Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Pdgfr-␣ mediates testis cord organization and fetal Leydig cell development in the XY gonad Jennifer Brennan,1,3 Christopher Tilmann,2,3 and Blanche Capel1,4 1Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA; 2Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA During testis development, the rapid morphological changes initiated by Sry require the coordinate integration of many signaling pathways. Based on the established role of the platelet-derived growth factor (PDGF) family of ligands and receptors in migration, proliferation, and differentiation of cells in various organ systems, we have investigated the role of PDGF in testis organogenesis. Analysis of expression patterns and characterization of the gonad phenotype in Pdgfr-␣−/− embryos identified PDGFR-␣ as a critical mediator of signaling in the early testis at multiple steps of testis development. Pdgfr-␣−/− XY gonads displayed disruptions in the organization of the vasculature and in the partitioning of interstitial and testis cord compartments. Closer examination revealed severe reductions in characteristic XY proliferation, mesonephric cell migration, and fetal Leydig cell differentiation. This work identifies PDGF signaling through the ␣ receptor as an important event downstream of Sry in testis organogenesis and Leydig cell differentiation. [Keywords: Sex determination; platelet-derived growth factor; Leydig cells; endothelial cells] Received October 22, 2002; revised version accepted January 24, 2003. Organogenesis of the gonad is unique in that both the an additional source of cells for the XY gonad. Migration testis and ovary are derived from an initially bipotential of cells from the mesonephros is dependent on Sry (Capel tissue, the genital ridge. Development diverges when Sry et al. 1999) and is required for organization of testis cords is expressed during a narrow window of time between (Buehr et al. 1993; Merchant-Larios et al. 1993; Martin- 10.5 and 12.5 days postcoitum (dpc) in the XY gonad, eau et al. 1997; Tilmann and Capel 1999). The migrating resulting in rapid morphological changes that produce a cell population includes peritubular myoid cells and characteristic testis morphology by 12.5 dpc. At this many endothelial and perivascular cells that contribute stage, the organization of testis cords defines two com- to divergent vascular development in the XY gonad partments of the testis. Inside testis cords are the Sertoli (Brennan et al. 2002). The origin of fetal Leydig cells has cells, which support the development of primordial germ not been clarified, but several sources have been sug- cells, are the cell type that expresses Sry, and are be- gested, which include the coelomic epithelium (Karl and lieved to orchestrate testis organogenesis. Peritubular Capel 1998), a migrating mesonephric population (Mer- myoid cells surround the cords and participate with Ser- chant-Larios and Moreno-Mendoza 1998; Nishino et al. toli cells in the production of basal lamina. The intersti- 2001) that may include neural crest cells (Middendorff et tial compartment lies between testis cords and contains al. 1993; Mayerhofer et al. 1996), or a common early steroid-secreting Leydig cells, fibroblasts, and the char- precursor of the adrenal steroid cells (Hatano et al. 1996). acteristic vasculature of the testis. Very little is known about the genetic signaling path- Although no direct targets of Sry have been identified, ways operating downstream of Sry that control the cell- several developmental processes in the gonad depend on ular mechanisms of testis organogenesis. Using a can- Sry and occur soon after Sry is expressed. One early event didate approach, we investigated the platelet-derived is an increase in proliferation of cells at the coelomic growth factor (PDGF) family of ligands and receptors, surface of the XY gonad (Schmahl et al. 2000). Prolifera- which have well-characterized roles in the proliferation tion occurs in two stages. The first stage of proliferation and migration of mesenchymal cell types, particularly occurs between 11.2 and 11.5 dpc and gives rise to Sertoli smooth muscle and endothelial cells (Betsholtz et al. cell precursors. The second stage of proliferation occurs 2001). There are two identified PDGF receptors, ␣ and ␤, between 11.5 and 12.0 dpc and gives rise to other, un- and four ligands, A, B, C, and D. Different combinations characterized somatic cells of the testis (Karl and Capel of ligands and receptors can homo- and heterodimerize 1998; Schmahl et al. 2000). The adjacent mesonephros is and activate several distinct and overlapping intracellu- lar pathways (for review, see Heldin and Westermark 3These authors contributed equally to this work. 1999; Klinghoffer et al. 2001). PDGFR-␣ has a broader 4Corresponding author. specificity for ligand binding and can bind PDGF-A, E-MAIL [email protected]; FAX (919) 684-5481. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ PDGF-B, and PDGF-C homodimers as well as PDGF-AB gad.1052503. heterodimers, whereas PDGFR-␤ can bind only PDGF-B 800 GENES & DEVELOPMENT 17:800–810 © 2003 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/03 $5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press XY-specific role of PDGFR-␣ and PDGF-D homodimers. Generally, Pdgf ligands are enchyme of the mesonephros at 11.5 dpc in both sexes expressed by epithelial or endothelial cells, whereas (Fig. 1A). High expression was detected in the coelomic mesenchymal cells express Pdgf receptors. Proliferation epithelium, and at the gonad–mesonephros border at and migration of mesenchymal cells in response to 11.5 dpc in both sexes (Fig. 1A, white arrows). However, PDGF signaling contribute to the morphogenesis and in- expression was seen on cells in the interior of the XY, tegrity of epithelial and endothelial structures as shown but not the XX gonad. By 12.5 dpc, expression was very by genetic disruptions that affect tissues such as the strong in the interstitial cells of the XY gonad, particu- lung, the kidney, and the intestine (Soriano 1994; larly in the region near the coelomic epithelium, Bostrom et al. 1996; Lindahl et al. 1998; Karlsson et al. whereas, in the XX gonad, there was expression in only a 2000). few scattered cells (Fig. 1A). Previous expression data (Gnessi et al. 1995) and re- Three ligands bind the PDGFR-␣ receptor: PDGF-A, cent genetic data have provided some insight into the PDGF-B, and PDGF-C. Expression of Pdgf-A was de- involvement of PDGFs in the postnatal development of tected in both XX and XY gonads and in the mesonephric the testis. In the Pdgf-A−/− testis, very few adult Leydig tubules (Fig. 1B, asterisk) at 11.5 dpc (Fig. 1B). By 12.5 cells develop, leading to a reduction in testis size and dpc, Pdgf-A was strongly expressed in Sertoli cells spermatogenic arrest by 32 days postnatal (dpn). Adult within testis cords of the XY gonad in contrast to a much Leydig cells are not derived from fetal Leydig cells, but lower level of expression detected in the XX gonad (Fig. instead arise as a separate, distinct cell population after 1B). Pdgf-B expression was also present in XX and XY birth (Ariyaratne et al. 2000). Although fetal stages were gonads at early stages, but at low levels in a small num- not examined, two pieces of evidence implied that fetal ber of cells, which could easily be identified as exclu- Leydig cell development was normal. First, normal num- sively endothelial by 13.5 dpc (Fig. 1C, arrow). Pdgf-C,a bers of fetal Leydig cells were reported in Pdgf-A−/− go- newly characterized ligand that binds and activates the nads at 10–25 dpn, just before they are replaced by adult Pdgfr-␣ receptor, was expressed in a similar, unique pat- Leydig cells. Second, testicular descent and masculiniza- tern in both sexes. At 11.5 dpc, we detected expression of tion occurred normally, indicating that fetal Leydig cells Pdgf-C in the coelomic epithelium as previously re- were present during fetal development and produced tes- ported (Ding et al. 2000), as well as strong expression at tosterone. (Gnessi et al. 2000). The role of PDGF signal- the gonad/mesonephros boundary and at lower levels in ing in the embryonic testis has not been investigated in a scattered population of cells within the gonad. By 12.5 detail. A recent study using the PDGFR tyrosine kinase dpc, expression was restricted to the mesonephros/gonad inhibitor, AG1296, implicated PDGF signaling in rat fe- boundary with little or no expression in the gonad itself tal testis cord development (Uzumcu et al. 2002). Large, (Fig. 1C, arrow). dilated cords were noted in the treated samples, although Because of the robust, sexually dimorphic expression the cellular and molecular basis for the defect was not of Pdgfr-␣ and Pdgf-A, they represent the strongest can- characterized, and the role of specific PDGF receptors didates for PDGF signaling in the testis. However, the and ligands was not addressed. nonoverlapping expression patterns of the three PDGF Our examination of gonads from Pdgfr-␤−/− mice (So- ligands suggest they may perform unique, restricted riano 1994) revealed no overt defects in early XY or XX functions during testis development. development at 11.5–13.5 dpc, ruling out a critical role for this receptor (K. Tilmann, unpubl.). These data led us Pdgfr-␣−/− XY gonads have defects in testis cord to focus our study on PDGFR-␣ and its ligands PDGF-A, formation and vascular development PDGF-B, and PDGF-C. We found that Pdgfr-␣ and Pdgf-A display a sexually dimorphic expression pattern in the To determine the role of PDGF signaling in testis devel- gonad.