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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Dev Dyn. 2014 May 6;243(8):1037–1045. doi: 10.1002/dvdy.24138

Novel domains of expression for orphan receptor tyrosine kinase Ror2 in the human and mouse reproductive system

Ripla Arora 1, Eran Altman 1, Nam D Tran 1, Diana J Laird 1,2,*
PMCID: PMC4258601  NIHMSID: NIHMS645865  PMID: 24753105

Abstract

Background

The non-canonical Wnt receptor and tyrosine kinase Ror2 has been associated with Recessive Robinow syndrome (RRS) and dominant Brachydactyly type B1. The phenotypes of mouse mutants implicate Ror2 in the development of the heart, lungs, bone and craniofacial structures, which are affected in RRS. Following a recently identified role of Ror2 in the migration of mouse primordial germ cells, we extensively characterized its expression throughout the fetal internal reproductive system and the postnatal ductal system.

Results

We show that Ror2 gene products are present in the germ cells and somatic cells of the testis and the ovary of both the mouse and human fetus. In reproductive tract structures, we find that Ror2 is expressed in the mesonephros, developing Wolffian and Müllerian ducts and later in their derivatives, the epididymal epithelium and uterine epithelium.

Conclusion

This study sets the stage to explore function for this tyrosine kinase receptor in novel regions of expression in the developing reproductive system in both mouse and human.

Keywords: Ror2, ovary, testis, germ cell, granulosa cell, Sertoli cell, Wolffian duct, Müllerian duct, Wnt5a

Introduction

Infertility affects approximately 14% of reproductive aged couples around the globe and about 22% of married women of reproductive age in developed countries (Chandra et al., 2013). Common causes of human infertility include defects in gametes (sperm or oocyte) or in the formation of the reproductive tract (Hull et al., 1985, 2012b; 2012a). Interactions between the gamete precursors in the embryo, also known as primordial germ cells (PGCs), and the surrounding somatic cells are essential for optimal sperm or oocyte production. This somatic environment varies greatly depending on the stage of PGC development (Wilhelm et al., 2007). Until its colonization by PGCs, the gonad is bipotential; genes that promote sex differentiation are upregulated thereafter (Kim and Capel, 2006). In the mouse, by embryonic day (E) 12.5, male and female gonads diverge as PGCs in the males aggregate, become surrounded by supporting Sertoli cells, and organize to form the testis cords; by contrast, in female gonads the PGCs remain dispersed amongst the other somatic cell types including the supporting granulosa cells. PGCs in the testes - termed spermatogonia - enter mitotic arrest around E13.5 and do not begin meiosis and differentiation to mature sperm until after birth (Goetz et al., 1984). In the female gonad, PGCs - now oogonia – enter meiosis at ~E13.5 and organize themselves into cyst-like structures during late fetal life. The oogonia are encapsulated by supporting granulosa cells to form the primordial follicles around birth in mice and during mid-gestation in humans; these follicles subsequently grow and are recruited cyclically and ovulated beginning at puberty (Pepling, 2012).

The embryonic precursors of the internal reproductive system include the Wolffian ducts, which proceed to form the male ductal structures including the epididymis, the vas deferens, and the seminal vesicles, and the Müllerian ducts that proceed to form the female ductal structures including the oviduct, the uterus and the upper part of the vagina. The Wolffian ducts are present as early as E10.5 and the Müllerian ducts form between E11.5 and E13.5. These two parallel structures persist in the mesonephros until E15.5, after which Müllerian ducts regress in males in response to anti Müllerian hormone (also known as Müllerian inhibitory substance) secreted by the Sertoli cells of the testis (Behringer et al., 1994; Mishina et al., 1996; Belville et al., 1999; Hoshiya et al., 2003). In the absence of anti Müllerian hormone, the Müllerian ducts persist and the Wolffian ducts regress in females (Staack et al., 2003).

