Fig. 7: Misexpression of Creb5 in all limb bud mesenchymal cells, but not in Sox9-expressing cells, profoundly blocks longitudinal growth in the stylopod and zeugopod cartilage elements and induces the perichondrium to develop as articular cartilage.

a Gene expression assayed by FISH in serial sections of forelimbs from either a P0 Prx1-Cre-iCreb5 mouse or a control littermate. Humerus (H) is indicated. b Alcian Blue/Alizarin Red whole mount staining of either a P0 Prx1-Cre-iCreb5 mouse or a control littermate (that lacks either the Prx1-Cre or the Rosa26^iCreb5-HA allele). c Immunofluorescence detection of either iCreb5-HA^(Prx1-Cre) (green), endogenous and exogenous Creb5 (red) and DAPI (to detect nuclei) in serial sections of the forelimb of a P0 Prx1-Cre-iCreb5 mouse. Red fluorescent signal in ectoderm (in right-most image) is an artifact and was also observed in the absence of anti-Creb5. d Alcian Blue/Alizarin Red whole mount staining of either an E16.5 Sox9-ires-Cre-iCreb5 embryo or a control littermate (that lacks either the Sox9-ires-Cre or the Rosa26^iCreb5-HA allele). e Immunofluorescence detection of iCreb5-HA^(Sox9-Cre), Sox9, and DAPI in the forelimb of an E16.5 Sox9-ires-Cre-iCreb5 embryo. Similar results were obtained in either 5 (a), 7 (b), 2 (c, e) or 4 (d) independent biological repeats. Scale bar equals 200 microns. f Our findings suggest that iCreb5-HA^(Prx1-Cre) expression in limb bud mesenchymal cells (other than in Sox9-expressing cells) promotes the generation of an articular perichondrium encasing the stylopod and zeugopod cartilage elements, blocks perichondrial expression of Wnt5a, Crabp1 and Runx2, and thus inhibits formation of both cortical bone and growth plates within these forming cartilage elements (which resemble tarsal/carpal-like bones).