Figure 5. Relative Gli3R activity levels in mouse 5’HoxdΔ/Δ mutant compared to WT.

(A) Whole mount in situ transcripts in wildtype (WT), 5’Hoxd mutant (5’HoxdΔ/Δ) and Gli3^−/− (Gli3KO) E10.75 forelimb buds were detected using HCR and quantitated with confocal microscopy. All limb buds oriented with anterior (A) at top, posterior (P) at bottom, and distal at right side of panel. 5’Hoxd mutants show reduced anterior Gli1 and Cdk6 expression compared to WT, in contrast to Gli3 KO in which levels of all three transcripts are elevated, as previously shown [21]. CyclinD1 changes did not reach significance between WT and 5’HoxdΔ/Δ. For each probe and each genotype, a minimum of 3 embryos were hybridized at the same time under uniform conditions and evaluated. Total fluorescence in equal-sized anterior domains across all confocal sections was calculated and intensities normalized relative to the average WT level. Graphs to right show mean fluorescence and error bars indicate SEM. Significance determined by standard 2-tailed T test. *, p<0.05; **, p<0.01; ***, p<0.001.

(B) Diagrammatic summary of model: The net Gli3R activity level, governed by Gli3–5’Hoxd mutual antagonism, regulates G1-S phase transit and onset of cell cycle arrest in the anterior limb bud. Cell cycle arrest enables response to differentiation cues leading to chondrogenic condensation (this paper; Lopez-Rios et al.[21]). Shifting the balance in favor of high Gli3R activity promotes earlier anterior cell cycle exit and leads to preaxial polarity in condensation appearance order, as seen normally in urodeles and in 5’Hoxd mutant mice; low Gli3R activity leads to postaxial dominance seen in most tetrapod vertebrates.

Related to Figures S5, S6.