A key mechanism in female sex determination is the Wnt signaling pathway. Mutations in Wnt4 and Wnt pathway members Rspo1 and β-catenin cause partial female to male sex reversal in the mouse (Kim et al., 2006; Maatouk et al., 2008; Chassot et al., 2011). Mutations in human Wnt4 are associated with two syndromes: Serkal syndrome (OMIM#611812), characterized by female to male sex reversal (Mandel et al., 2008), and Mayer-Rokitansky-Kuster-Hauser Syndrome (OMIM#277000), characterized by uterine hypoplasia and Müllerian duct anomalies (Sultan et al., 2009). Despite the importance of the Wnt signaling pathway in development of female gonad and ducts, the receptor for Wnt signaling in the internal reproductive system has not yet been identified.

The Ror2 gene encodes a receptor tyrosine kinase and has been implicated as a receptor for the non-canonical Wnt ligand Wnt5a (Oishi et al., 2003; Minami et al., 2010). The Ror2 domain structure comprises of a N-terminal signal peptide, Ig-like extracellular domain, cysteine-rich frizzled-like domain (the Wnt ligand binding domain), kringle domain, and single transmembrane domain; within the cytosolic tail reside a receptor tyrosine kinase domain, serine threonine-rich cytosolic domain, and a proline-rich intracellular domain (Masiakowski and Carroll, 1992). Mutations in the Ror2 gene have been associated with two major human syndromes: dominant Brachydactyly type B1 (BDB1, OMIM#113000) (Afzal and Jeffery, 2003) and Recessive Robinow Syndrome (RRS, OMIM#268310) (van Bokhoven et al., 2000). BDB, which involves shortening of the digits and fusion of the middle digits, is associated with specific gain of function mutations in the C terminus of the Ror2 protein, primarily the serine threonine-rich or the tyrosine kinase domains. The phenotype is autosomal dominant and thus occurs in the heterozygous state. By contrast, RRS associated mutations occur throughout the protein and generally cause loss of function phenotypes when homozygous. Clinical features of RRS include dysmorphic facial structure, dwarfism, brachydactyly with shortening of limbs, congenital heart defects in some cases, and hypoplastic external genitalia (Afzal et al., 2000; Oldridge et al., 2000; van Bokhoven et al., 2000; Schwarzer et al., 2009).

Owing to the implication of Ror2 in RRS and BDB, its expression has been extensively studied in major organs such as heart, limbs, bone and cartilage (Al-Shawi et al., 2001; Matsuda et al., 2001) however the expression pattern has not been analyzed in the developing reproductive system. Furthermore, the hypoplastic genitalia associated with RRS syndrome has probably precluded any analysis of fertility or germ cells in these patients. Mice homozygous for null alleles of Ror2 are perinatal lethal and thus limit analysis of the contribution of this gene to reproductive function (DeChiara et al., 2000; Takeuchi et al., 2000). The role of Ror2 in the developing reproductive system was recognized recently where it was shown to function earlier in germ cell migration (Laird et al., 2011) in the mouse. In another study, male mice homozygous for a Ror2 point mutation, Ror2W749X were subfertile, although the cause is not clear as sperm derived from these mice were motile and functional (Raz et al., 2008). In light of these reproductive phenotypes in two Ror2 mouse mutants, here we systematically examine the expression pattern of Ror2 in the developing gametes and the reproductive system at the time of sex differentiation and later. In both mouse and human, we show that Ror2 is dynamically expressed in male and female germ cells during embryonic development. Ror2 is also detected in the nascent ductal structures embryonically, in adult uterus of both mice and humans, and in the epithelium of the epididymis in postnatal mice. Our expression studies set the stage for functional studies of this protein in the developing reproductive system.

