Fig. 4: Spatial plasticity of the ancient CRM2 and CRM5 enhancers during evolutionary diversification of mammalian limb development.

a Multiple sequence alignments using the mouse genome as reference reveals the deep evolutionary conservation of the Grem1 TAD in jawed vertebrates (Gnathostomata). The CRM enhancers active in the mouse are indicated in blue and all others CRMs in grey. Regions with ≥70% conservation are shaded in light red. Black arrowheads indicate the conserved Fmn1 exons. A: amphibians. Genomes from the following species are included. Mouse: Mus musculus (reference genome); rabbit: Oryctolagus cuniculus; pig: Sus scrofa; bovine: Bos taurus; opossum: Monodelphis domestica; chicken: Gallus gallus; lizard: Anolis carolinensis; python: Python bivittatus; frog: Nanorana parkeri; coelacanth: Latimeria chalumnae; medaka: Oryzias latipes; spotted gar: Lepisosteus oculatus; elephant shark: Callorhinchus milii, bamboo shark: Chiloscyllium punctatum. b Conservation plots reveal the evolutionary conservation of the relevant mammalian CRM2 and CRM5 regions in comparison to the mouse. All highly conserved regions (≥70%, shaded light red) were included in the LacZ reporter constructs. Black arrowhead indicates Fmn1 exon 22 that is an integral part of CRM2 in all species. c Evolutionary diversification of the Grem1 expression domains and spatial activities of CRM2 and CRM5 in pentadactyl (mouse, rabbit) and artiodactyl (pig, bovine) species. Left panels: Grem1 expression in mouse (E11.0), rabbit (gestational day D12), pig (D23) and bovine (D34) forelimb buds (n ≥ 3 independent embryos analysed). Middle and right panels: CRM2 and CRM5 enhancer activities from the different species as determined by LacZ reporter assays in transgenic mouse limb buds mouse CRM2: n = 7/11, CRM5 n = 7/13 (see Fig. 1c); rabbit CRM2 n = 10/11 and CRM5 n = 4/6 with highly variable activities; pig CRM2 n = 3/3 and CRM5 n = 5/5, bovine CRM2 n = 4/4 and CRM5 n = 5/5. Black arrowheads indicate the anterior expansion of Grem1 expression/enhancer activities compared to the mouse. Bovine CRM2: open arrowhead indicates the loss in posterior activity bias. The transgenic founder embryos that express LacZ in forelimb buds are indicated as the fraction of all embryos with LacZ expression in limb and non-limb tissues. Ant: anterior, Dist: distal, Post: posterior, Prox: proximal. Scale bars: 250 μm.