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, 9 (1), 1511

Matrix Stiffness Controls Lymphatic Vessel Formation Through Regulation of a GATA2-dependent Transcriptional Program

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Matrix Stiffness Controls Lymphatic Vessel Formation Through Regulation of a GATA2-dependent Transcriptional Program

Maike Frye et al. Nat Commun.

Abstract

Tissue and vessel wall stiffening alters endothelial cell properties and contributes to vascular dysfunction. However, whether extracellular matrix (ECM) stiffness impacts vascular development is not known. Here we show that matrix stiffness controls lymphatic vascular morphogenesis. Atomic force microscopy measurements in mouse embryos reveal that venous lymphatic endothelial cell (LEC) progenitors experience a decrease in substrate stiffness upon migration out of the cardinal vein, which induces a GATA2-dependent transcriptional program required to form the first lymphatic vessels. Transcriptome analysis shows that LECs grown on a soft matrix exhibit increased GATA2 expression and a GATA2-dependent upregulation of genes involved in cell migration and lymphangiogenesis, including VEGFR3. Analyses of mouse models demonstrate a cell-autonomous function of GATA2 in regulating LEC responsiveness to VEGF-C and in controlling LEC migration and sprouting in vivo. Our study thus uncovers a mechanism by which ECM stiffness dictates the migratory behavior of LECs during early lymphatic development.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Venous LEC progenitors experience lower matrix stiffness upon migration out of the cardinal vein. a, b Immunofluorescence of sagittal (a) and transverse (b) vibratome sections of E11 Prox1-GFP embryos using antibodies against PROX1 (magenta), GFP (green, Prox1 reporter) and Collagen I (single channel images in B only). In a, note the round nuclear shape of PROX1+ LECs in the cardinal vein (CV) (arrows) compared to elongated nuclei of the migrating PROX1+ LECs (arrowheads) in the surrounding tissue. In b, aorta (A) and blood capillaries (BC) show higher Collagen I levels compared to CV (arrow) and migrating LECs (arrowheads). c Quantification of Collagen I density in the respective vessel types and avascular tissue in E11 embryos. Data represent mean integrated density values of corrected total cell fluorescence ± s.e.m. (unpaired Student’s t-test) quantified from n = 10 images taken from two embryos. d Immunofluorescence of transverse vibratome sections of E11 wild type embryos using antibodies against Emcn (magenta; marker of venous EC) and LaminB (green, marker of nuclear envelope). Single plane image for LaminB staining is shown as a close-up (grey). Nuclear morphology is distorted in cells outside the CV (arrow), as opposed to a round morphology in cells of the CV vessel wall (arrowhead). e Quantification of nuclear circularity of Emcn+ venous ECs (CV wall) and Emcn cells of the surrounding tissue (outside CV). Horizontal lines represent mean (n = 30 (CV wall) and n = 34 (outside CV) from two embryos). p value, unpaired Student’s t-test. f Ex vivo AFM measurements (graph on the left) in transverse vibratome sections (image on the right) of E11 Prox1-GFP embryos. Young’s Modulus (kPa) is a measure for the actual tissue stiffness. Horizontal lines represent mean (n = 10 measurements from one embryo (CV) and n = 114 measurements from three embryos (outside CV)). p value, unpaired Student’s t-test. Measurements were done on the dorsal side of the CV (arrow) and the area of LEC migration (boxed area, outside CV). Prox1-GFP+ spinal cord (SC), dorsal root ganglion (DRG) and nerve (N) were used for orientation. Scale bars: 100 µm
Fig. 2
Fig. 2
GATA2 regulation by matrix stiffness in LECs. a, b qRT-PCR analysis of GATA2 in human LECs (a) and primary mouse LECs (b) grown on soft (0.2 kPa) or stiff (25 kPa) matrix. n = 3 experiments, mean ± s.e.m. p value, one-sample t-test. c Immunofluorescence of human LECs grown on stiff and soft matrix using antibodies against GATA2 (green) and VE-cadherin (red), and for DAPI to show nuclei (grey). LECs grown on soft matrix exhibit an overall higher expression of GATA2 as indicated by higher immunofluorescence intensity in both the nucleus and cytoplasm and have an elongated shape and a distorted nucleus. d Quantification of nuclear and cytoplasmic GATA2 protein in human LECs grown on soft (0.2 kPa) or stiff (25 kPa) matrix. Data represent mean pixel intensity (n = 8 images with 8–24 cells per image (soft), and n = 8 images with 21–37 cells per image (stiff) from 3 experiments) ± s.e.m. p value, unpaired Student’s t-test. Scale bars: 50 µm
Fig. 3
Fig. 3
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)
Fig. 4
Fig. 4
