Retinoic acid signaling regulates sonic hedgehog and bone morphogenetic protein signalings during genital tubercle development

Abstract

Retinoic acid (RA) plays pivotal roles in organogenesis, and both excessive and reduced amounts of RA cause developmental abnormalities. Reproductive organs are susceptible to teratogen toxigenicity, and the genital tubercle (GT) is one such representative organ. The physiological function of endogenous RA signaling and the mechanisms of RA-induced teratogenicity are poorly understood during the GT development. The objective of this study is to understand the developmental and teratogenic roles of RA during GT development by analyzing genetically modified mouse models. We found dynamic patterns of gene expression for the RA-synthesizing enzyme, Raldh2, and for the RA-catabolizing enzyme, Cyp26b1, during GT development. Rarb, an indicator gene for RA signaling, starts its expression in the prospective corpus cavernosum penis and in the urethral plate epithelium (UE), which plays central roles during GT development. Excessive RA signaling in Cyp26b1(-/-) mutants leads to abnormal extents of cell proliferation and differentiation during GT development, and also upregulates expression of growth factor signalings. They include Sonic hedgehog (Shh) signaling and Bone morphogenetic protein (Bmp) signaling, which are expressed in the UE and its bilateral mesenchyme. RA signaling positively regulatesShh and Bmp4 expression during GT development as testified also by the experiment of RA administration and analyses of loss-of-function of RA signaling mutants. Thus, RA signaling is involved in the developmental cascade necessary for UE formation and GT development.

Keywords: Bone morphogenetic protein (Bmp); Sonic hedgehog (Shh); genital tubercle; retinoic acid; urethral plate epithelium.

Fig. 1

RA signaling activities are detected during GT development. A–L are ventral views of GT; A′–L′ are midline sagittal sections of the caudal urogenital region. A–D and A′–D′: Raldh2 is expressed in the caudal ventral region adjacent to the GT at E10.5, but not in the urethral plate epithelium (UE, enlarged view in A′) (A and A′); its expression begins in the UE from E12.5 (B and B′), becoming stronger in the UE at E13.5 and E14.5 (C, C′, D and D′). Arrows in A–D indicate the UE. The areas in between the dashed lines in A′–D′ indicate the UE. E–H and E′–H′: Cyp26b1 is expressed in the mesenchyme adjacent to the distal ectodermal epithelium of the GT at E11.5 (E and E′), becomes broadly expressed in the mesenchyme at E12.5 (F and F′); and its expression becomes restricted to the mesenchyme dorsal of the UE in the proximal region and in the distal mesenchyme of the GT at E13.5 and E14.5 (G, G′, H, and H′). Black arrowheads indicate Cyp26b1 expression in the proximal GT. I–L and I′–L′: Rarb is expressed in the caudal region adjacent to the proximal GT at E11.5 and E12.5 (I, I′, J, and J′). Its expression begins in the UE and mesenchyme of the prospective corpus cavernosum penis in the proximal GT areas at E13.5 to E14.5 (K, K′, L, and L′). White arrowheads indicate Rarb expression in the prospective corpus cavernosum penis of the GT. Scale bar, 400 μm. Arrows with dashed stems in D′, H′, and L′ indicate the ventral–dorsal direction of the GT. M: schematic picture for ventral view of E14.5 GT. N: schematic picture for midline sagittal view of E14.5 GT. GT, genital tubercle; RA, retinoic acid.

Fig. 2

Abnormal cell proliferation and differentiation in the GTs of Cyp26b1^−/− mutants. A and B: Frontal view of an E18.5 female GT in a control embryo and Cyp26b1^−/− mutant. The size of the GT in the mutant is enlarged compared with that of the control. C, C′, D, and D′: Transverse section of the proximal GT in an E18.5 female; C′ and D′ are enlarged views of the framed area (dashed black line) in C and D. The development of the preputial gland (black arrowhead) and hair follicles (white arrowheads) under the genital skin is impaired in the Cyp26b1^−/− mutants; mesenchymal condensation of the prospective corpus cavernosum penis is abnormal in the mutants (white arrow); ectopic blood vessels (area indicated by white frame) develop in the bilateral and dorsal mesenchyme of the UE in the mutants (C′ and D′); epithelium (black arrow) between the glans and prepuce is absent in the mutant (C, C′, D, and D′). E and F: Blood vessels indicated by Laminin immunostaining developed ectopically (hollow arrow) in the dorsal and bilateral mesenchyme of the UE at E14.5 in the mutant. To show the images of the mesenchyme on the dorsal side of the UE, the picture is displayed with an angled orientation, indicated by arrows. G and H: BrdU immunostaining of an E14.5 GT section. I: Cell proliferation assay. Framed area in G and H was counted, and the ratio of BrdU-positive cells was compared between the control and the mutant at E13.5 (n = 6) and E14.5 (n = 6). The asterisk (*) indicates significant statistical differences. All images of the cross-section are placed ventral side down unless otherwise indicated. Arrow with dashed stem in E indicates the ventral–dorsal direction of the GT. Scale bar, 200 μm. GT, genital tubercle; UE, urethral plate epithelium; BrdU, 5-BromodeoxyUridine.

