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Fig. 6. Detection of Pax3 (part I), Met (part II), Lbx-1 (part III) and Pax7 (part IV) by whole-mount in situ hybridization on E10.5 heterozygous and dKO embryos. (Part I) Pax3 expression in heterozygous (A,C,E,G,I,K,M,O,Q) and dKO embryos (B,D,F,H,J,L,N,P,R). Pax3 is still expressed in the dKO (B). At the caudal level, in youngest somites Pax3 expression is initiated similarly in both control and dKO embryo (C,D) and its dorsoventral expression domain is comparable, as revealed by vibratome sections (E,F). At the hindlimb level, ventral expression of Pax3 is perturbed (G-J) and no Pax3 myogenic precursor delaminate to reach the limb bud. At the interlimb level (K-N), Pax3 expression is restricted to the posterior lip in the dKO embryo (arrow in L); in ventral (arrow in N) and dorsal lips, expression is progressively lost (L,N) when compared with wild-type expression (K,M). More rostrally at the forelimb level Pax3 is only detected in the caudal lip of dKO embryos (P), and completely lost in anterior, dorsal and ventral lips (P,R) when compared with heterozygous embryos (O,Q). No myogenic precursor is detected in the limb bud (R). Dorsal neural tube expression of Pax3 is conserved in dKO embryos along the rostrocaudal level (F,J,N,R). In occipital somites, Pax3 expression is very low in dKO embryos. (Part II) Comparison of Met expression in heterozygous embryos (A,C,E,G), with its caudal expression in the top right-hand corner of A, and in dKO embryos (B,D,F,H), with its caudal expression in the bottom left-hand corner in B. Although Met expression is correct in the two first caudal somites of dKO embryos, most expression is lost in more rostral somites, and ectodermal expression is detected at the interlimb level in dKO embryos (arrow in B). Transverse sections through embryos at hindlimb level (C,D), interlimb levels (E,F) and forelimb bud levels (G,H). At the hindlimb level, Met can be detected in dorsal and ventral dermomyotomal lips (D) and few cells are detected in the hindlimb bud – in caudal-most areas of this limb bud (arrow in D); however, Met expression is lost more rostrally from these hindlimb somites. The central somitic expression of Met is faint and restricted (H) at the forelimb level (arrowhead). Epaxial Met expression (white arrow in A) is lost in dKO embryos (white arrow in B). (Part III) Lbx1 expression in wild-type (A,C,E) or KO (B,D,F) embryos. Lbx1 expressed at the hypoglossal chord (arrow in C and D) and forelimb bud levels (asterisks in C and D) is abrogated in dKO embryos but still faintly detected in a few somites at the hindlimb level (arrowhead in F). (G) Bandshift assays performed with recombinant Eya1, Six1 or Six1+Eya1 proteins on a potential MEF3 site present in the promoter of mouse Lbx1 gene showing formation of a MEF3^Lbx-Six1 or a MEF3^Lbx-Six1-Eya1 DNA protein complex. (Part IV) Pax7 expression in heterozygous (A,C,E,G,I) and KO (B,D,F,H,J) embryos. Pax7 is expressed in dermomyotome and its expression pattern is mainly unchanged in KO somites (C,D) at the hindlimb level, as revealed by vibratome sections (E,F), and at the interlimb level (G,H). At the forelimb bud level, we noticed that dorsoventral extension of the expression domain of Pax7 was reduced (I,J).

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