QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 28 January 2009
doi: 10.1242/dev.029942

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Le Guen, L. Articles by de Santa Barbara, P. Search for Related Content PubMed PubMed Citation Articles by Le Guen, L. Articles by de Santa Barbara, P. Social Bookmarking                         What's this?

Intermuscular tendons are essential for the development of vertebrate stomach

INSERM ERI 25, Muscle and Pathologies, 34295 Montpellier Cedex 05, France and University Montpellier I, EA4202, 34295 Montpellier Cedex 05, France.

View larger version (67K): [in this window] [in a new window]   Fig. 1. Intermuscular tendons are present in the embryonic stomach. (A) Relative expression level of transcripts in E6 and E9 chick gizzards; the highest signals are in red, lowest in green. At E9, smooth muscle and tendon markers are expressed at higher levels than at E6. (B) Expression pattern of Scleraxis in the stomach by whole-mount in situ hybridization using an antisense Scleraxis riboprobe. Scleraxis expression is restricted to two newly identified domains located on the dorsal and ventral sides of the gizzard. From E6 to E9, the Scleraxis expression domain widens. At E12, Scleraxis expression is restricted to the boundaries of these initial domains. (C) Whole-mount in situ hybridization on E9 stomachs. SMA is expressed in SMC and Sox10 in ENS cells. Scleraxis, Type I Collagen (ColI), Tenomodulin and Four jointed (Fjx) are expressed mainly in the tendon domains. All tendon markers show a pattern of expression that overlaps with that of Scleraxis, whereas they are absent from the smooth muscle domains and ENS cells. (D) Histology of E6 and E12 gizzards. At E6 the visceral mesenchyme is homogenous (arrow), in spite of the presence of migrating enteric nervous cells on the outer layer (arrowhead). At E12, the gizzard is composed of two well-differentiated smooth muscle structures (black arrows) adjacent to two domains composed of connective tissue (red arrows). (E) Serial transversal sections of an E9 gizzard. Scleraxis, Tenomodulin and Type I Collagen are detected by in situ hybridization, and SMA and Decorin by immunostaining. Two differentiated smooth muscle structures are associated with the two emerging domains of connective tissues characterized by the specific expression of Scleraxis and of the tendon cell markers Type I Collagen, Decorin and Tenomodulin. SMA labels the two smooth muscle areas as well as the monolayer of smooth muscle tissue surrounding the vasculature (arrowheads). In situ hybridization of Scleraxis or Tenomodulin followed by SMA immunodetection on the same sections demonstrated the presence of two tendon structures closely associated with the visceral smooth muscle structures of the gizzard. (F) Left panel: schematic representation of avian E9 stomach indicating the presence of intermuscular tendons, the visceral SMC domain (brown area), and the well-organized tendon domain (green area). Right panels: in situ hybridization on mouse E13 stomachs using a mouse Scleraxis antisense riboprobe (whole-mount and section). Two Scleraxis expression domains (red arrows) are visible.

