Figure 3. Aggregation rates of limb mesenchyme ex vivo are similar for wildtype and 5’HoxdΔ/Δ, and preaxial polarity of condensation order in vivo is unaltered by reducing Bmp activity in 5’HoxdΔ/Δ.

(A,B) Aggregation rates in high density cultures (HDC) measured by live cell fluorescence imaging in genetically tagged (EYFP, TdTomato) wildtype control (WT) and 5’HoxdΔ/Δ cells from anterior or posterior hindlimb bud halves in mixed HDCs. Left diagrams summarize experimental strategies. Middle panels show representative static images of mixed control vs mutant mesenchyme in HDC at different times post-plating. Scale bar for each set of panels is 200 μm. Right graphs show normalized data and p values comparing cell aggregation rates for control and mutant 5’HoxdΔ/Δ in mixed HDC as indicated in diagrams. (N=4 independent experiments for each type of mixed HDC imaged). See also Figures S3, S4.

(A) Aggregation rates in HDC comparing mixed anterior (upper panels) or mixed posterior (lower panels) hindlimb bud halves from either WT/WT or WT/5’HoxdΔ/Δ limb mesenchyme.

(B) Aggregation rates in HDC comparing mixed anterior and posterior hindlimb bud halves from either WT/WT or 5’HoxdΔ/Δ/5’HoxdΔ/Δ limb mesenchyme.

(C) NogginLacZ staining of forming limb condensations in control and 5’HoxdΔ/Δ hindlimb buds from stages E11.25 - E11.75, from embryos also carrying a Bmpr1a or Bmpr1b mutant allele to reduce Bmp receptor gene dosage (Bmpr1a^+/-; Bmpr1b^+/-).

(D) NogginLacZ staining of forming limb condensations in control and 5’HoxdΔ/Δ hindlimb buds from stages E11.25 - E11.75, from embryos also carrying conditional, Cre-dependent RosaGrem1 transgene and Hoxb6CreER alleles, and tamoxifen treated at E10.5 to activate Grem1 expression and reduce net Bmp activity.

Related to Figures S3, S4.