Figure 3.

Comparison between SOX9/lacZ expression derived from the 350-kb and 600-kb YACs. (a–f) β-Galactosidase staining of embryos from line A46 (one copy of the 600-kb YAC), line A45.1 (one copy of the short construct), and line A75 (multiple copies of the 350-kb YAC). (g–n) Microtome sections performed on 10.5- to 12.5-dpc embryos from lines A46 and A75. ba, branchial arches; ov, otic vesicle; lb, long bones; hp, hand plate; fp, foot plate; r, ribs; ea, endolymphatic duct; ut, utriculus; md, mandibular process of the first branchial arch; mx, maxillary process; h, humeral mesenchymal condensation; r/u, radio-ulna mesenchymal condensation. Throughout development, expression in line A45.1 is found dramatically decreased in all tissues normally expressing SOX9/lacZ (line A46). Expression in the neuroectoderm, however, is only mildly affected. (g, h, k, and l) Expression derived from the 350-kb YAC occurs at the correct location in the developing ear, but a significant delay in the establishment of the expression pattern takes place: SOX9/lacZ expression in the endolymphatic and utricular portions of the otocyst is only detected from 12.5 dpc instead of 11.5 dpc with the 600-kb YAC. This delay may correspond to the time necessary for SOX9/LacZ proteins to accumulate in the cells to obtain detectable levels of β-galactosidase activity. (i and m) In the developing branchial arches, the deletion of SOX9 upstream sequences results in SOX9/lacZ expression only in the maxillary process of the first branchial arch. This suggests the involvement of multiple elements in the control of SOX9 expression in distinct regions of the branchial arches. (j and n) In contrast to line A46, where expression in the developing long bones precedes expression in the hand and foot plates, expression derived from the 350-kb YAC is induced from 11.5 dpc in the hand and foot plates, with the expression in the long bones remaining weak. The 5′ deletion therefore alters onset and level of expression in particular regions of the limb bud.