Vertebrate limb bud formation is initiated by localized epithelial-to-mesenchymal transition

Abstract

Vertebrate limbs first emerge as small buds at specific locations along the trunk. Although a fair amount is known about the molecular regulation of limb initiation and outgrowth, the cellular events underlying these processes have remained less clear. We show that the mesenchymal limb progenitors arise through localized epithelial-to-mesenchymal transition (EMT) of the coelomic epithelium specifically within the presumptive limb fields. This EMT is regulated at least in part by Tbx5 and Fgf10, two genes known to control limb initiation. This work shows that limb buds initiate earlier than previously thought, as a result of localized EMT rather than differential proliferation rates.

Fig. 1. Limb progenitors arise from EMT of the epithelial somatopleure

(A to C) Transverse sections of stage 13 (A), 15 (B) and 19 (C) chick embryos, at the forelimb level. Sections were stained with Dapi (blue), F-actin (red) and with anti-laminin antibody (green). (D to F) are higher magnifications (A–C), white arrows point at laminin basement membrane breakdown. (G to I) Transverse sections of chick embryos electroporated at stage 13 and harvested after 3h, 12h and 24h. Sections were stained with Phalloidin (red) and with anti GFP antibody (Green). (J) Proportion of cells in epithelial or mesenchymal state at the forelimb and trunk level. (n=3723 cells, 5 embryos; Mann-Whitney U test ***p-value=2.4×10⁻⁹. (K) Percentage of proliferating cells (nBrdU+/nDapi+) in the somatopleure of chick embryos at stage 15, 16, 17 and 18 at the level of the forelimb (blue), Trunk (red) and hindlimb (green); (n=5 embryos for each stage, over 250,000 cells were counted in total; Mann-Whitney U test, n.s: non-significant p-value>0.05; ***p-value <10⁻⁷. The timing of EMT in relation to proliferation is represented in yellow. Errors bars indicate the SEM. Scale bars represent 50μm in (A–C, G–I,) and 10μm in (D–F). nt: neural tube; no: notochord; en: endoderm; so: somite; im: intermediate mesoderm; ec: ectoderm; spp: splanchnopleure; sp: somatopleure.

Fig. 2. Epithelial-to-Mesenchymal Transition of the somatopleure is a necessary step in limb bud initiation

(A, E) Dorsal view of stage 21 chick embryos 36 h after electroporation of GFP or GFP/RhoA. (B to D and F to H) Transverse section of chick embryo electroporated with GFP or GFP/RhoA at the forelimb level and stained with Phalloidin (red), GFP (green) and Laminin antibody (blue). (J to L) higher magnification of (F to L); Note the over-stabilization of F-actin (arrows) and the presence of large aggregates of laminin (arrowheads). (I) Cartoon representing the extent of RhoA/GFP electroporation as shown in (E) and highlighting the absence of limb formation in the RhoA electroporated region, red arrow. Scale bars represent 50μm in (B to D, and F to H) and 10μm in (J to L)

Fig. 3. Tbx5/FGF10 participates in the regulation of the EMT of the somatopleure

(A) FGF10/GFP electroporated chick embryos showing ectopic swellings (red arrowheads). (B, C) transverse section of FGF10/GFP electroporated embryo, (C) is a higher magnification of (B). (D) Proportion of cells in mesenchymal or epithelial state (n= 1912 (FGF10/GFP), 3 embryos; Mann-Whitney U test ***p-value <0.0001; n.s: non-significant, p-value=0.38). (E, H, K) Transverse sections of mouse embryos at the forelimb level stained with Dapi (Blue), β–catenin (Green) and aPkc (red) antibody. (F, I, L) are Higher magnification of (E, H, K). (G, J, M) Transverse sections stained with Dapi (blue) and Laminin antibody (green) focusing on the somatopleure epithelium. (N) Proportion of cells in mesenchymal or epithelial state (n= 2962 (WT), 10894 (Tbx5^−/−) and 3115 (FGF10^−/−) cells, 3 embryos for each genotype; Mann-Whitney U test ***p-value <0.0001). Scale bars represent 50 μm in (B, C, E, H, K) and 10 μm in (F, I, L, G, J, M). Coe: coelomic cavity; asterisks: separation between epithelium and mesenchyme.