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First published online 22 March 2006
doi: 10.1242/dev.02336

Development 133, 1635-1644 (2006)
Published by The Company of Biologists 2006
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Integrin {alpha}6ß1-laminin interactions regulate early myotome formation in the mouse embryo

Fernanda Bajanca1,2, Marta Luz1,2,*, Karine Raymond3, Gabriel G. Martins1,2, Arnoud Sonnenberg3, Shahragim Tajbakhsh4, Margaret Buckingham5 and Sólveig Thorsteinsdóttir1,2,{dagger}

1 Department of Animal Biology and Centre for Environmental Biology, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal.
2 Gulbenkian Institute of Science, 2781-901 Oeiras, Portugal.
3 Department of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
4 Stem Cells and Development, Department of Developmental Biology, CNRS URA 2578, Pasteur Institute, 25 rue du Dr Roux, 75724 Paris Cedex 15, France.
5 Molecular Genetics of Development, Department of Developmental Biology, CNRS URA 2578, Pasteur Institute, 25 rue du Dr Roux, 75724 Paris Cedex 15, France.


Figure 1
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  		Fig. 1. Characterisation of laminin matrix assembly in the myotome and its interaction with myotomal cells. (A-D) A Myf5nlacZ/+ embryo at E9.5 processed for whole-mount immunostaining for ß-gal (A,C) and laminin (A-D; polyclonal antibody). Confocal imaging showing a sagittal plane of the myotome in caudal (A,B) and interlimb (C,D) somites. z-series projection of: (A) 10x1 µm, (B) 6x1 µm (starting 1 µm ventral to last section in A) and (C,D) 24x1 µm optical sections. Shortly after the myotome (white cells in A) begins to form, a laminin matrix appears under these cells (B). In an interlimb somite of the same embryo, more cells have entered the myotome (C) and the laminin matrix accompanies this growth (C,D). (E-M) Transverse (E-G), sagittal (H-J) and longitudinal (K-M) cryostat sections of interlimb myotomes from wild-type embryos at E9.5-10.0. (E-G) Immunoreactivity for the {alpha}5 (E), {alpha}1 (F) and ß1 (G) laminin chains delimits the dermomyotome dorsally under the surface ectoderm (arrowheads), and at the myotome-sclerotome interface (arrows). Myf5-expressing myotomal cells are frequently seen in contact with the myotomal laminin matrix (arrows in F,G). (H-J) Confocal imaging of the myotome in a thick (30 µm) sagittal cryostat section immunostained for Myf5 and myogenin. Three optical sections are shown: (H) immediately below the dermomyotome; (I) deeper into the myotome, near the myotome-sclerotome interface; and (J) showing the medioventral limit of the myotome immediately before entering the sclerotome. Myf5-positive cells are observed in the dermomyotomal lips. Closest to the dermomyotome (H), only a few Myf5-positive cells are detected in the myotome, while many myogenin-positive nuclei are centrally aligned. Numerous Myf5-positive cells are observed close to the sclerotome (I,J), most of these (arrows in I) located rostral and caudal to the central myogenin-positive nuclei. A few nuclei in the central myotome stain for both Myf5 and myogenin (arrowheads in I,J). (K-M) Longitudinal sections (plane shown in J) show the relative position of differentiating cells within the myotome. (K) Young, Myf5-positive, myogenin-negative cells are located rostral or caudal to the centrally located myogenin-positive cells. (L) Fully elongated myocytes expressing myosin (long red cells; between brackets) are located closest to the dermomyotome, while the myogenin-positive cells (red nuclei, no cytoplasmic extensions) located at the interface with the sclerotome are myosin-negative (arrowheads). They are located centrally, close to discontinuous laminin (arrowheads). A continuous sheet of laminin delimits the rostral and caudal areas of the myotome (arrows). (M) Below the fully elongated myocytes (between brackets), a few elongating myocytes, the extremities of which are desmin labelled, contact the laminin matrix at their tips (arrows). ep, epaxial; hyp, hypaxial; nt, neural tube; scl, sclerotome; lam, laminin chain; laminin, immunoreactivity with polyclonal antibody; myog, myogenin. Scale bars: 50 µm in A-D,H-M; 100 µm in E-G.

 

Figure 2
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  		Fig. 2. The major components of the myotomal basement membrane are produced in Myf5nlacZ/nlacZ embryos, but fail to assemble into a continuous matrix. Whole-mount Myf5nlacZ/+ (A) and Myf5nlacZ/nLacZ (B) embryos at E9.5 (seen through the dermomyotome towards the sclerotome), transverse sections of E10.0 Myf5nlacZ/+ (D,F,H) and Myf5nlacZ/nLacZ embryos (E,E',G,G',I,I') and longitudinal section of a Myf5nlacZ/nLacZ embryo at E10.5 (C), immunolabelled for ß-gal and laminin (A-E,E'), type IV collagen (F,G,G') or perlecan (H,I,I'). (A-E') Laminin immunoreactivity in Myf5nlacZ/+ embryos shows that laminin is organised into a sheet-like matrix between the ß-gal-positive myotomal cells and the mesenchymal sclerotome (arrows in D). In Myf5nlacZ/nlacZ embryos, laminin-immunoreactivity appears absent at low magnification (B); at higher magnification, this immunoreactivity is limited to dots and lines (arrows in C,E,E'). (F-I') The myotomal basement membrane of Myf5nlacZ/+ embryos also contains collagen type IV (F) and perlecan (H). In Myf5nlacZ/nlacZ embryos, collagen type IV (arrows in G) and perlecan (arrows in I) immunoreactivity presents a patchy organisation, but ß-gal-positive cells are not delimited by this matrix. Patches of collagen IV and perlecan, are also visible near the dermomyotome (arrows in G',I'). ep, epaxial; hyp, hypaxial; nt, neural tube. Scale bars: 100 µm in A-B,D-I'; 50 µm in C.