Results and Discussion

Ror2 is expressed in the developing mouse gonads and ducts

Using whole mount in situ hybridization (ISH), Ror2 was robustly detected in the mesonephros and the Wolffian duct of both male and female E11.5 mouse embryos (Figure 1A, A′). At E12.5, when morphological sex differentiation becomes apparent, Ror2 transcript was detected in both testes and ovaries (black dashed lines in Figure 1C, D), the mesonephros, Wolffian duct (Figure 1C′, D′, blue arrows) and Müllerian duct (Figure 1C′, D′, pink arrows). At E13.5 increased expression is detected in the ovaries and testes compared to E12.5 (Figure 1E, F). Expression in the female gonads appeared homogeneous, whereas the pattern of expression in male gonads resembled that of the testis cords (Figure 1E, F). Ror2 persists in Wolffian as well as Müllerian ducts in both males and females at E13.5 (Figure 1E′, F′ blue and pink arrows).

Figure 1.

Figure 1

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Expression of Ror2 in the developing mouse gonad and ducts. A–F′ Ror2 expression was assessed using whole mount ISH. A′ is a hand cut slice and C′,D′,E′,F′ are 100μm vibratome sections through the mesonephros (yellow dotted lines) following whole mount ISH. The gonads are demarcated from the mesonephros by a black dotted line in all panels. At E11.5, Ror2 expression was detected most highly in the developing mesonephros (below the outlined gonad in A) and the Wolffian duct (A′). Ror2 sense control probe was used on male gonads at E13.5 and no expression was detected (B). At E12.5 expression is seen in the gonad, mesonephros, Wolffian ducts and Müllerian ducts in both the female (C,C′) and male (D,D′). At E13.5 similar expression is seen in the female and male gonads and in the ducts throughout the middle of the mesonephros (E,E′,F,F′). Black dotted line: gonad; blue arrows: Wolffian duct; pink arrows: Müllerian duct.

Ror2 is localized to the membrane of transfected HEK 293Tcells using different antibodies

Multiple antibodies have been generated against hROR2 but few have been tested on tissue sections for immunohistochemistry. We used 3 different antibodies: SantaCruz HX07 (Cat# 80329), a monoclonal antibody that is raised against an epitope in the extracellular domain of hROR2; SantaCruz H1 (Cat# 374174), a monoclonal antibody raised against amino acids 868-943 in the intracellular domain of hROR2; and Sigma (Cat# HPA021868), a polyclonal antibody raised against amino acids 796-927 in the intracellular domain of hROR2. We first validated these antibodies in Human Embryonic Kidney (HEK) - 293T cells transfected with a vector overexpressing hROR2 cDNA. No ROR2 signal was detected in HEK-293T cells alone (Figure 2A, C, E). By contrast, in the presence of the vector (as characterized by the expression of mCherry throughout the cell) all three antibodies localized to the cell membrane (Figure 2B,D,F). Ror2 protein sequence is highly conserved among vertebrates, with 92% overall similarity between human and mouse and 87% similarity with chicken. Given the precedent for cross-reaction between hROR2 antibody and chick Ror2 (Stricker et al., 2006), we tested these antibodies in E12.5 lung tissues, which express high levels of Ror2 transcript (Al-Shawi et al., 2001; Matsuda et al., 2001). The Sigma Rabbit anti-human antibody labeled the membrane of branching E-Cadherin positive epithelial cells of the lung (Figure 3A) and was completely absent from the epithelium of the Ror2 null lungs (Figure 3B). This antibody was hereafter used to characterize Ror2 expression in the embryonic and post natal mouse gonadal sections. Both mouse anti-hROR2 antibodies were used to characterize expression of Ror2 in the human fetal gonadal sections and adult human uterus (Figure 6 and Figure 7)

Figure 2.

Figure 2

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Validation of Ror2 antibodies using HEK-293T cells overexpressing Ror2. A–F HEK 293T cells alone or HEK 293Tcells transfected with ROR2-T2A-mCherry were cultured on slides. Santa Cruz HX07 mouse anti-human ROR2 (A,B), Santa Cruz H1mouse anti-human ROR2 (C,D) and Sigma rabbit anti-human ROR2 (E,F) were added to 293T cells alone (A,C,E) or 293T cells expressing ROR2mCherry (B,D,F). Anti-hROR2 antibodies are shown in green, nuclei in blue (Hoechst) and mCherry expression is shown in red. Scale bar: 50μm

Figure 3.

