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First published online 16 December 2004
doi: 10.1242/dev.01537

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Gli2 and Gli3 have redundant and context-dependent function in skeletal muscle formation

¹ Department of Biomedical Science, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK
² Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
³ Department of Molecular and Medical Genetics, University of Toronto and Program in Developmental Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada
⁴ Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA

View larger version (100K): [in a new window]   Fig. 1. Gli gene expression in mouse somites. The pattern of expression of Gli1 (A,D,G), Gli2 (B,E,H) and Gli3 (C,F,I) was compared to that of the sclerotomal marker Pax1 (J), the syndetomal marker scleraxis (K), and the myotomal marker Myf5 (L) using whole-mount in situ hybridisation in E9.5 mouse embryos. Gli1 expression is more abundant in the sclerotome (sc) and excluded from the dermamyotome, whereas Gli2 and Gli3, although initially expressed throughout the somite, rapidly become restricted to the myotome and ventral dermomyotome. Black and white arrows point to Gli expression in the sclerotome (sc), which expresses Pax1, the dorsal medial lip (dml) and ventral lateral lip (vll) of the dermamyotome, which express Myf5, and the syndetome (syn), which expresses scleraxis.

View larger version (74K): [in a new window]   Fig. 2. Gli1 expression in somites requires Gli2 or Gli3. Gli1 expression was investigated in E9.5 wild-type (A,G,M), Gli3–/– (B,H,N), Gli2–/– (C,I,O), Gli2+/–Gli3–/– (D,J,P), Gli2–/–Gli3+/– (E,K,Q) and Gli2–/–Gli3–/– (F,L,R) embryos using whole-mount in situ hybridisation. Transverse sections were performed at the level indicated by double black arrows. Black arrows indicate domains of Gli1 expression that are unchanged or upregulated. Black arrowheads indicate loss of Gli1 expression.

View larger version (100K): [in a new window]   Fig. 3. Myf5 activation is impaired in Gli2–/–Gli3–/– embryos. Myf5 expression was analysed by whole-mount in situ hybridisation at E8.5 (A-L) and E9.5 (M-X) in wild-type, Gli2–/–Gli3+/–, Gli2+/–Gli3–/– and Gli2–/–Gli3–/– embryos. Transverse sections were performed at the level indicated by black arrows. Black arrowheads point to the disrupted Myf5 expression. Brackets indicate loss of Myf5 expression in posterior somites. Myf5 expression is not activated in posterior somites of Gli2–/–Gli3–/– embryos and is reduced or delayed in Gli2–/–Gli3+/– and Gli2+/–Gli3–/– embryos at E8.5 and E9.5. In addition, upregulation and ventral expansion is observed in E8.5 anterior somites and in E9.5 interlimb somites of Gli2–/–Gli3+/– and Gli2–/–Gli3–/– embryos.

View larger version (147K): [in a new window]   Fig. 5. Gli2–/–Gli3–/– embryos exhibit abnormalities in the formation of the myotome and hypaxial muscle progenitor cells. Expression of myogenin (A-A'''), paraxis (B-B'''), Pax7 (C-C'''), Pax3 (D-D'''), Myod1 (E-E'''), Lbx1 (F-F'''), Noggin (G-G'''), and Sim1 (H-H''') was assessed by whole-mount in situ hybridization in wild-type, Gli2–/–Gli3+/–, Gli2+/–Gli3–/–, and Gli2–/–Gli3–/– embryos ranging from E9.0 to E10.0. Red lines indicate the position of transverse sections shown below the whole-mount image. Black arrowheads point to upregulation and/or misexpression of the gene studied. Note that Noggin expression is lost in the notochord of Gli2–/–Gli3–/– embryos.

View larger version (89K): [in a new window]   Fig. 4. Gli2–/–Gli3–/– embryos fail to activate the Myf5 EE enhancer/lacZ transgene. X-gal staining of E9.5 wild-type (A,C,E,G) and Gli2–/–Gli3–/– (B,D,F,H) embryos was performed to assess the activity of the Myf5 early epaxial enhancer. (A,B) Whole-mount X-gal staining shows that expression is lost in posterior somites (bracket). (C-H) Transverse sections were performed at the level indicated by the black line in posterior somites (C,D), interlimb somites (E,F) and anterior somites (G,H). In Gli2–/–Gli3–/– embryos, ß-gal+ cells are absent from posterior somites (black arrowheads) and greatly reduced in anterior somites.

View larger version (85K): [in a new window]   Fig. 6. Shh signalling converts Gli3, but not Gli2 into a transcriptional activator in somites. E9.5 wild-type (A,E,I,M,Q), Shh–/– (B,F,J,N,R,V,V'), Gli3+/–Shh–/– (C,G,K,O,S), Gli3–/–Shh–/– (D,H,L,P,T), Gli2+/–Shh–/– (W,W') and Gli2–/–Shh–/– (X,X') embryos were analysed by whole-mount in situ hybridisation for myogenin (A-D) and Myf5 (E-X') expression. Transverse sections were performed as indicated by red lines. Black arrows indicate loss of expression. Black arrowheads indicate domains of altered gene expression. Asterisks indicate the ventromedial expansion of Myf5 expression.

View larger version (71K): [in a new window]   Fig. 7. Model for Gli2 and Gli3 function in somite myogenesis. Dorsoventral and mediolateral patterning of the somite is dictated in part by the opposing action of a ventral gradient of Hedgehog signalling (in red) and a lateral gradient of Bmp4 signalling (in blue). In the medial somite, the combined activity of Gli2A and Gli3A, converted from Gli3R into Gli3A by Shh, leads to the activation of Myf5 in DML cells and the activation of Pax1 in sclerotomal cells. Myf5 is also a target of Gli2A and Gli3A in the epaxial myotome, although a repressor function of Gli3 is revealed in Gli2–/–Gli3+/– embryos. In the lateral somite, characterised by low levels of Shh signalling and high levels of Bmp4 signalling, Myod1 and Lbx1 expression is controlled by Gli3R and Smad. We suggest that Gli3R could directly control Lbx1, Myf5 and Myod1 levels, and/or could interfere with Bmp4 signalling via an interaction with Smad in the control of Lbx1, Myf5 and Myod1 levels in the VLL and hypaxial myotome. Additional Gli/Shh-independent mechanisms of Myf5 activation are active in both the medial and lateral somite, possibly mediated by Wnt and/or FGF signalling.

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