Fig. 3 | Nature Communications

Fig. 3

From: Matrix stiffness controls lymphatic vessel formation through regulation of a GATA2-dependent transcriptional program

Fig. 3The alternative text for this image may have been generated using AI.

Defective migration of venous-derived LEC progenitors upon loss of endothelial Gata2 expression. a Relative expression levels of Gata2, the LEC markers Vegfr3 and Pdpn and the pan-endothelial marker Erg in ECs within the CV (Prox1-GFP+PDPN−) compared to LECs outside of the CV (Prox1-GFP+PDPN+). ECs were freshly isolated from E11 Prox1-GFP embryos (n = 3 independent samples with 4 pooled embryos in each, from two different litters). Horizontal lines represent mean ± s.e.m. p value, unpaired Student’s t-test. b Immunofluorescence of transverse cryosections of E11 embryos using antibodies against GFP (green; Prox1-GFP), GATA2 (magenta) and VEGFR3 (red). Single channel images are shown. The IMARIS surface mask depicts in yellow the surface area used to extract GATA2 and VEGFR3 signal intensity. The surface mask was generated based on Prox1-GFP expression of VEGFR3+ LECs within and outside of the CV. c Quantification of GATA2 and VEGFR3 staining intensity in ECs within (n = 35) and outside (n = 37 measurements) of the CV. Data represent mean ± s.e.m. p value, unpaired Student’s t-test. d Schematic of the genetic constructs and analyzed embryonic stages. The time frames for pTD + PLLV (i.e. ‘jugular lymph sac’) and dermal lymphatic vessel formation are indicated. e Immunofluorescence of transverse vibratome sections of E12.5 embryos using antibodies against PROX1 (green) and Emcn (red; marker of venous EC). Single channel images for Emcn staining are shown. Note smaller jugular lymph sac (JLS) and the presence of PROX1+ Emcn− LECs (arrowhead) within the CV in the mutant embryo. f Maximum intensity projections of E12.5 embryos (sagittal view) stained whole-mount for indicated proteins and imaged using light sheet microscopy. Single channel images for PROX1 staining are shown. Arrowheads point to lymphovenous valves that are deformed in the mutant embryos. g E13.5 embryos (panels on the right) and whole-mount upper thoracic dorsal skin stained for Nrp2 (green) and Emcn (red). Nrp2+ dermal lymphatic vessels are absent in the mutant. Scale bars: 20 μm (b) 100 μm (e, f, g skins), 1 mm (g, embryos)

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