Figure 3

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Validating Sigma rabbit anti-hROR2 antibody for use on mouse tissue sections. A,B IF staining of wild type and Ror2−/− lung sections at E12.5 with Sigma Rabbit anti-hROR2 antibody. Ror2 expression (red) is detected in the E-Cad (grey) positive epithelium of the lungs of wild type embryo (A) but not in the epithelium of the Ror2−/− lungs. Scale bar: 50μm

Figure 6.

Figure 6

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Expression of Ror2 in the epithelium of developing ducts. A,B IF on cryosections of P0 and P21 male epididymis. Inset in A shows Ror2 expression in the epithelium of the epididymal ducts. C,D IF on cryosections of adult female ductal structures derived from the Müllerian duct. Similar to the male, Ror2 expression (red) is seen in the epithelium of the ducts colocalizing with E-Cad expression (C,D, grey). Inset in C shows membrane epithelial staining in the ductal derivative. E IF on cryosections of gestational day 11 pregnant female uterus. E′ Magnified region marked with yellow box in E. Expression is seen in the epithelium of the uterus (E,E′). Expression is higher on the basal side (yellow arrow) of the epithelium as compared to the luminal side. Expression is also seen in the glands (white arrow) beneath the epithelium. F IF on cryosections of human adult uterus. Similar to the mouse, ROR2 expression is higher on the basal side (yellow arrow) of the epithelium as compared to the luminal side. Scale bar in A,B,C,D,E,F,G is 50μm, and F′ is 10μm.

Figure 7.

Figure 7

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Expression of ROR2 in the human embryonic gonads and ducts. A Cryosection of a single testis cord in the 18week human male fetus immunostained with ROR2 (red), VASA (grey, germ cells) and Hoechst (nuclei). ROR2 expression is seen on the membrane of all VASA positive germ cells in the testis cords. B Cryosection of a 16week human ovary stained with ROR2 (red), VASA (grey, germ cells) and Hoechst (nuclei). Expression is seen in some but not all VASA positive germ cells. Yellow asterisks: ROR2 and VASA double positive germ cell, Yellow arrows: ROR2 negative and VASA positive germ cell. C,D IF on crysoections of 20week human epididymis and 21week human uterus. Similar to the mouse, ROR2 expression is seen in the epithelium of the epididymis (C) and uterus (D). Scale bar in A,B is 10μm and C,D is 50μm.

Ror2 is expressed in the mouse fetal testis and ovary

Among the signaling mechanisms implicated downstream of Ror2 are the β-catenin dependent (or canonical) and several arms of the non-canonical Wnt pathway including planar cell polarity (PCP) (Minami et al., 2010). Wnt5a remains the only ligand shown to bind Ror2 by in vivo evidence (Oishi et al., 2003), although biochemical association has been demonstrated with other Wnts via the Ror2 cysteine rich domain (Mikels and Nusse, 2006; Yamamoto et al., 2008). Wnt5a is upregulated by interstitial cells of the developing mouse testis but not the ovary at the time of sex differentiation (Chawengsaksophak et al., 2012); this suggests that Wnt5a might act as a ligand for Ror2 in the mouse testis. Wnt4, which has been shown to act through both canonical and non-canonical pathways, is expressed in the granulosa cells (Kim et al., 2006) and is therefore a potential Ror2 ligand in the fetal ovary (Liu et al., 2010; Naillat et al., 2010).

