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First published online 1 August 2007
doi: 10.1242/dev.003665

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Redefining the role of ectoderm in somitogenesis: a player in the formation of the fibronectin matrix of presomitic mesoderm

Pedro Rifes^1,2,*, Lara Carvalho^1,*,, Catarina Lopes¹, Raquel P. Andrade³, Gabriela Rodrigues^1,2, Isabel Palmeirim³ and Sólveig Thorsteinsdóttir^1,2,

¹ Departamento de Biologia Animal e Centro de Biologia Ambiental, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal.
² Instituto Gulbenkian de Ciência, 2781-901 Oeiras, Portugal.
³ Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057 Braga, Portugal.

View larger version (102K): [in this window] [in a new window]   Fig. 1. Distribution of fibronectin in the chick PSM and surrounding tissues. (A,B) Longitudinal (A) and sagittal (B) sections reveal short strands of fibronectin immunoreactivity within the PSM (arrowheads in A,B), and an extensive fibronectin immunoreactivity surrounding the anterior PSM. This immunoreactivity is distinguishable from that of the neural tube, ectoderm and endoderm (arrows in A,B). (C) Non-enzymatic removal of ectoderm exposes fibrillar fibronectin immunoreactivity on the PSM. (D,E) By contrast, PSMs isolated with pancreatin (D) or dispase (E) are completely negative. (F) When collagenase is used an intact fibronectin matrix is maintained. Polyclonal anti-fibronectin (A,B) and monoclonal anti-cellular fibronectin (C-F) antibodies were used. fn, fibronectin. Scale bars: 100 µm in A,B; 25 µm in C-F.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Collagenase-isolated chick PSMs form somites in the absence of both ectoderm and endoderm. All explant types were either fixed immediately (0 hours, 0h) and labelled for fibronectin or cultured for 6 hours (6h) and processed for fibronectin and F-actin staining. (A-I) Pancreatin-isolated explants. Isolated PSMs are fibronectin-negative at 0 hours (A). At 6 hours, fibronectin staining is visible (B) but somites have not formed (C). In PSM+endoderm explants, some fibronectin immunoreactivity is present between endoderm and PSM at 0 hours (arrows in D). These explants show fibronectin immunoreactivity at 6 hours (E), but no somites have formed (arrow in F). PSM+ectoderm have fibronectin immunoreactivity between ectoderm and PSM (arrows in G), whereas the remaining PSM is negative (arrowheads in G). At 6 hours, clefts (asterisks in H,I) containing fibronectin immuoreactivity (H) and rings of F-actin staining (arrows in I) are observed. (J-R) Collagenase-isolated explants. All explants isolated with collagenase retain their endogenous, fibrillar (see insert in J) fibronectin matrix (J,M,P). At 6 hours, all explant types have clefts (asterisks in K,L,N,O,Q,R) containing fibronectin (K,N,Q) and several epithelialised somites (arrows in L,O,R). Anterior, right. psm, presomitic mesoderm; endo and en, endoderm; ecto and ec, ectoderm. Scale bars: 100 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Quantitative representation of the capacity of all PSM explant types treated with pancreatin or collagenase to form somites. The average number of somites formed for each explant type and enzyme treatment is depicted, and the proportion of explants forming 0, 1, 2, 3, 4 and 5 somites during the 6-hour culture period is indicated with a colour code. Note that the somites formed in PSM and PSM+endoderm explants isolated with pancreatin (marked with ) were frequently less compacted and had incomplete clefts as compared with all other experimental situations. A factorial ANOVA (results depicted at the bottom of the figure) followed by a contrast analysis of predicted differences (results shown in graph) were performed to test for differences among treatments. ns, not significant. Error bars represent 95% confidence limits.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Paraxis expression is maintained in both pancreatin- and collagenase-isolated chick PSMs cultured for 6 hours. After 6 hours of culture, control sides (intact halves, A,C) strongly express Paraxis in the anterior PSM in a pattern identical to that of the contralateral isolated PSMs, regardless of whether these were isolated with pancreatin (B) or collagenase (D). Only the collagenase-isolated explants formed somites (asterisks in D). Anterior, top. nt, neural tube; lp, lateral plate; psm, presomitic mesoderm; panc, pancreatin-isolated PSM; coll, collagenase-isolated PSM. Scale bars: 100 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 5. Partial rescue of somite formation in pancreatin-isolated PSMs cultured with exogenous fibronectin. (A) Pancreatin-isolated chick PSMs present immunoreactivity for cellular fibronectin after 6 hours in culture. (B-D) z-projection of confocal images of dispase-isolated PSMs cultured for 2 hours (B) show faint fibronectin immunoreactivity, whereas after 4 (C) and 6 (D) hours it is more extensive. (E) Average fluorescence labelling intensity relative to background in dispase-isolated PSMs cultured for 2 (n=7), 4 (n=4) and 6 hours (n=6). (F-I) Contralateral pancreatin-isolated PSMs, cultured in control (F,G) or fibronectin-supplemented (H,I) medium and stained for F-actin (F,H) and fibronectin (G,I). (J) Bar chart of the average number of somites formed (n=20 pairs) showing that PSMs cultured with fibronectin form significantly more somites (P=0.002). Error bars represent 95% confidence limits. cFN, cellular fibronectin; +FN, PSM cultured in fibronectin-supplemented medium. Scale bars: 100 µm in A,F-I; 50 µm in A insert, B-D.

