Fig 2: Zmiz1 regulates Prox1 expression in vivo and in vitro and genetic deletion of Zmiz1 results in decreased valve number.(A) Western blot analysis for PROX1 on control and ZMIZ1 siRNA HDLECs. (B) Densitometric quantification of (A). (C) qPCR analysis for PROX1 expression levels in control and Zmiz1 siRNA HDLECs. n = 3–5 for each experimental sample. (D) Graphical representation of Cre-LoxP system components and tamoxifen injection procedure for postnatal deletion of Zmiz1. (E) Wholemount immunostaining of PROX1 (green) and CD31 (magenta) in Zmiz1 WT and mutant postnatal day (P) 8 mesentery indicating reduction in PROX1 expression. Scale bars: 1000 µm (B), 200 µm (a, b). (F) PROX1 high valve area intensity quantification in WT and Zmiz1-KO mesentery. (G) Fluorescence imaging of the morphology of the P8 mesenteric lymphatic vasculature indicated by RFP expression. RFP positive valves defects are represented by green arrow. Scale bars: 1000 µm. (H) Valves per millimeter from WT and Zmiz1-KO P8 mesentery. (I) A schematic representation of ZMIZ1 regulation of Prox1 and lymphatic mesenteric valve. At least three controls and 3 knockout mesenteries were used in each analysis, n=3–5. All values are mean ± SEM. **P <0.01, ***P < 0.001, ****P < 0.0001 calculated by unpaired Student’s t test.

Fig 3: Loss of Zmiz1 in LECs impairs the expression of lymph vessel development genes.(A) Immunofluorescence staining for ZMIZ1 (green) and DAPI (blue) showing nuclear localization of ZMIZ1 in HDLECs. Scale bar: 10 µm. (B) Zmiz1 mRNA expression in ECs and LEC from mouse lungs (adapted from single cell data on http://betsholtzlab.org/VascularSingleCells/database.html). (C) Zmiz1 mRNA expression in various LEC subtypes (adapted from single cell RNA sequencing dataset of adult mouse mesenteric lymphatic endothelial cells22). Capillary (capLECs), collecting (collLECs), precollecting and collecting (pre/collLECs), valve (vLECs), proliferative (prolifLECs), IFN (IfnLECs), endothelial cells (EC) and lymphatic endothelial cells (LEC). (D) Outline of the workflow used to knockdown (KD) Zmiz1 in HDLECs for RNA sequencing of control and Zmiz1 siRNA treated HDLECs (n=3). (E) MA plot of differentially expressed genes (DEGs) between control and Zmiz1 siRNA HDLECs. (F) Top Gene Ontology (GO) biological process terms enriched in downregulated genes in Zmiz1 siRNA (FDR ≤ 0.05). (G-I) Representative clustered heatmaps of gene count Z scores for lymph vessel development genes (G), cellular response to vascular endothelial growth factor stimulus (H), and heart valve development genes (I).

Fig 4: Zmiz1 facilitates chromatin accessibility in LECs.(A) ATAC sequencing experimental paradigm on HDLECs treated with control and Zmiz1 siRNA. (B) Heatmap depicting the ATAC-seq profile around protein coding transcription start sites (TSS). (C) Genomic distribution of ATAC-seq peaks. (D) Volcano plot of differentially accessible regions (DAR) between Zmiz1 and control siRNA HDLECs. PROX1 is highlighted in red. (E) Top enriched motif sequences. (F) Venn diagram depicting gene overlap among ATAC-seq downDARs and upDARs, and RNA-seq down and upregulated DEGs. (G) GO for biological process in overlapped genes between ATAC-seq DARs and RNA-seq DEGs. (H) Representative ATAC-seq tracks on the human PROX1 gene. ATAC-seq peaks for Zmiz1 (orange) and control (blue) siRNA HDLECs. Red dotted box, Prox1 enhancer; green dotted box, Prox1 promoter. ChIP-seq peaks for GATA2, PROX1, FOXC2, and NFATC1 in the PROX1 enhancer are depicted in grayscale.

Fig 5: Loss of Zmiz1 in LECs impairs the expression of lymph vessel development genes.(A) Immunofluorescence staining for ZMIZ1 (green) and DAPI (blue) showing nuclear localization of ZMIZ1 in HDLECs. Scale bar: 10 μm. (B) Zmiz1 mRNA expression in ECs and LEC from mouse lungs (adapted from single cell data on http://betsholtzlab.org/VascularSingleCells/database.html). EC1 and EC2 are different populations of cell clusters that are distinct from artery, vein and capillary ECs. (C) Zmiz1 mRNA expression in various LEC subtypes (adapted from single cell RNA sequencing dataset of adult mouse mesenteric lymphatic endothelial cells [28]). Capillary (capLECs), collecting (collLECs), precollecting and collecting (pre/collLECs), valve (vLECs), proliferative (prolifLECs), IFN (IfnLECs), endothelial cells (EC) and lymphatic endothelial cells (LEC). (D) Outline of the workflow used to knockdown (KD) ZMIZ1 in HDLECs for RNA sequencing of control and ZMIZ1 siRNA treated HDLECs (n = 3). (E) MA plot of differentially expressed genes (DEGs) between control and ZMIZ1 siRNA HDLECs. (F) Top Gene Ontology (GO) biological process terms enriched in downregulated genes in ZMIZ1 siRNA (FDR ≤ 0.05). (G-I) Representative clustered heatmaps of gene count Z scores for lymph vessel development genes (G), cellular response to vascular endothelial growth factor stimulus (H), and heart valve development genes (I).