Fig. 2: Two enhancer clusters in the Grem1 TAD control the spatio-temporal dynamics of limb bud mesenchymal Grem1 expression. | Nature Communications

Fig. 2: Two enhancer clusters in the Grem1 TAD control the spatio-temporal dynamics of limb bud mesenchymal Grem1 expression.

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

Fig. 2

a Chromatin conformation capture (4C) using the Grem1 promoter as viewpoint (VP, also indicated by a black arrowhead) to reveal its interactions with the Grem1-Fmn1 landscape. The 4C profiles of forelimb buds lacking both EC1 and EC2 or EC2 and EC1 alone were compared to their respective wild-type controls (upper Wt: control for EC1Δ/ΔEC2Δ/Δ and EC2Δ/Δ, lower Wt: control for EC1Δ/Δ forelimb buds). Subtraction after normalization reveals that the deletions do not affect the interactions of the Grem1 promoter with the remainder of the Grem1-Fmn1 TAD (subtraction, green: reduction or loss of interactions, red: gain of interactions). The position of Grem1 TAD is indicated at the bottom of the panel. b–e Left panels (except b): RT-qPCR was used to determine the relative Grem1 transcript levels in wild-type and homozygous mutant forelimb buds (n = 7 independent biological replicates at E11.0, 40–42 somites per genotype). Bars represent mean values +/− SEM. P-values were determined using the two-tailed Mann–Whitney test: ***p = 0.000583 (panels c, e), *p = 0.017483 (panel d). Middle panels: In situ hybridisation shows the spatio-temporal Grem1 distribution in wild-type (Wt) and mutant forelimb buds at three developmental stages (n = 4 embryos analysed per genotype and stage from different litters and in minimally two independent experiments). E10.5: 35–37 somites; E11.0: 40–42 somites, E12.0: staged by developmental time). Ant: anterior, Dist: distal, Post: posterior, Prox: proximal. Scale bars: 250 μm. Right panels: limb skeletons at ~E14.5 (blue: cartilage, red: ossification centres in radius and ulna). Digits are shown from anterior (digit 1) to posterior (digit 5). Only three rudimentary digits form in EC1Δ/ΔEC2Δ/Δ forelimb buds (indicated by asterisks, n = 5). In contrast, pentadactyly is perfectly maintained in EC2Δ/Δ forelimbs (n = 3), while 64% of all EC1Δ/Δ (n = 9/14) forelimb skeletons display variable fusions of digits 2 and 3 (asterisk). Scale bar: 1 mm. RT-qPCR source data are provided in a Source data file.

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