Fig. 3

Altered expression of the homeobox genes, Shh and Bmp signaling factors in the GTs of Cyp26b1^−/− mutants. A and B: Rarb expression is upregulated in the dorsal and bilateral mesenchyme of the UE as well as in the prospective corpus cavernosum penis in Cyp26b1^−/− mutants. C and D: Expression level of Hoxa13 is upregulated in the mesenchyme around the urethra in the Cyp26b1^−/− mutants compared with control. E and F: Expression level of Hoxd13 is increased in the mesenchyme of the GT. G and H:Meis2 expression level is decreased in the ventral bilateral mesenchyme of the UE (indicated by black arrows) in Cyp26b1^−/− mutants. I–L: Shh expression level in the UE is increased in the mutant, both in mRNA (I and J) and in protein (K and L) levels. M and N: Patched1 (Ptc1) expression is upregulated in the bilateral mesenchyme of the UE in the mutant. O and P: Bmp4 expression is upregulated in the bilateral mesenchyme of the UE in the mutant. Q and R: pSmad1/5/8 expression is upregulated in the ventral region of mutant GTs. All images of the GT section are placed ventral side down. Scale bar, 400 μm. UE, urethral plate epithelium; GT, genital tubercle; Shh, Sonic hedgehog; Bmp, bone morphogenetic protein.

Fig. 4

Prompt alterations of expression for Shh and Bmp4 induced by RA administration. A–E: Rarb expression level is upregulated 2 hr after RA administration (A and B). Such upregulated expression continued for 4 hr (C), and decreased 6 hr (D and E) after RA treatment. F–I: Shh expression level is increased 2 hr (F and G) after RA treatment, sustained at high levels for 6 hr (H and I), and slightly decreased 8 hr after RA treatment (J). K–O: Ptc1 expression is unaltered within 4 hr of RA treatment (K–M), then prominently increased 6 and 8 hr after RA treatment (N and O). P–T: Bmp4 expression is upregulated 2 hr after RA treatment, and sustained with increased level until 8 hr after RA treatment. U and V: Cyp26b1 expression is upregulated in the GT mesenchyme and ectoderm epithelium 6 hr after RA administration. W and X: Meis2 expression in the ventral bilateral mesenchyme of the UE (indicated by black arrows) is reduced 6 hr after RA treatment. All images are placed ventral side down. Scale bar, 400 μm. GT, genital tubercle; RA, retinoic acid; UE, urethral plate epithelium.

Fig. 5

Impaired RA signaling reduces Shh and Bmp4 expression levels in the GT. A: The Isl1^MerCreMer mouse line shows Tamoxifen (TM)-induced Cre expression in the UE. B and C: Rarb expression in the UE is reduced in the Isl1^MerCreMer/+:RARaDN^F/F mutant. D and E: Shh expression in the UE is reduced in the Isl1^MerCreMer/+:RARaDN^F/F mutant. F and G: Ptc1 expression in the bilateral mesenchyme of the UE is reduced in the Isl1^MerCreMer/+:RARaDN^F/F mutant. H and I: Rarb expression in the GT mesenchyme is reduced in the Gli1^CreERT2/+:RARaDN^F/F mutant. J and K: Bmp4 expression in bilateral mesenchyme of the UE is reduced in the Gli1^CreERT2/+: RARaDN^F/F mutant. L and M: Ptc1 expression in the bilateral mesenchyme of the UE is unaltered in the Gli1^CreERT2/+: RARaDN^F/F mutant. All images of the cross-section are placed ventral side down. Scale bar, 200 μm. GT, genital tubercle; RA, retinoic acid; UE, urethral plate epithelium; Bmp, bone morphogenetic protein.