Fig. 4. In vitro culture of forelimb and hindlimb buds from 10.5 dpc embryos. (A) Forelimb tissue explanted at 10.5 dpc. Dashed lines indicate planes of dissection; gray area represents tissue explanted. (B,C) Forelimb buds dissected from normal and mutant embryos, respectively, immediately after explantation. (D,E) Forelimb explants from control and mutant embryos, respectively, show an increase in distance between the distal limb margins (arrowheads) and the development of a paddle-shaped autopod after 3 days in culture. (F) Explanted hindlimb tissue. (G,H) Hindlimb buds from normal and mutant embryos, respectively, immediately after dissection. (I) Control hindlimb buds also exhibit increased distance between limb margins (arrowheads) and autopod development after 3 days in culture. (J) By contrast, explants from Tbx4-mutant embryos have no visible limb structures remaining after the same time period. (K,L) Limb explants hybridized with a Tbx4 probe from outside the region deleted in the mutant. The wild type (K) expresses Tbx4 in the morphologically apparent hindlimb buds, whereas the mutant explant (L) shows only faint Tbx4 expression, and regions of Tbx4 expression show no morphological limb structure. All panels are at the same magnification.

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