View larger version (67K): [in this window] [in a new window]   Fig. 2. The FGF signaling pathway is necessary and sufficient to establish the stomach tendon structures. (A) Semi-quantitative RT-PCR analysis of FGF ligand and receptor expression in E6 gizzards. Shown are the mean values±s.e.m. relative to the 18S ribosomal RNA control. Each measurement was done on four independent cDNA preparations (n=4). (B) Whole-mount in situ hybridization on E6 and E9 stomachs. At E6, Fgf7, Fgf10, Fgfr1 and Fgfr2 are widely expressed in the gizzard mesenchyme. At E9, Fgf7 and Fgfr1 expression becomes restricted to the mesenchyme close to the edge of the tendon domains, and Fgf10 and Fgfr2 are both expressed in ENS cells; in addition, Fgfr2 expression defines the tendon borders. Red arrows indicate the tendon area, black arrows the mesenchyme or smooth muscle domain, arrowhead the ENS cells. (C) Inhibition of the FGF signaling pathway in the stomach affects Scleraxis expression. Whole-mount in situ hybridization with an antisense Scleraxis riboprobe on E9 stomachs in which GFP (RCAS-GFP) and sFgfR2b (RCAS-sFgfR2b) are misexpressed. GFP misexpression does not alter stomach morphology, while sFgfR2b misexpression affects slightly the size of the gizzard. The Scleraxis expression domain is downregulated in sFgfR2b-misexpressing stomachs (red arrow), although it is not affected in GFP-misexpressing stomachs (black arrow). (D) Inhibition of the FGF signaling pathway in the stomach affects Scleraxis and Fgf10 but not Fgfr1 and Fgfr2 expression. Quantitative RT-PCR experiments on E7 stomachs in which RCAS-GFP (control) and RCAS-sFgfR2b were misexpressed. sFgfR2b infected stomachs express 60% less Scleraxis and 90% less Fgf10 than do controls. Each measurement was done on two independent cDNA preparations (n=4). (E) Activation of the FGF signaling pathway in the stomach affects the expression of Scleraxis and specific tendon markers. Whole-mount in situ hybridization using an antisense Scleraxis riboprobe on RCAS-GFP and RCAS-Fgf8 E9 stomachs. Ectopic activation of the FGF pathway through Fgf8 misexpression induces ectopic mesenchymal buddings associated with ectopic expression of Scleraxis in the proventriculus (red arrows). Serial adjacent longitudinal sections of a RCAS-Fgf8 E9 stomach analyzed by in situ hybridization with specific antisense riboprobes directed against Env, Scleraxis, Tenomodulin and Type I Collagen, and by immunohistochemistry with an anti-SMA antibody. Longitudinal section of a RCAS-GFP E9 stomach analyzed by immunohistochemistry with an anti-SMA antibody. Areas with RCAS-Fgf8-expressing cells, detected by Env, are characterized by the presence of Scleraxis and other tendon markers, such as Tenomodulin and Type I Collagen, and by reduction of the SMA-positive domain. Black arrows indicate endogenous tendon structures; red arrows, ectopic tendons. (F) Serial longitudinal sections of E9 stomach infected with RCAS-Fgf8 and RCAS-GFP retroviruses and analyzed by in situ hybridization with an Env riboprobe and by immunohistochemistry with an anti-PH3 antibody. Positive signals were counted in three comparable sections. Misexpression of Fgf8 in the gizzard had no effect on proliferation.

View larger version (66K): [in this window] [in a new window]   Fig. 3. Inhibition of Scleraxis expression in the stomach impairs its normal development. (A) Sequence of the sense oligonucleotide (21 red nucleotides) from the avian Scleraxis-coding sequence that makes the hairpin and specifically inhibits its expression. (B) To deliver ShScleraxis into the chick stomach in vivo, the hairpin, the expression of which was under the control of the mouse U6 promoter, was cloned into the RCAS viral vector. (C) Real-Time RT-PCR amplification of dissected E5 gizzard mesenchymal-derived cells cultured for 3 days after infection with RCAS-ShScleraxis. Infected cells express 85% less Scleraxis transcripts than do control cells (RCAS-GFP). Each measurement was done on two independent cDNA preparations (n=4). (D) Inhibition of Scleraxis expression in RCAS-ShScleraxis stomachs. Whole-mount in situ hybridization using the antisense Scleraxis riboprobe on E6 stomachs in which RCAS-GFP and RCAS-ShScleraxis are misexpressed. RCAS-ShScleraxis stomachs present a slight defect characterized by thinner gizzard and straight proventriculus compared with RCAS-GFP controls. These defects are associated with a diminution of Scleraxis expression. Black arrows indicate normal Scleraxis expression in the tendon structures and red arrows show the decrease of Scleraxis expression in the tendon domains. (E) Serial longitudinal sections of an E9 RCAS-ShScleraxis stomach analyzed by in situ hybridization with specific antisense riboprobes directed against Env, Scleraxis, Type I Collagen and Tenomodulin, and by immunohistochemistry with an anti-SMA antibody. Longitudinal section of a RCAS-GFP E9 stomach analyzed by immunohistochemistry with an anti-SMA antibody. Areas with RCAS-ShScleraxis-expressing cells, detected with the Env probe, are characterized by a strong decrease in the expression of Scleraxis, Type I Collagen and Tenomodulin, and an increase of the SMA-positive domain. Note the aberrant localization of a few Scleraxis- and Type I Collagen-expressing cells in the submucosal layer close to the gastric epithelium (red arrowheads). Black arrows indicate endogenous tendon structures; red arrows, areas where RCAS-ShScleraxis is strongly expressed.