 

Figure 3
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  		Fig. 3. The {alpha}6ß1 integrin is not expressed on the surface of Myf5-null MPCs. (A-D) Co-immunohistochemistry for ß1 (A,B) or {alpha}6 (C,D) and ß-gal (A-D) on transverse cryosections of E9.5 embryos. Inserts show a higher magnification of each epaxial lip. (A,C) Myf5nlacZ/+ embryos express both ß1 (A) and {alpha}6 (C) on the surface of dermomyotomal (arrowheads) and ß-gal expressing myotomal cells (arrows). (B,D) Myf5nlacZ/nlacZ embryos express both ß1 (B) and {alpha}6 (D) on dermomyotomal cells (arrowheads), but most delaminated ß-gal positive MPCs are negative (arrows). ep, epaxial; nt, neural tube; scl, sclerotome. Scale bars: 100 µm.

 

Figure 4
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  		Fig. 4. Incubation of embryo explants with GoH3 perturbs myotome formation. (A-D) Transverse cryostat sections of E8.0 (A,B) and E9.5 (C,D) wild-type embryo explants cultured for 14 hours under control conditions (A,C) or with GoH3 (B,D). In E8.0 (+14 hours) control explants, a few Myf5-positive cells are localised under the dermomyotome in a typical myotomal position (arrow in A). In E8.0 (+14 hours) explants cultured with GoH3, Myf5-positive cells are found near the neural tube (arrows in B). Myf5 expression is present in the dermomyotome (asterisks in B). Laminin immunoreactivity at the myotome-sclerotome interface is discontinuous in GoH3-treated embryos (arrows in D; also see amplification in inserts), and laminin staining is interrupted at the epaxial lip (arrowheads). (E,F) X-gal staining of Myf5nlacZ/+ embryos (E) shows normal distribution of reporter-expressing cells in the myotome, while in Myf5nlacZ/nlacZ embryos (F), these cells disperse. (G-L) Whole mount co-immunohistochemistry of E9.5 embryo explants cultured under control conditions for 24 hours show a normal expression pattern of Myf5 (G,I) and desmin (H,I), while the presence of GoH3 resulted in abnormal dispersion of Myf5-positive cells (J,L), particularly in interlimb-level somites. Furthermore, Myf5-positive cells fail to invade the myotome (absence in the area indicated by brackets). These effects are very similar to the defect observed in Myf5nlacZ/nlacZ embryos (compare J with F). A disorganised pattern of desmin immunoreactivity (K,L) is also observed (area indicated by the brackets and patches among dispersed cells). Anterior is towards the right in E-L. ep, epaxial; hyp, hypaxial; nt, neural tube; fl, forelimb. Scale bars: 100 µm in A-D; 80 µm in G-L.

 

Figure 5
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  		Fig. 5. Incubation of embryo explants with GoH3 leads to precocious myogenic differentiation in the dermomyotome. (A-D) Co-immunohistochemistry for Myf5 and myogenin on cryostat sections of E9.5 wild-type embryo explants cultured for 14 hours (A,B, transverse; C,D, longitudinal). Control explants (A,C) show normal expression of Myf5 and myogenin, including a few Myf5-expressing cells in the dermomyotomal lips (arrowheads in C). In the presence of GoH3 (B,D), myogenin is ectopically expressed in the dermomyotome (arrows) and Myf5-expression has extended (arrowheads). This effect is particularly evident in interlimb-level somites. (E,F) Graphical representation of the increase in fluorescence intensity for Myf5 and myogenin in the dermomyotome compared with the neural tube in control versus GoH3-treated explants. This value was obtained for whole dermomyotomes (E) and for dermomyotomes excluding lips (F). ep, epaxial; nt, neural tube; dm, dermomyotome; myog, myogenin. *P<=0.05; **P<=0.01; bars represent s.d. of means. Scale bars: 100 µm in A-D.

 

Figure 6
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  		Fig. 6. Potential roles of {alpha}6ß1-laminin interactions during early myotome formation in the mouse. (A) (1) Our results strongly suggest that {alpha}6ß1-laminin interactions repress myogenesis in the dermomyotome (yellow cells). This repression is lifted when {alpha}6ß1-laminin binding is broken, an event that normally occurs at the epaxial lip (green cells). (2) {alpha}6ß1 expression is maintained on early Myf5-expressing MPCs and our results demonstrate that {alpha}6ß1 on early MPCs is required for the assembly of the first myotomal laminin matrix. (B) (3) At slightly later stages, Myf5-positive MPCs continue to colonise the myotomal space and their guidance into this space is also dependent on {alpha}6ß1-laminin interactions. The majority of these cells stay in close contact with laminin and do not proceed further in their differentiation programme until they reach the central area of the myotome. (4) This central area near the myotome-sclerotome interface is the area where the laminin matrix is discontinuous and cells expressing Myf5 and myogenin are found. (5) With the addition of younger cells at the myotome-sclerotome interface, elongating myocytes lose contact with laminin except at their caudal and cranial extremities. This laminin matrix may play a role in supporting their elongation.

 

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© The Company of Biologists Ltd 2006