FIG. 7.

Ectopic expression of Msx1, but not Msx1(R31P), alters chicken embryonic limb morphology. (A) The replication-competent retroviral vector RCASBP (9, 14) provides a vehicle with which to introduce Msx1, Msx1(R31P), or Msx1(R31A) into the limb and to drive their expression. Note that the murine genes are Myc tagged, but the chicken genes are not (see Materials and Methods). The retroviruses that contain the murine Msx1 sequences allow for detection of the exogenous genes as distinguished from the endogenous chicken gene (GMsx1) by in situ hybridization. (B) Whole-mount in situ hybridization shows expression of endogenous GMsx1 at stage 26 in the embryonic limb buds, where it is confined primarily to the distal mesenchyme (progress zone; arrows). At this stage, GMsx1 is also expressed in the dorsal neural tube (white arrowhead) and branchial arches (not shown). (C) Whole-mount in situ hybridization shows ectopic expression of murine Msx1 in the forelimb bud at stage 26 following retroviral infection at stage 17. Exogenous Msx1 is expressed throughout the forelimb, but not the hindlimb, on the infected side (black arrows) and not in the forelimb of the uninfected (control) side (white arrowhead) or in other regions of endogenous GMsx1 expression. A similar pattern of ectopic expression was observed for GMsx1 and GMsx1(R31P) (data not shown). Whole-mount in situ hybridization was performed with a chicken (B) or murine (C) Msx1 antisense riboprobe as described in reference 34. (D to F) Ectopic expression of Msx1 (D) or Msx1(R31A) (F), but not Msx1(R31P) (E), in the forelimb produces a smaller wing on the infected side (upper right) relative to the wing on the uninfected side (control, lower left). (G) Ectopic expression of Msx1 together with Msx1(R31P) produces a smaller wing comparable to that seen with ectopic expression of Msx1 alone. (H to I) Ectopic expression of GMsx1 (H), but not GMsx1(R31P) (I), in the forelimb produces a smaller wing and smaller feathers on the infected side relative to the uninfected one (control). In panels D to I, infection was achieved by injection of high-titer virus (10⁸ CFU/ml) into the region of the prospective forelimb (right side) at stage 10 (H and I) or stage 17 (D to G); embryos were staged according to the method of Hamburger and Hamilton (13). Infection was generally restricted to the site of injection (Fig. 7C); however, blood-borne virus sometimes led to ectopic expression in the heart (not shown). Two retroviral vectors encoding alternative viral envelope proteins were used [RCASBP(A) and RCASBP(B)]. Ectopic expression of Msx1 by using either retroviral vector produced equivalent results. In panel G, coinfection of Msx1 and Msx1(R31P) was achieved with a 1:1 mixture of Msx1-RCASBP(A) and Msx1(R31P)-RCASBP(B). Panels D to I show representative wings (stages 36 to 39) with the ventral surface facing up. Data analysis is provided in Table 1. R, radius; U, ulna; H, humerus; D, digit III.