View larger version (123K): [in this window] [in a new window]   Fig. 6. F-actin and N-cadherin distribution in dispase-isolated PSMs cultured with and without fibronectin. Somite III regions (i.e. somite -II at 0 hours) of dispase-isolated chick PSMs after 6 hours of culture in control medium show only small foci of F-actin (A) and N-cadherin (D) clustering. By contrast, the same region of dispase-isolated PSMs cultured with fibronectin shows a prominent apical enrichment of F-actin (B) and N-cadherin (E) and a nuclear alignment and elongation typical in epithelial somites (B,E). Interestingly, this cellular arrangement is very similar to that observed in collagenase-treated PSMs (C,F). Anterior, left. Scale bars: 25 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 7. mRNA expression pattern of fibronectin (Fn1) and its integrin receptor (Itga5) in the PSM and surrounding tissues. (A-D) In situ hybridisation for Fn1 on HH12-14 stage chick embryos shows that Fn1 mRNA is strongly expressed in the ectoderm overlying the PSM (A-D) and in the posterior part of epithelial somites (A,B,D). Endoderm is negative posteriorly but expression is detected at the level of anterior PSM and epithelial somites (B-D). PSM appears negative for Fn1 mRNA except for a faint staining posteriorly (B,C). (E-I) Removal of ectoderm before fixation confirms this pattern and shows that Fn1 expression is very strong in the intermediate mesoderm (E,F,G). (J) However, Fn1 expression is detectable in both the anterior third and posterior two-thirds of the PSM by RT-PCR. (K) Negative hybridisation control with Fn1 sense probe. (L-N) In situ hybridisation for Itga5 shows that it is strongly expressed in the posterior two-thirds of the PSM and in epithelial somites. Insert (L) shows sense probe control. Sections of Itga5-hybridised embryos show that the signal in PSM is faint anteriorly (M), but strong posteriorly (N). Itga5 is also expressed in notochord, intermediate (arrows) and lateral mesoderm (M,N), and extraembryonic blood vessels (arrows in L). Faint staining is detectable in neural tube and ectoderm (arrowheads, M,N). Anterior, up. *, Epithelial somite; im, intermediate mesoderm; n, notochord; som, somites; ant PSM, anterior PSM; post PSM, posterior PSM; Ctrl+, positive control (whole embryo cDNA); RNA, reverse-transcription omitted; water, cDNA omitted. Scale bars: 500 µm in A,B,E,L; 100 µm in C,D,F-K,M,N.

View larger version (63K): [in this window] [in a new window]   Fig. 8. A 70 kDa peptide containing the fibronectin assembly domain inhibits somitogenesis in embryo explants cultured for 12 hours. (A-D) Ventral view of chick embryo explants cultured with BSA (control) or the 70 kDa fibronectin fragment. Endoderm was removed on the left while the other side was kept intact. Embryos incubated with BSA form the expected number of somites in 12 hours (A,C), whereas embryos cultured with the 70 kDa fibronectin fragment form fewer somites (B,D). When embryos are cultured with 100 µg/ml of the peptide, somitogenesis is practically abolished (D). (E) Bar chart of the results obtained in A-D. Error bars represent 95% confidence limits. *, Somites formed in culture; BSA, bovine serum albumin; 70 kDa, 70 kDa fibronectin fragment; ns, not significant; **, P0.005. Scale bars: 100 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 9. Model of fibronectin assembly in the chick PSM. Sagittal view of PSM and first epithelial somite, showing ectoderm dorsally and endoderm ventrally. Based on our data, we suggest that PSM fibronectin matrix assembly occurs primarily in the posterior two-thirds of the PSM and that the fibronectin matrix in the anterior-most PSM is (if left unperturbed) sufficiently extensive to support somite formation. We propose that ectoderm is the major source of fibronectin protein for this matrix, and that only a minor contribution comes from the PSM itself. The PSM does however express 5ß1 integrin, which is essential for the assembly process. Although Itga5 mRNA is not detected in the anterior PSM, we hypothesise that the 5ß1 protein remains on its surface. Itga5 mRNA is again detected in epithelial somites, whereas Fn1 is expressed in their posterior half, suggesting a potential role in the stabilisation of somite clefts (Koshida et al., 2005). According to our model, ectoderm ablation over the posterior two-thirds of the PSM compromises fibronectin matrix assembly and the formation of somites by the underlying PSM. By contrast, ectoderm ablation over the anterior-most PSM, if performed without destroying the endogenous fibronectin matrix, does not affect somite formation.