Fig. 3: Interactions among CRMs provide the spatially dynamic Grem1 expression and pentadactyly with cis-regulatory robustness. | Nature Communications

Fig. 3: Interactions among CRMs provide the spatially dynamic Grem1 expression and pentadactyly with cis-regulatory robustness.

From: Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity

Fig. 3

a Left panel: indication of the relevant limb bud axes. Ant: anterior, Dist: distal, Post: posterior, Prox: proximal. b–e Left panels: RT-qPCR was used to determine the relative Grem1 transcript levels by comparing wild-type and CRM2Δ/Δ (b), CRM5Δ/Δ (c), CRM2Δ/ΔCRM5Δ/Δ (d) and EC1Δ/ΔCRM5Δ/Δ forelimb buds (e; n = 7 independent biological replicates at E11.0, 40–42 somites for all genotype). Bars represent mean values +/− SEM. P-values were determined using the two-tailed Mann–Whitney test: ***p = 0.000583 (panels b, d, e) and *p = 0.011072 (panel c). Middle panels: spatial Grem1 expression in wild-type (a) and the different single and compound mutant forelimb buds (b–e) at the developmental stages indicated (n = 4 embryos analysed per genotype and stage from different litters and in minimally two independent experiments). Scale bars: 250 μm. Right panels: limb skeletons at ~E14.5 (blue: cartilage, red: ossification centre in radius and ulna). Digits are shown from anterior (digit 1) to posterior (digit 5). Pentadactyly is maintained in CRM2Δ/Δ (n = 4 embryos), CRM5Δ/Δ (n = 8) and CRM2Δ/ΔCRM5Δ/Δ (n = 3) forelimb skeletons. In contrast, all EC1Δ/ΔCRM5Δ/Δ (n = 8) forelimb skeletons are tetradactyl with symmetrical middle digits of equal length (indicated by asterisks). Scale bar: 1 mm. f Direct comparison of the Grem1 expression domain in forelimb buds of the most relevant CRM loss-of-function alleles (E11.0, 40–42 somites). These forelimb buds belong to the same group of biological replicates as the ones shown in Figs. 2 and 3. Arrowheads indicate the posterior domain. Bar-ended lines point to the stunted crescent domain. The forelimb buds are representative of the spatial distributions in the respective genotype. The relative Grem1 transcript levels in comparison to the wild-type (set at 100%, see before) and schematics of the distal limb skeletons are shown below. Skeletons in black: pentadactyly maintained, grey: pentadactyly altered/disrupted. RT-qPCR source data are provided in a Source data file.

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