(A1–B3) E11.0 and E11.5 embryos showing Myf5 (A1–A3) and MyoD (B1–B3) expression in the developing epaxial and hypaxial muscles. (A1 and B1) Normal embryos. Loss of Shh leads to a lack of Myf5 (A2) and MyoD (B2) in the epaxial region (thin line) but not the hypaxial region (thick line). Shh;Apaf1 DKO embryos also display a loss of Myf5 (A3) and MyoD (B3) in epaxial but not hypaxial progenitors. (C1–C3) Analysis of Pax1 expression at E9.0. (C1) Pax1 expression in normal embryos. (C2) Pax1 expression in the sclerotome of Shh KO embryos. (C3) Shh;Apaf1 DKO embryos also express Pax1 in a similar pattern. (D1–D3) Analysis of Pax9 expression at E9.5. (D1) Pax9 is expressed in the sclerotome of normal embryos. (D2). Shh KO embryos do not have Pax9 expression in the sclerotome. Note Pax9 expression in the brachial arches (arrowheads). (D3) Shh;Apaf1 DKO embryos exhibit a similar pattern. (E1–F3) Analysis of Foxc2 expression at E9.0 and E10.0. Foxc2 is expressed throughout the developing somite at E9.0 (E1) and is restricted to the sclerotome by E10.5 (F1) in normal embryos. Foxc2 expression is detected in similar patterns in both the Shh KO (E2, F2) and the Shh;Apaf1 DKO (E3, F3) embryos at both E9.0 and after de-epithelialization of the thoracic somites (E10.0). Brackets outline comparable somite regions, curved line indicates forelimb bud location. (G) Pax1 expression at E9.0 in Apaf1 KO embryos is similar to controls. Compare to C1. (H) Pax9 expression at E9.5 in Apaf1 KO embryos is similar to controls. Compare to D1. Tail truncated due to collection for genotyping. (I) Foxc2 expression at E10.0 in Apaf1 KO embryos is similar to controls. Compare to F1. Brackets outline comparable somite regions, curved line indicates forelimb bud location. Phenotypes were consistently observed in at least 3/3 animals of each genotype. When null for skeletal muscle genes, embryos can also display skeletal abnormalities, including rib fusions, truncations and sternal defects (Pax3: [Henderson et al., 1999; Vivian et al., 2000]). In Shh KO embryos epaxial progenitors are reportedly absent, while the hypaxial progenitors are reduced (Borycki et al., 1999; Gustafsson et al., 2002) and our results looking at Myf5 and MyoD expression confirmed this observation (A2, B2). Perhaps, absence of the epaxial compartment explains the failure of the proximal rib to form in both situations while additional defects in the hypaxial compartment are responsible for lost distal segments in the Shh;Apaf1 DKO embryos. However, we found that Shh;Apaf1 DKO embryos displayed the same pattern as Shh KO embryos (A3, B3) suggesting that a failure to specify the hypaxial muscle compartment is not the cause of the Shh;Apaf1 DKO phenotype. Pax9 and Pax1 are expressed in similar patterns in the sclerotome and are required for rib and vertebrae development (Furumoto et al., 1999; Peters et al., 1999; Rodrigo et al., 2003). Previous studies have shown that Pax1 is initially expressed in the Shh KO embryo but then lost (Chiang et al., 1996; Borycki et al., 1998; Zhang et al., 2001). As previously reported, we found Pax1 expressed in a smaller domain in Shh KO embryos compared to normal or Apaf1 KO embryos at E9.0 (C2). However, in Shh;Apaf1 DKO embryos, Pax1 expression was similar suggesting that some sclerotome specification still occurs (C3). As development continues, Pax1 expression is eventually lost in Shh KO embryos as well as in the Shh;Apaf1 DKO embryos. Neither Shh KO nor Shh;Apaf1 DKO embryos exhibited Pax9 expression in somites at E9.5 (B2–B3). We then determined if the expression of Foxc2, a winged helix/forkhead transcription factor required for axial skeletal development (Kume et al., 2001; Wilm et al., 2004) is altered. Foxc2, which is expressed in the early unsegmented paraxial mesoderm and later becomes restricted to the sclerotome, may be a more comprehensive sclerotome marker than Pax1/9 (C1, D1) (Furumoto et al., 1999). Like Pax1, Foxc2 was expressed in a reduced domain in both Shh KO and the Shh;Apaf1 DKO embryos compared to normal embryos prior to E9.0, (E2, E3). However, in contrast to Pax1, the smaller domain of Foxc2 expression continued to be present after E10.0 in both Shh KO embryos and somite-matched Shh;Apaf1 DKO embryos (F2, F3). In summary, the loss of Shh correlates with smaller Pax1 and Foxc2 expression domains. In addition, the Pax1 domain disappears while the smaller FoxC2 expression domain persists, suggesting that at least a portion of the sclerotome is maintained in both contexts. Compared to control embryos, Apaf1 KO embryos did not display disruptions in sclerotome patterning as assessed by RNA in situ marker expression analysis (G, H, I).