In the developing male gonads at E14.5, Ror2 expression was detected using immunofluorescence (IF) in Oct4-ΔPE-GFP and E-Cadherin (E-Cad) expressing germ cells present in the testis cords (Figure 4A, A′, yellow asterisks). Ror2 was also expressed in the Sertoli cells surrounding the germ cells in the testis cords (Figure 4A′ yellow arrows). This expression pattern persisted at E18.5, where Ror2 was seen in both germ cells and Sertoli cells using ISH and IF (Figure 4B,B′,C,C′ germ cells: yellow asterisks; Sertoli cells: yellow arrows). Expression was not detected on the membrane of either cell type in Ror2 null E18.5 testis sections (Figure 4D,D′), confirming the utility of the Sigma anti-hROR2 antibody for mouse tissue sections. In E12.5 females, Ror2 expression was detected by IF in SSEA1-positive germ cells as well as in SSEA1-negative support cells in the mesenchyme (Figure 5A,A′). We validated the absence of Ror2 staining in Ror2 null E14.5 ovary sections (Figure 5C), as compared to wild-type E14.5 ovary sections (Figure 5D). Closer examination revealed co-localization of Ror2 in Foxl2 positive granulosa cells and some Oct4-ΔPE-GFP positive germ cells in E14.5 ovaries (Figure 5D,D′, germ cells: yellow asterisks, granulosa cells: yellow arrows). Interestingly, the expression pattern of Ror2 seemed to demarcate the ovigerous cords since it marks Foxl2 positive granulosa cells surrounding germ cell cysts (Figure 5D′). One day later, at E15.5 expression was down regulated in the E-Cad positive germ cells and remained only in the Foxl2 positive granulosa cells (Figure 5E,E′).

Figure 4.

Figure 4

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Expression of Ror2 in the mouse embryonic testis. A Sagittal section of E14.5 XY embryo stained with Hoechst (blue, nuclei), endogenous Oct4-ΔPE-GFP (green, germ cells), E-Cad (grey, germ cells) and Ror2 (red). A′ Magnified region marked with yellow box in A. Ror2 protein is detected in E-Cad positive germ cells (yellow asterisks) and E-Cad negative Sertoli cells surrounding the germ cells (yellow arrows) in the testis cords. B ISH on cryosection of E18.5 testis with a Ror2 antisense probe showing Ror2 transcript in the testis cords. Cryosection is counterstained with nuclear fast red. B′ Magnified region marked with a black box in B. C Ror2 (red) antibody staining on cryosection of E18.5 wild type testis. D Ror2 (red) antibody staining on cryosection of E18.5 Ror2−/− testis. Similar to E14.5, Ror2 expression is seen on the membrane of germ cells (yellow asterisks) and Sertoli cells (yellow arrows) in the wild type testis (C) but not in the mutant testis (D). C′ and D′ are magnified regions marked with yellow box in C and D respectively. Scale bar in A,B,C,D is 50μm and A′,B′, C′, D′ is 10μm.

Figure 5.

Figure 5

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Expression of Ror2 in the mouse fetal ovary. A Sagittal section of E12.5 XX embryo stained with Hoechst (blue, nuclei), Ror2 (red) and SSEA1 (grey, germ cells). A′ Magnified region marked with yellow box in A. Expression is seen on the membrane of germ cells but also on the surrounding cells in a salt and pepper-like pattern. B Ror2 (red) antibody staining on cryosection of E14.5 wild type ovary. C Ror2 (red) antibody staining on cryosection of E14.5 Ror2−/− ovary. D Sagittal section of E14.5 XX embryo stained with Hoechst (blue, nuclei), endogenous Oct4-ΔPE-GFP (green, germ cells), Ror2 (red) and Foxl2 (grey, granulosa cells). D′ Magnified region marked with yellow box in B. Ror2 expression is seen on the membrane of Foxl2 positive granulosa cells (yellow arrows) and Oct4-ΔPE-GFP positive germ cells (yellow asterisks). C Sagittal section of E15.5 XX embryo stained with Hoechst (blue, nuclei), E-Cad (green, germ cells), Ror2 (red) and Foxl2 (grey, granulosa cells). C′ Magnified region marked with yellow box in C. Ror2 expression is only seen on the membrane of Foxl2 positive granulosa cells (yellow arrows) but not on the E-Cad positive germ cells (yellow asterisks). Scale bar in A, B, C, D,E is 50μm and A′, D′, E′ is 10μm.

