model of morphological somite formation and morphogenetic movements.
A) Schematic representation of a “hypothetical” coronal slice representing stages of somite formation. Medial is up and rostral to the right. The different morphogenetic movements that contribute to the assembly of the somite epithelium are represented: Medial cells first become cuboidal with basal nuclei (dark green cells), then these cells elongate and recruit other cells to the epitheloid layer via accretion (red cells); accretion spreads ventrally and dorsally (not shown), and eventually to the caudal and rostral side. Simultaneously, cells egress from the core into the epitheloid layer (green cells), and, finally, at the lateral side of the somite, cells elongate, intercalate and condense (yellow cells). In s0, the rostral and lateral sides epithelialization is only completed after the somite separates from the PSM. The FN matrix is depicted in green. A′ depicts how the accretion and condensation “spread” through the whole somite to complete the “spherical” ball of epitheloid cells. B) When the assembly of the FN matrix is disrupted, PSM cells (especially caudal cells) do not polarize or orient centripetally and core cells fail to egress into the epithelium.