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

Abstract

Background: The noncanonical 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.

Conclusions: 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: Müllerian duct; Ror2; Sertoli cell; Wnt5a; Wolffian duct; germ cell; granulosa cell; ovary; testis.

Figure 1

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.

Figure 2

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

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 4

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

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.

Figure 6

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

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.