The expression pattern of Ror2 in the somatic cells of the gonad may provide a useful marker for purification of novel cell populations in addition to yielding biologic insights. In early differentiating mouse testes and ovaries, Ror2 delineates the testis cords and the ovigerous cords respectively. Although testis cords can be distinguished solely on the basis of morphology as early as E12.5, this is not the case with the ovigerous cords, and so far no marker has been identified that allows easy identification of these structures (Pepling, 2012). With the presence of Ror2 positive somatic cells surrounding aggregations of oogonia at E14.5 and E15.5, Ror2 has the potential to be used as a marker for nascent ovigerous cords. By contrast, heterogenous Ror2 expression may define important developmental states in the germ cell compartment. At E14.5 we observed Ror2 in few Oct4-ΔPE-GFP positive germ cells, and at E15.5 Ror2 was completely absent in E-Cad positive germ cells. This observation raises the question: do Ror2 positive germ cells form a distinct population from the Ror2-negative ones? Alternatively, it is possible that all germ cells proceed through a Ror2 positive state as part of a developmental progression, such as the entry into meiosis. The consistent expression of Ror2 in male germ cells, contrasted with more dynamic and heterogeneous expression in female germ cells coincident with meiotic initiation raises questions of its role during germ cell sex differentiation that will be approachable with conditional knockouts.

Ror2 is detected in the epithelium of developing ducts postnatally

Ror2 was observed in the epithelium of the male epididymis in P0 and P21 males by IF (Figure 6A,B). In females, expression was also seen in in the epithelium of the adult uterus and other duct-derived epithelia surrounding the ovary (Figure 6C,D). Consistent with a previously published report, Ror2 expression was present in the glands of the uterus at gestational day (GD) 11 (Figure 6E′ white arrow). Closer inspection revealed enrichment of ROR2 was higher on the basal side of the uterine epithelium (Figure 6E′ yellow arrow) compared to lower although still detectable level on the luminal side of the epithelium (Figure 6E,E′). ROR2 expression on the luminal side of the uterus was not reported in the previous study (Hatta et al., 2010). This small discrepancy in expression patterns may be attributable to the sensitivity of detection for the different antibodies used in the two studies, with the Sigma rabbit antibody used here being more sensitive to lower levels of the Ror2 protein. We further analyzed ROR2 expression in human adult uterus using a different antibody (anti-hROR2 SantaCruz H1). Similar to the mouse GD11 uterus, we observed enrichment of ROR2 on the basal side of the hUterus epithelial cells and lower level of expression on the luminal side (Figure 6F,G).

Development of the female reproductive tract (FRT) involves a number of non-canonical Wnt ligands including Wnt4, Wnt5a, Wnt7a and Wnt9b. Wnt7a-deficient females are infertile owing to abnormal development of the oviduct and uterus, both of which are Müllerian duct derivatives (Parr and McMahon, 1998). Mutation in Vangl2, a key component of the PCP and non-canonical Wnt signaling pathways, leads to an FRT phenotype similar to that of Wnt7a mutants. In the Vangl2Looptail mutants, uterine horns fail to fuse at the cervix, oviduct coiling is lost, and uterine epithelia are disorganized (Vandenberg and Sassoon, 2009). FRT defects have also been reported in the absence of other PCP components including, Celsr1, where deletion leads to sterility and Celsr2, where mutant females have vaginal atresia (Boutin et al., 2012). Despite the involvement of multiple Wnt receptors including Fzd3 and Fzd6 in PCP signaling in other organs, none has been implicated in embryonic or post natal development of the FRT (Wang et al., 2006; Stuebner et al., 2010). PCP phenotypes have been observed in the mouse cochlea, neural tube and xenopus gastrula in Ror2 deficient embryos (Schambony and Wedlich, 2007; Gao et al., 2011) making Ror2 a likely candidate receptor for mediating non canonical Wnt-PCP signaling. In male reproductive development, infertility is reported in one third of males carrying two copies of a hypomorphic allele for Vangl2 gene, despite apparently normal copulatory plugs and sperm appearance (Guyot et al., 2011). Similarly, male mice homozygous for a Ror2 point mutation, Ror2W749X, present with partially penetrant infertility despite visibly unaffected sperm quality (Raz et al., 2008). The resemblance of phenotypes amongst Ror2 and Vangl2 mutants in the males, and non-canonical Wnt ligand mutants and Vangl2 mutants in females could suggest these genes operate in a common Ror2 mediated PCP signaling pathway in reproductive tract development.