View larger version (54K): [in this window] [in a new window]   Fig. 4. Misexpression of Scleraxis in the whole stomach inhibits SMC differentiation. (A) Whole-mount in situ hybridization using the Scleraxis and SMA riboprobes on E9 stomachs misexpressing GFP or Scleraxis. Ectopic Scleraxis expression (RCAS-Scleraxis) induces a gross phenotype characterized by dilated gizzard associated with a curved proventriculus, compared with RCAS-GFP controls. Furthermore, RCAS-Scleraxis inhibits the expression of the SMC marker SMA. Upper black arrows indicate endogenous tendon structures; lower black arrows indicate normal SMC domain; red arrows, perturbation of the gizzard mesoderm. (B) Serial longitudinal sections of E9 RCAS-Scleraxis and RCAS-GFP stomachs analyzed by in situ hybridization with specific Scleraxis and Type I Collagen ripobrobes, and by immunohistochemistry with anti-GAG (3C2) and anti-SMA antibodies. Areas with many RCAS-Scleraxis-expressing cells, detected by the anti-GAG antibody, are associated with inhibition of SMA expression, but not with an induction of Type I Collagen expression. Black arrows indicate endogenous tendon structures; red arrows, ectopic Scleraxis expression domains. (C) Serial longitudinal sections of E9 stomach infected with RCAS-Scleraxis-Engrailed or RCAS-GFP retroviruses analyzed by in situ hybridization with Env and Type I Collagen riboprobes, and by immunohistochemistry with anti-SMA and 3C2 antibodies. The misexpression of Scleraxis-Engrailed in tendon domains is associated with the repression of Type I Collagen expression in the infected tendon and defective tendon structures. Misexpression of GFP had no effect on tendon development. Black arrows indicate endogenous tendon structures; red arrows, Scleraxis-Engrailed expression domains. (D) Scleraxis misexpression in the gizzard increases Tenomodulin expression. Quantitative-RT-PCR experiments on E9 stomachs in which RCAS-GFP (control), RCAS-Scleraxis or RCAS-Scleraxis-Engrailed were misexpressed. RCAS-Scleraxis infected gizzards express 4.2-fold more Tenomodulin than do controls, whereas RCAS-Scleraxis-Engrailed infected gizzards express 90% less Tenomodulin transcripts than do controls. Ectopic expression of Scleraxis in the proventriculus does not induce Tenomodulin in this tissue. Each measurement was done on three independent cDNA preparations (n=4).

View larger version (89K): [in this window] [in a new window]   Fig. 5. Ultrastructural analysis of E9 gizzard sections following retroviral misexpression. (A) Analysis of E9 gizzard misexpressing GFP, Fgf8 or Scleraxis. Control (RCAS-GFP) SMCs are rounder with few mitochondria and have actin filaments in the cytoplasm. Control tendon cells are aligned and surrounded by collagen fibrils. In addition, they show predominantly rough endoplasmic reticulum and mitochondria. RCAS-Fgf8 SMCs present aberrant filopodia, no intracellular actin formation and numerous organelles. RCAS-Scleraxis SMC also show aberrant filopodia, disorganized intracellular actin and an increased number of organelles. Fgf8 misexpression is also associated with ectopic collagen fibrils in the intercellular region that are not observed in RCAS-Scleraxis SMC. Black arrows indicate extracellular collagen fibrils; black arrowheads, intracellular actin formation; blue arrows, the absence of extracellular matrix; red arrows, ectopic collagen fibrils; red arrowheads, the absence of intracellular actin formation. (B) Analysis of tendon cells from E9 gizzards misexpressing sFgfR2b, ShScleraxis or Scleraxis-Engrailed. RCAS-sFgfR2b tendon cells present aberrant morphology with dark cytoplasm (arrowhead), and without collagen fibrils (blue arrows). RCAS-ShScleraxis tendon cells present a moderate phenotype with a strong decrease of collagen fibril production (blue and black arrows), dark cytoplasm and rounder morphology (arrowhead). RCAS-Scleraxis-Engrailed gizzards also have tendon cells with dark cytoplasm (arrowhead) and a strong inhibition of collagen fibril production (blue arrow). Scale bars: 2 µm (upper panels) and 500 nm (lower panels).