GATA2 regulates a matrix stiffness-induced transcriptional program in the LECs. a Schematic overview of microarray gene expression analysis comparing Ctrl and GATA2 siRNA-treated human LECs grown on stiff (25 kPa) or soft (0.2 kPa) matrix. Number of genes regulated by matrix stiffness (n = 6 biological replicates) and the proportion of those showing GATA2-regulated expression (n = 2 biological replicates) are shown. b Selected TOP genes that are upregulated or downregulated on soft (0.2 kPa) in comparison to stiff (25 kPa) matrix and affected by GATA2 depletion. Heatmap color coding shows maximum for log2 fold change >1 or <−1. c Verification of microarray data in 2 additional independent experiments by qRT-PCR analysis shows the expected downregulation of markers of proliferation (CCNB1) and YAP/TAZ dependent mechanosignalling (CTGF, ANKRD1) in LECs grown on soft matrix. d Selected significantly enriched GO terms in LECs grown on soft matrix. The fold enrichment of upregulated (red bars) and downregulated (green bars) is shown. Genes associated with cell–matrix adhesion, cell migration and vascular development are upregulated on soft matrix, whereas genes associated with cell proliferation are downregulated. Notably, GO terms associated with cell migration, locomotion and motility (highlighted in red) are upregulated in a GATA2 dependent manner. e Verification of selected matrix stiffness regulated genes from the microarray expression analysis by qRT-PCR analysis in two independent experiments. Horizontal lines represent mean
Fig. 5
Fig. 5
GATA2 regulates VEGFR3 expression and LEC responsiveness to VEGF-C. a Relative mRNA expression levels of Gata2, Vegfr3, Efnb2 and Fgfr3 in freshly isolated LECs from E15.5 Gata2 mutant (n = 4 or n = 5) and Cre littermate control (n = 4) embryos. Horizontal lines represent mean ± s.e.m. p value, unpaired Student’s t-test. b, c qRT-PCR analysis of GATA2 and VEGFR3 in control (Ctrl) and GATA2 siRNA-treated human LECs grown on soft (0.2 kPa) or stiff (25 kPa) matrix. n = 4 experiments, mean ± s.e.m. p value, one-sample t-test (for siCtrl stiff vs soft) or unpaired Student’s t-test. d Occupancy of chromatin at the FLT4 (encoding VEGFR3) locus as viewed in UCSC Human Genome Browser (http://genome.ucsc.edu/). GATA2 ChIP-seq profile demonstrating binding at the first intron of the FLT4 locus in human dermal LECs. Co-occupancy with H3K27Ac and sites of DNase hypersensitivity, both marks of active enhancer elements, are shown in ENCODE tracks (HUVEC, light blue peaks). e qRT-PCR and western blot analysis of Gata2 and Vegfr3 in control and 4-OHT treated (Gata2 deleted) primary mouse LECs. For qRT-PCR: n = 3 experiments, mean ± s.e.m. p value, one-sample t-test. Main band (125 kDa) quantification is shown in red. f Vehicle (Ctrl) and 4-OHT (KO) treated primary mouse LEC spheres in the absence (−) or presence of VEGF-C. Asterisks indicate sprouts. g Proportion of control (Ctrl) and Gata2 deficient (KO) LEC spheres forming sprouts in the absence (−) or presence of VEGF-C. n is indicated; p value, Fisher’s exact test. h Quantification of sprout length. Dots represent individual sprouts. Horizontal lines represent mean. n = 20–30 beads (from two independent experiments). p value, unpaired Student’s t-test
Fig. 6
Fig. 6
LEC autonomous function of GATA2 in dermal lymphatic vessel sprouting and patterning. a Schematic of the genetic constructs, 4-OHT treatments (red arrowheads; red bars represent expected time frame of 4-OHT activity) and analyzed embryonic stages. The time frames for pTD + PLLV and dermal lymphatic vessel formation are indicated. b Whole-mount immunofluorescence of E15.5 and E17.5 upper thoracic dorsal skins using antibodies against VEGFR3 (green) and PROX1 (red), or VEGFR3 alone (single channel images on the right). Note reduced VEGFR3 levels in the mutant. c Whole-mount immunofluorescence of E17.5 upper thoracic dorsal skins using antibodies against PROX1 (green) and LYVE1 (red). Note developing valves consisting of clusters of highly PROX1 positive cells (arrowheads) that are present in control skin only. df Quantification of lymphatic vessel diameter (d), branch points (e) and the distance between contralateral sprouts (open midline; f) in E15.5 and E17.5 dorsal skin in Gata2 mutant (KO) and Cre littermate control embryos. Horizontal lines represent mean (n = 16 (E15.5 Ctrl), n = 12 (E15.5 KO), n = 5 (E17.5 Ctrl), n = 11 (E17.5 KO)). p value, unpaired Student’s t-test. g Morphology of lymphatic vessel sprouts in E17.5 skin. h, i Quantification of filopodia numbers (h) and length (i) in E17.5 dermal lymphatic vessel sprouts. Horizontal lines represent mean (n = 9 (Ctrl) and n = 10 (KO) embryos), p value, unpaired Student’s t-test. j Quantification of LEC nuclei in E17.5 dorsal skin. Horizontal lines represent mean (n = 4 (Ctrl) and n = 7 (KO) embryos). p value, unpaired Student’s t-test. k Relative mRNA expression levels of the proliferation markers Mik67 and Ccnb1 in dermal LECs sorted from E15.5 Gata2 mutant (KO; n = 4 or n = 5) and Cre littermate control (Ctrl; n = 4) embryos. Horizontal lines represent mean ± s.e.m. p value, unpaired Student’s t-test. Scale bars: 100 μm (double-stained images in b, c), 50 μm (single channel images in b, g)
Fig. 7
Fig. 7
Common and unique mechanoresponses during lymphatic vascular morphogenesis. a Area-proportional Venn diagrams of up- or downregulated genes in LECs grown on stiff or soft matrix (mimicking conditions of venous-derived LEC migration during the formation of first lymphatic vessels), and LECs subjected to static or oscillatory flow (mimicking initiation of valve formation,) conditions. Common TOP genes and selected GO terms are listed. b Schematic of venous-derived LEC migration out the CV. Exposure of venous-derived LECs to soft matrix induces GATA2-dependent increase in VEGFR3 expression and LEC migration. TOP regulated genes (red, up; green, down) are indicated

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