ROR2 expression is detected in human embryonic gonads and ducts

Gametogenesis in the human, although less understood than in mouse, shares many common cellular and molecular mechanisms. Human germ cell development is more protracted than mouse, with mitotic arrest in males and meiotic entry in females occurring at 10 weeks compared to mouse E13.5. The conserved RNA helicase VASA is expressed by human PGCs during migration, in the gonadal ridge, and through mitotic arrest or meiotic initiation, although it is downregulated in a subset of less differentiated PGCs around 11 weeks (Anderson et al., 2007; Gkountela et al., 2013). We used mouse anti-hROR2 antibodies to examine expression in second trimester human embryonic gonads. In the 18wk human testis, ROR2 was detected on the membrane in the VASA positive germ cells located within the cords; a lower level of ROR2 expression was present in other cells within the testis cord and in the surrounding support cells (Figure 7A). In the 16wk human ovary, ROR2 immunostaining was detected in some but not all VASA positive germ cells as well as in other cells (Figure 7B). This reinforces the previous observation that second trimester human germ cells are a heterogenous population, and leaves open the possibility of ROR2 expression in a VASA negative germ cell population (Gkountela et al., 2013) or in non-germ cells. Thus similar to the mouse, ROR2 expression was detected in germ cells of the developing gonads in both males and females with expression in the female germ cell population being heterogeneous. Additionally ROR2 expression was detected in the epithelium of 20wk human epididymis (Figure 7C) and 21wk human uterus (Figure 7D), which is comparable to the ROR2 ductal expression observed in the mouse in Figure 6. Previous work showing similar expression patterns of Ror2 between mouse and chicken (Stricker et al., 2006) raises the possibility of its conserved function in vertebrates. Here we show that in the context of the developing gonads and ducts, the expression pattern is similar between mouse and human. These observations also pose important questions regarding the reproductive function in BDB and RRS patients.

Our analysis of the Ror2 RNA transcript and protein uncovers multiple new domains of expression and lays the foundation to ask important questions pertaining to the function of the PCP and non-canonical Wnt signaling pathways in the developing gametes, support cells of the gonads and ducts of the reproductive tract. Functional analysis will then provide insight into the etiology of human disorders including but not limited to RRS, BDB, Serkal’s and the Müllerian duct anomalies.

Experimental Procedures

Mice

Ror2−/− mice were generated from the Ror2flox/flox {JAX#018354} strain using the ZP3Cre line {JAX#003650}. Control CD1 female mice were mated with homozygous Oct4-ΔPE-GFP males (Szabo et al., 2002; Boiani et al., 2004). Embryos were dissected from timed matings and yolk sacs or tails were removed for PCR genotyping. Males and females were distinguished based on PCR genotyping as described previously (Clapcote and Roder, 2005). The dark period was 19.00 to 05.00 h and noon on the day of a mating plug was identified as E0.5. All mouse work was carried out under University of California San Francisco Institutional Animal Care and Use Committee guidelines, in an AAALAC approved facility.