View larger version (80K): [in this window] [in a new window]   Fig. 6. Characterization of distinct cell populations in primary cell cultures derived from E5 gizzard mesenchyme. (A) Enzymatic dissociation and microdissection of an E5 gizzard into mesenchyme and endoderm. The undifferentiated mesenchymal cells will be cultured for one (E5+1D) and three days (E5+3D). (B) In cellulo in situ hybridization using the antisense Scleraxis riboprobe on E5+1D and E5+3D mesenchymal-derived cells. Nuclei were visualized with DAPI. At E5+1D, Scleraxis-positive cells are isolated (upper panel), whereas at E5+3D they have aggregated into colonies (lower panel). Scleraxis expression is colored using the Photoshop software. (C) Analysis of E5+3D mesenchymal-derived cells by in cellulo in situ hybridization using the antisense Scleraxis riboprobe (upper panel) and Methyl Violet staining (lower panel). Clusters of cells expressing Scleraxis are also positive for Methyl Violet, a dye that labels cells containing high-level organelles. (D) Characterization of E5+3D mesenchymal-derived cells by immunofluorescence. Nuclei were visualized with DAPI. The tendon-specific markers Tenascin and Type I Collagen were used, as well as the SMC marker Caldesmon, the mesenchymal cell marker Desmin, and the chondrocyte marker Sox9. Colonies were all positive for the tendons markers, but not for Caldesmon or Sox9. The scattered cells adjacent to the colonies presented strong expression of muscle markers, but no expression of tendon or chondrocyte markers. These results demonstrate that the mesenchymal-derived cultures contain two types of cells: tendon cells aggregated into colonies, and SMC, represented by the scattered cells. (E) Colony-forming efficiency assessed by Methyl Violet staining of E5+3D primary cells derived from the mesenchyme of gizzards and infected with RCAS-GFP, RCAS-Scleraxis, RCAS-ShScleraxis, RCAS-Fgf8, RCAS-sFgfR2b or RCAS-Scleraxis-Engrailed for three days. Fgf8 and Scleraxis misexpression have a strong positive effect on the number of colonies, while ShScleraxis misexpression moderately inhibits their formation. Scleraxis-Engrailed and sFgfR2 are both potent inhibitors of cell cluster formation. Each measurement was done on three independent experiments (n=3). (F) Analysis of Pcna expression by quantitative RT-PCR amplification of E5+3D culture cells following infection with different retroviral constructs. Each measurement was done on two independent cDNA preparations (n=4). Perturbation of Scleraxis expression by different means has a moderate impact on Pcna expression.

View larger version (55K): [in this window] [in a new window]   Fig. 7. Model of the molecular pathways and the tissue interactions during stomach development. (A) Schematic representations of avian E6 stomach and the molecular pathways involved in the determination of the tendon domains. At E6, the mesenchymal layer is composed of undifferentiated visceral mesenchyme and two emerging committed tendon cell domains (green areas). The cells of these domains, upon mesenchymal FGF activation, express Scleraxis and become committed to the tendon lineage, while their differentiation into SMC is inhibited. (B) Schematic representations of avian E9 stomach and the molecular pathways involved in the differentiation of the intermuscular tendons. At E9, the mesenchymal layer is divided in two differentiated visceral SMC domains (brown areas), submucosa (gray area) and two well-organized tendon domains (green areas). Mesenchymal FGF activation allows the differentiation of the tendons that express Scleraxis. Scleraxis with additional transcription factors or interacting partners (X factor) might ensure the differentiation of tendon cells.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter