Vascular-mesenchymal cross-talk through Vegf and Pdgf drives organ patterning

Abstract

The initiation of de novo testis cord organization in the fetal gonad is poorly understood. Endothelial cell migration into XY gonads initiates testis morphogenesis. However, neither the signals that regulate vascularization of the gonad nor the mechanisms through which vessels affect tissue morphogenesis are known. Here, we show that Vegf signaling is required for gonad vascularization and cord morphogenesis. We establish that interstitial cells express Vegfa and respond, by proliferation, to endothelial migration. In the absence of vasculature, four-dimensional imaging of whole organs revealed that interstitial proliferation is reduced and prevents formation of wedge-like structures that partition the gonad into cord-forming domains. Antagonizing vessel maturation also reduced proliferation. However, proliferation of mesenchymal cells was rescued by the addition of PDGF-BB. These results suggest a pathway that integrates initiation of vascular development and testis cord morphogenesis, and lead to a model in which undifferentiated mesenchyme recruits blood vessels, proliferates in response, and performs a primary function in the morphogenesis and patterning of the developing organ.

Fig. 1.

Vegfa and its receptors are expressed in XX and XY gonads. Whole-mount E12.0 Vegfa-lacZ gonads stained with X-gal (blue). (A) X-gal staining of XY E12.0 gonads shows broad expression throughout the gonad including the coelomic domain. Expression of Vegfa extends to the surface of the gonad (A′, brackets) and is reduced at the gonad/mesonephros border (A′, arrowhead). (B) XX gonads also express Vegfa, although expression is absent from the coelomic domain (B′, brackets) and is enriched along the gonad/mesonephros border (B′, arrowhead). (C) VEGFA receptor expression was examined relative to PECAM-1, which labels both germ and endothelial cells in the gonad. Both NRP1 (D) and Flk1-mCherry (E) localize specifically to PECAM-1–positive endothelial cells (F). (G) Schematic representation of the early gonad shows the expression domain of various transgenic reporter lines expressed by gonadal subpopulations at E12.5. Key regions of the XY gonad including the coelomic domain (CD, brackets) and coelomic vessel (CV, arrow) are also indicated. (H) mRNA was extracted from FACS-sorted populations and expression of Vegfa was compared across XY cell types after normalizing to expression in whole E12.5 XY gonads. Bar colors represent analysis of specific populations as indicated in the schematic (G). Pink bar indicates Vegfa expression levels of whole XX gonads relative to whole XY gonads (*P < 0.05, **P < 0.005, ***P < 0.0005).

Fig. 2.

Inhibition of VEGF blocks vascular remodeling in the gonad but not male-specific lineage specification. (A) VEGF Trap and rhodamine lectins were delivered to the gonad by injection into the embryonic heart. (A′) Injected gonads were explanted to culture and the delivery of fluorescent lectins was evaluated for each sample. Dotted line indicates gonad/mesonephric boundary in A′ and B–D. (B) After 24 to 36 h of culture, control gonads, injected only with rhodamine lectins, showed normal development of a coelomic vessel (arrowhead) and germ cell aggregation inside testis cords. (C and D) In XY organs injected with VEGF Trap, male-specific vasculature was very limited or absent. (C) The presence of some endothelial cells in injected samples correlated with sporadic cord-like structures (arrowhead/dotted lines). (D) Robust delivery of VEGF Trap completely blocked male-specific vascular development and testis morphogenesis. (E–J) Markers of distinct gonadal cell types were specified but mislocalized after vascular inhibition (Right) compared with controls injected with lectins alone (Left). (E–J) Dotted lines define the surface epithelium and bars indicate the coelomic domain in I and J. (E and F) SOX9-positive (red) and AMH-positive (green) Sertoli cells did not aggregate into testis cords. (G and H) 3β-HSD–positive Leydig cell (red) localization was severely disrupted after Vegf inhibition. (G′) In controls, Leydig cells (red) are in close proximity to PECAM-1–positive (green) endothelial cells (arrow). (H′) Injection of VEGF Trap randomized Leydig localization. (I) αSMA-EYFP–positive (green) interstitial protrusions typically extend into the gonad at regular intervals and surround testis cords. (J) Inhibiting VEGF blocked extension of interstitial protrusions between testis cords.

Fig. 3.

Interstitial expansion fails after VEGF block. (A–D) αSma-EYFP–positive gonads were imaged in real time to characterize interstitial dynamics. (E–H) Littermates were injected with VEGF Trap and imaged in parallel. (A and B) After 6 to 12 h of culture, control organs display increased αSma-EYFP expression (Movie S1). (C and D) Within 18 to 24 h, controls develop clear testis cord domains that are segregated from the interstitium (asterisk). (E–H) Cord structures never appear and αSma-EYFP–positive cells do not expand into the interior of the gonad in the absence of robust vascular remodeling (Movie S2).

Fig. 4.

Vascular migration is necessary and sufficient for interstitial proliferation. (A–G) Male-specific proliferation was quantified by counting pHH3-positive dividing cells (green) in control and cases in which vasculature is blocked or misregulated. (A–C) Gonads at E11.25 to E11.5 were cultured for 24 h. Endothelial cells were visualized by using PECAM-1 (red). (A and B) Proliferation in the coelomic domain (CD; boxes designated with dotted lines) was compared between WT and VEGF Trap-injected embryos. (A) In WT XY gonads, proliferation is abundant and up-regulated in somatic cells within the epithelium and surrounding vessels (arrowheads). (B) Blocking vascular development inhibits male-specific somatic cell proliferation in the CD. (C) BV13-treated gonads develop disorganized vasculature with reduced proliferation in the CD. (D) Quantification of pHH3-positive cells showed reduced proliferation after VEGF Trap injection and BV13 treatment. (E–G) To determine if vasculature was sufficient to induce proliferation, XX Wnt4^−/− mutants were compared with littermate controls. Blue nuclei are false colored, and indicate PECAM-1/pHH3 double-positive proliferating germ cells not included in this analysis. In XY Wnt4^+/− gonads (E), proliferation is higher than in XX Wnt4^+/− littermates (F). (G) However, XX Wnt4^−/− gonads develop male-specific vasculature and exhibit proliferation levels similar to XY littermates and significantly higher than XX gonads, suggesting the presence of vasculature is sufficient to drive proliferation. Quantification of these results is presented in D (***P < 0.0005).

Fig. 5.

PDGF-BB rescues defective vascular development. (A) Expression of Pdgfb, but not Pdgfa or Fgf9, was reduced after VEGF Trap and BV13 treatment. (B–D) Reduced proliferation after VEGF Trap injection was rescued by addition of rPDGF-BB to cultures. All organs were stained with PECAM-1 (red), NRP1 (blue), and pHH3 (green) in order to assess vascular remodeling and proliferation. (B and C) Injected cultures showed a loss of proliferation relative to controls. (D) Proliferation was restored in injected cultures treated with rPDGF-BB. (E) Although addition of rPDGF-BB to WT gonads had no significant effect on proliferation, quantification of pHH3-positive cells confirmed that the significant decrease in proliferation after VEGF Trap injection, and to a lesser extent BV13, was rescued by the addition of rPDGF-BB (*P < 0.05, ***P < 0.0005).