In situ hybridization and Immunofluorescence

Whole-mount ISH, IF and ISH on cryosections was performed as described previously (Wilkinson and Nieto, 1993; Grieshammer et al., 2004). Briefly, for IF histology, embryos or gonads fixed in 4% paraformaldehyde were embedded in OCT and cryosectioned at 8 μm for mouse embryo sections and 10 μm for human fetal sections. Sections were permeabilized with 0.5% Triton in PBS, blocked 1 h in 5% donkey serum + 0.1%Triton in PBS, stained overnight at 4°C in the blocking buffer, washed in PBS, incubated with secondary antibody for 1 hour at room temperature, washed with PBS and cover slipped with Vectashield (Vector labs, Cat# H-1000). Primary antibodies used were rabbit anti-human Ror2 (Sigma, Cat# HPA021868), mouse anti-human Ror2 (Santa Cruz H1, Cat# 374174), mouse anti-human Ror2 (Santa Cruz HX07, Cat# 80329), goat anti-human VASA (R & D, Cat# AF2030), Rat anti-E-Cadherin (Invitrogen, Cat# 13-1900), mouse anti-SSEA1 IgM (Developmental studies hybridoma bank, Cat# MC-480), and Goat anti-mouse Foxl2 (Novus Biologicals, Cat# NB100-1277). All secondary antibodies were fluorescently conjugated Alexa Fluor IgG or IgM from Invitrogen. Brightfield imaging was performed on an Olympus MVX10 stereomicroscope. Confocal imaging was carried out with a 10X, 20X or 63X objective on a Leica SP5 TCS microscope equipped with 405, 488, 543, 594, and 633 nm lasers. Image stacks were analyzed using Volocity (Improvision).

HEK-293T cells culture and transfection

Human embryonic Kidney 293T cells were cultured and maintained in DMEM with 10% FBS, 2mM L-glutamine and 0.1mM non essential amino acids. For transfection, human ROR2 (hROR2) cDNA was cloned downstream of an EF1α promoter, followed by a T2A driving mCherry in a lentiviral vector (Gateway). Transfected cells were flow-cytometrically purified based on mCherry expression and plated for expansion. For antibody validation experiments, HEK-293T cells alone or HEK-293T cells with hROR2-mCherry were plated on an 8 chambered glass slide (Lab-Tek, Cat# 70415) and cultured until confluent. The cells were then fixed in 4% paraformaldehyde for 10 minutes, washed with PBS and then used for IF.

Human fetal tissue collection

Human fetal ovaries, testes and uteri were obtained following elective termination of pregnancy after eligible participants were informed of the studies, which were approved by the University of California, San Francisco Institutional Review Board (IRB). None of the terminations were for reasons of fetal abnormality, and all fetuses appeared to be morphologically normal. Gestational age was determined by ultrasound and confirmed by subsequent measurement of foot length. Tissues were dissected and processed for IF. Human adult uterine samples were obtained from the NIH UCSF Human Endometrial Tissue and DNA Bank, which contains samples of women undergoing endometrial biopsy or hysterectomy for non-malignant indications. The samples were collected under approved IRB protocols and informed consent to participate in the study was obtained from the patients.

Key Findings.

  • Non canonical Wnt signaling receptor Ror2 is expressed in human and mouse germ cells

  • Ror2 is also expressed in support cells of both male and female mouse gonads

  • Expression is detected in epithelium of the ductal structures of the gonads and the adult uterus of both mouse and human

Acknowledgments

We thank M. Conti and members of the Laird lab for discussion and critical review of the manuscript. We also thank Yanhua Xu and Ralph Marcucio for the Ror2 RNA probe and Henry Ho and Michael Greenberg for the Ror2 conditional mice.

Grant Sponsor: California Institute of Regenerative Medicine and National Institutes of Health

Grant Number: CIRM grant# TG2 01153 to R.A. and N.D.T, NIH 1DP2OD007420 to D.J.L

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