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Fig. 3. Gli3-Hoxd expression overlap and interaction between endogenous Gli3-Hoxd12
proteins. (A) Expression of Gli3, and of Hoxd10/11/12/13 RNA
in nested posterior domains of E10 (left panel) and interdigits of E12 (right
panel) forelimb buds (digit I-V, AP, indicated for Gli3). (B) Western blot
comparing Gli3 protein in lysates from early chick (stage 22) anterior (A) or
posterior (P) limb bud with late stage (27/28) distal digit arch region
containing interdigit (ID) mesenchyme, either intact or separated into A and P
parts. Lower panels show Hoxd12 and Hoxd13 proteins detected in the same
lysates. Note these stages are comparable to mouse E10.5/E11 (early) and
E12/E12.5 (late); chick and mouse RNA and protein expression profiles are
generally similar (see Dolle et al.,
1989; Nelson et al.,
1996; Mo et al.,
1997; Schweitzer et al.,
2000; Wang et al.,
2000; Litingtung et al.,
2002). The ratio of the short-repressor form of Gli3 protein (83
kDa) relative to full length (190 kDa) in late interdigits is similar to the
anterior early limb bud profile, consistent with lack of Shh
expression at this stage. In posterior early limb buds, Shh activity results
in a high ratio of full-length to repressor form of Gli3. p75kD is a
Gli-related antigen of uncertain identity
(Wang et al., 2000). (C)
Co-immunoprecipitation (IP) of Gli3 and Hoxd12 from early (stage 22, upper
panels) and late distal (stage 27/28, lower panel) chick limb bud lysates,
using immobilized anti-Hoxd12 or control purified IgG for immunoprecipitation,
and anti-Gli3 for detection of bound proteins. Endogenous Hoxd12 binds Gli3
from early limb bud, when both full-length (190kD) and repressor forms (83kD)
of Gli3 are expressed, and from later interdigital zones, when mainly the
repressor form of Gli3 protein is expressed.
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