DEVELOPMENTAL DYNAMICS 236:951–960, 2007 RESEARCH ARTICLE HOXA13 Directly Regulates EphA6 and EphA7 Expression in the Genital Tubercle Vascular Endothelia Carley A. Shaut,1† Chie Saneyoshi,1† Emily A. Morgan,1 Wendy M. Knosp,2 Diane R. Sexton,3 and H. Scott Stadler1,3* Hypospadias, a common defect affecting the growth and closure of the external genitalia, is often accompanied by gross enlargements of the genital tubercle (GT) vasculature. Because Hoxa13 homozygous mutant mice also exhibit hypospadias and GT vessel expansion, we examined whether genes playing a role in angiogenesis exhibit reduced expression in the GT. From this analysis, reductions in EphA6 and EphA7 were detected. Characterization of EphA6 and EphA7 expression in the GT confirmed colocalization with HOXA13 in the GT vascular endothelia. Analysis of the EphA6 and EphA7 promoter regions revealed a series of highly conserved cis-regulatory elements bound by HOXA13 with high affinity. GT chromatin immunoprecipitation confirmed that HOXA13 binds these gene-regulatory elements in vivo. In vitro, HOXA13 activates gene expression through the EphA6 and EphA7 gene-regulatory elements. Together these findings indicate that HOXA13 directly regulates EphA6 and EphA7 in the developing GT and identifies the GT vascular endothelia as a novel site for HOXA13-dependent expression of EphA6 and EphA7. Developmental Dynamics 236:951–960, 2007. © 2007 Wiley-Liss, Inc. Key words: Hoxa13; Hypospadias; genital tubercle; vascular endothelia; EphA6; EphA7 Accepted 4 January 2007 INTRODUCTION 2003). One phenotype commonly as- and Eichmann, 2005; Davy and So- sociated with hypospadias is the en- riano, 2005). Indeed, Eph–ephrin Hypospadias, a defect affecting the largement of blood vessels supplying signaling is essential for the pattern- growth and closure of the external the glans or prepuce (Baskin et al., ing of multiple tissues and cell types, genitalia, is highly prevalent in the birth populations of industrialized 1998; Baskin, 2000). At present, no including vascular endothelial cell nations, including the United States, molecular link between hypospadias assembly, cell migration, mesenchy- United Kingdom, Sweden, and Ja- and vessel enlargement in the geni- mal cell condensation, vascular bed pan (Giwercman et al., 1993; Pau- talia has been identified; however, formation, tumor neovasculariza- lozzi et al., 1997; Gallentine et al., studies examining perturbations in tion, and the closure of the external 2001). While the frequency of hypo- Eph–ephrin signaling may provide genitalia (Wang et al., 1998; Ogawa spadias ranges as high as 1 in 125 important clues toward understand- et al., 2000; Stadler et al., 2001; live births, the molecular mecha- ing the pathology of hypospadias and Chan et al., 2001; Dravis et al., 2004; nisms underlying this defect are its associated vascular malforma- Davy et al., 2004; Marquardt et al., poorly understood (Svensson et al., tions (reviewed by Eichmann et al., 2005; Egea et al., 2005). 1997; Paulozzi et al., 1997; Stadler, 2005a,b; Hinck, 2004; Klagsbrun Recently, we and others have 1Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon 2UCSF-Mission Bay Campus, San Francisco, California 3Shriners Hospital for Children Research Division, Portland, Oregon Grant sponsor: the National Institutes of Health; Grant number: HSS R01 DK66539; Grant sponsor: the American Heart Association. †Drs. Shaut and Saneyoshi contributed equally to this work. *Correspondence to: H. Scott Stadler, Shriners Hospital for Children Research Division, Portland, OR 97239. E-mail: [email protected] DOI 10.1002/dvdy.21077 Published online 15 February 2007 in Wiley InterScience (www.interscience.wiley.com). © 2007 Wiley-Liss, Inc. 952 SHAUT ET AL. shown that HOXA13 function is nec- essary for EphA7 expression in the developing limb (Stadler et al., 2001; Salsi and Zappavigna, 2006). Recog- nizing that Hoxa13-deficient mice also exhibit hypospadias and capil- lary vessel enlargement (Morgan et al., 2003), we hypothesized that HOXA13 may regulate Eph receptor expression in the genital tubercle (GT) and its vasculature. Testing this hypothesis, we report that HOXA13 directly regulates EphA6 and EphA7 expression in the GT vas- cular endothelia. Analysis of the EphA6 and EphA7 promoter regions revealed a conserved series of DNA sequences bound with high affinity by the HOXA13 DNA binding do- main (A13). In vivo, direct interac- tions between HOXA13 and the EphA6 and EphA7 promoter ele- ments were detected in the GT using Hoxa13-directed ChIP. In vitro, HOXA13 can use the bound gene- regulatory elements in the EphA6 and EphA7 promoters to direct gene expression. Together these findings indicate that EphA6 and EphA7 are direct transcriptional targets of HOXA13 in the GT vascular endo- thelia, providing new insight into the cell-signaling mechanisms func- tioning during the growth and devel- opment of the external genitalia. RESULTS Fig. 1. Hoxa13-deficient mice display enlarged blood vessels in the developing genital tubercle. A–L: Hematoxylin and eosin staining of sectioned genital tubercles from wild-type (A–D), homozygous Vessel Expansion Is Present mutant male (E–H), and homozygous mutant female (I–L) embryos at embryonic days (E) 12.5–15.5. Throughout GT Development Arrows denote a typical example of normal vessel diameters in wild-type embryos (A–D) or enlarged vessels in homozygous mutants (E–L). UPE, urethral plate epithelium. Scale bar ϭ 100 ␮m. in Hoxa13–Green Fluorescent Protein Homozygous Mutants Analysis of the distal GT from em- bryonic days (E) 12.5 to 15.5 re- vealed average vessel diameters of 15 ␮m(Ϯ 0.5 ␮m; n ϭ 12 indepen- dent samples) in Hoxa13-GFP (GFP, green fluorescent protein) wild-type and heterozygous mutants. In con- trast, the GT vasculature of age- matched homozygous mutants ex- hibited greatly enlarged vessels with average diameters of 85 ␮m(Ϯ 15 ␮m; n ϭ 12 independent samples; Fig. 1). Of interest, embryonic sex Fig. 7. Genital tubercle (GT) vessel diameter and endothelial cell identity are maintained in EphA7 did not influence the presentation of homozygous mutants. A: Typical GT vessel diameters and endothelial cell adhesion molecule-1 (PECAM-1) expression in embryonic day (E) 13.5 wild-type control embryos. B,C: The GT vascu- the vascular phenotype as homozy- lature of EphA7 homozygous mutants (B) does not exhibit vessel enlargement or alteration in gous mutant male and female em- PECAM-1 expression when compared with the enlarged GT vasculature of Hoxa13-GFP homozy- bryos exhibited similar increases in gous mutants (C). Scale bar ϭ 50 ␮m. HOXA13 REGULATES EPHA6,7 EXPRESSION IN THE GT 953 Fig. 2. Immunohistochemical localization of EPHA6 and EPHA7 in the genital tubercle (GT) of E13.5 Hoxa13-green fluorescent protein (GFP) mice. A,C: EPHA6 and EPHA7 (red signal) are expressed in the GT mesenchyme (MES) as well as the endothelial cells lining the GT vasculature (arrows) of heterozygous Hoxa13-GFP embryos. B,D: EPHA6 and EPHA7 expression is reduced in the enlarged GT vessels of age-matched Hoxa13 homozygous mutants (arrows). E,G: Colocalization (yellow signal) of Hoxa13-GFP with EPHA6 and EPHA7 in the GT vascular endothelia of heterozygous embryos. Arrows denote colocalization in the GT endothelia. F,H: Reduced expression of EPHA6 and EPHA7 in the homozygous mutant vascular endothelia. Arrows denote the enlarged vasculature. I–L: Higher magnification images of the heterozygous control and homozygous mutant vessels depicted in E–H. M,N: Platelet endothelial cell adhesion molecule-1 (PECAM-1) expression (red) is maintained in the GT vascular endothelia of Hoxa13-GFP (green) heterozygous mutant controls and age-matched homozygous mutants. UPE, urethral plate epithelium; MES, mesenchymal tissue; R, red blood cells. For clarity, only male GTs are shown, although similar vascular defects were observed in mutant female GTs. Scale bars ϭ 140 ␮m in A–H, 20 ␮m in I–L, 70 ␮m in M–P. GT vessel diameter (Fig. 1, compare with perturbations in Eph–ephrin both EPHA6 and EPHA7 were con- E–H with I–L). signaling, we examined whether the sistently reduced in the GT vascular affected GT vasculature exhibited endothelia compared with heterozy- EphA6 and EphA7 Are changes in the expression of Eph re- gous mutant controls (Fig. 2A–D), Reduced in the GT Vascular ceptors or their ephrin ligands. which do not exhibit a GT phenotype Endothelia of Hoxa13 While no changes in EPHA2, (Morgan et al., 2003). In the vascu- EPHA4, EPHA5, EPHB2, EPHRIN lature, HOXA13-GFP, EPHA6, and Homozygous Mutants A2, or EPHRIN A5 expression were EPHA7 were strongly colocalized in Recognizing that defects in vascular detected in the Hoxa13-GFP ho- the endothelial layer of heterozygous patterning are strongly associated mozygous mutants (data not shown), controls, whereas the reduced levels 954 SHAUT ET AL. of EPHA6 and EPHA7 in the ho- mozygous mutants minimized our detection of colocalization in the ex- panded GT vasculature (Fig. 2E–L). Platelet endothelial cell adhesion molecule-1 (PECAM-1) expression was also present in the affected GT vasculature, suggesting that endo- thelial cell identity was not affected by the loss of HOXA13 function (Fig. 2M–N). Semiquantitative reverse transcrip- tase-polymerase chain reaction (RT- PCR) analysis confirmed the levels of EphA6 and EphA7 expression detected by immunohistochemistry. In particu- Fig. 3. Quantitation of Hoxa13, EphA6, and EphA7 expression in the genital tubercle (GT) mesenchyme and vascular endothelia. A: Semiquantitative RT-PCR of EphA6 and EphA7 tran- lar, a uniform level of EphA6 and scripts revealed no differences in EphA6 and EphA7 expression in the whole distal GT. B: Hoxa13, EphA7 expression was detected EphA6, and EphA7 are coexpressed in the GT vascular endothelium as determined by semiquan- throughout the GT mesenchyme in titative reverse transcriptase-polymerase chain reaction on platelet endothelial cell adhesion mol- Hoxa13-GFP heterozygous and ho- ecule (PECAM) -positive endothelial cell isolates. Furthermore, both EphA6 and EphA7 expression mozygous mutant embryos (Fig. 3A). levels were reduced in vascular endothelial cells of Hoxa13 homozygous mutant GT compared with Hoxa13 heterozygous controls.