Retinoic acid signaling determines the fate of uterine stroma in the mouse Müllerian

Tadaaki Nakajimaa,b, Taisen Iguchia,c, and Tomomi Satoa,1

aGraduate School of Nanobioscience, Yokohama City University, Yokohama 236-0027, Japan; bDepartment of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585, Japan; and cOkazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki 444-8787, Japan

Edited by Jan-Åke Gustafsson, University of Houston, Houston, Texas, and approved November 3, 2016 (received for review June 1, 2016)

The Müllerian duct develops into the oviduct, , and , promoted by cytochrome P450 26A1 (CYP26A1), CYP26B1, and all of which are quite distinct in their morphology and function. CYP26C1 (17–19). In adult mice, Dhrs9, Aldh1a1, Cyp26,and The epithelial fate of these female reproductive organs in developing Aldh1a2 are expressed in the uterus (20–22), whereas Aldh1a1, mice is determined by factors secreted from the stroma; however, Aldh1a2,andCyp26 are not detected in the vagina (20). These how stromal differentiation occurs in the female reproductive organs data suggest that RA signaling may act in the adult uterus. derived from the Müllerian duct is still unclear. In the present study, The necessity of vitamin A and its derivatives in Müllerian duct roles of retinoic acid (RA) signaling in developing female reproduc- development was first observed when fetuses from pregnant rat dams reared on vitamin A-deficient diets exhibited agenesis or incomplete tive tracts were investigated. Retinol dehydrogenase 10 (RDH10) and − − development of the Müllerian ducts (23). Müllerian ducts in Rara / / aldehyde dehydrogenase family 1 subfamily A2 (ALDH1A2) mRNAs − − − − − − Rarb2 / or Rara / /Rxra / mice and the caudal Müllerian ducts of and and transactivation activity of endogenous RA were − − − − Rara / /Rarg / mice were absent at embryonic day 12.5 (E12.5) and found in the stroma of proximal Müllerian ducts and gradually de- E13.5 (24, 25), suggesting that RA signaling through RARs, partic- creased from the proximal to caudal regions in fetal mice. In organ- ularly RARα, is essential for Müllerian duct development. Feeding cultured Müllerian ducts, retinaldehyde or RA treatment induced vitamin A-deficient diets for 10 or 14 wk from birth causes squamous uterine epithelial differentiation, defined as a layer of columnar ep- metaplasia in the uterine (26–28), suggesting a role for ithelial cells negative for oviductal and vaginal epithelial markers. In RA signaling in maintaining the . However, the contrast, inhibition of RA (RAR) signaling induced vaginal role of RA signaling in the differentiation of the female reproductive epithelial differentiation, characterized as vaginal epithelial marker tracts from the Müllerian duct has not been elucidated. –positive stratified epithelium. Grafting experiments of the In the present study, we investigated the role of RA signaling in organ-cultured Müllerian duct revealed irreversible epithelial fate the developing mouse female reproductive tracts. First, ontogenic determination. Although RAR did not directly bind to the mRNA and expression of the RA-signaling pathway com- A10 (Hoxa10) promoter region, RA–RAR signaling stimulated Hoxa10 ponents together with the transactivation activity of endogenous RA expression. Thus, RA–RAR signaling in the Müllerian duct determines in the female reproductive tracts from embryonic to adult stages were the fate of stroma to form the future uterus and vagina. examined. To study the roles of RA signaling in developing female reproductive tracts, effects of RA or RAR antagonists on organ- Müllerian duct | oviduct | uterus | vagina | retinoic acid cultured female reproductive tracts were investigated. Finally, RAR binding to Hoxa promoter was examined to determine whether Hoxa genes were candidates for downstream genes of RA signaling. n female mice, Müllerian ducts remain and differentiate into Ioviducts, uterus, and vagina; however, they are morphologi- Results cally and functionally distinct. The oviductal epithelium consists Developmental mRNA and Protein Expression of the RA-Synthesizing of ciliated cells and secretory cells (1), whereas the uterine epithe- Enzymes, RARs, and RA-Metabolizing Enzymes in Female Reproductive lium is composed of simple columnar luminal and glandular epi- Tracts. Expression of components of the RA-signaling pathway thelia (2). The develops into a stratified cuboidal components in the neonatal uterus and vagina was investigated epithelium (3, 4). Tissue recombination experiments between the by RT-PCR. Rdh10 was expressed in the uterus and vagina at epithelium and stroma have been performed previously to investi- gate the mechanisms underlying differentiation of the epithelia from Significance the Müllerian ducts (3–5). Data from these studies indicate that factors secreted from the stroma determine the fate, differentiation, and growth of the overlying epithelia into the oviduct, uterus, and Oviduct, uterus, and vagina are all derived from the Müllerian vagina of developing mice. The epithelial fate-determining factors duct. Epithelial fate of these female reproductive organs in de- have been identified in the female reproductive tracts (4, 6–9), and veloping mice is determined by factors secreted from the stroma; posterior Hoxa genes, which are differentially expressed along the however, how the preceding stromal differentiation of female A–P axis, are important for development of the female reproductive reproductive tracts from the Müllerian duct occurs is still unclear. tracts (10); however, earlier steps in the stromal differentiation of the This study showed that retinoic acid (RA) signaling was activated female reproductive tracts from the Müllerian ducts remain unclear. in the proximal Müllerian duct, which develops into oviduct and DNA microarray analysis (11, 12) revealed that the expression uterus. Furthermore, RA treatment induced uterine stromal dif- of several genes involved in retinoic acid (RA) signaling is higher ferentiation, whereas inhibition of RA receptor signaling induced in the uterus of neonatal mice than that in the vagina. In the first vaginal stromal differentiation. Therefore, we concluded that RA step of RA synthesis, retinol is oxidized to retinaldehyde by al- establishes a border between the stroma of uterus and vagina. cohol dehydrogenases (ADHs) and retinol dehydrogenases (RDHs), mainly including RDH1, 10 and dehydrogenase/reductase (SDR Author contributions: T.N. and T.S. designed research; T.N. performed research; T.N. and family)member9(DHRS9)(13–15). RA oxidized from retinaldehyde T.S. analyzed data; and T.N., T.I., and T.S. wrote the paper. by aldehyde dehydrogenase family 1 subfamily A (ALDH1As) binds The authors declare no conflict of interest. to retinoic acid receptors (RARs). RARs form obligate hetero- This article is a PNAS Direct Submission. dimers with the retinoid “X” receptors (RXRs). The RAR/RXR 1To whom correspondence should be addressed. Email: [email protected]. heterodimer binds to RA-responsive elements (RARE) and reg- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ulates transcription of the target genes (16). Degradation of RA is 1073/pnas.1608808113/-/DCSupplemental.

14354–14359 | PNAS | December 13, 2016 | vol. 113 | no. 50 www.pnas.org/cgi/doi/10.1073/pnas.1608808113 Downloaded by guest on September 24, 2021 expression in the uterus and vagina did not show a marked change with age (Fig. S1). Rarb expression in the uterus and vagina de- creased with age. Thus, Rara and Rarg wereexpressedinboththe uterus and vagina throughout the ages examined. Because expres- sion of Rdh10 and Aldh1a2 was high in the middle Müllerian ducts at E17.5 (Fig. 2), the expression pattern in the Müllerian ducts at E14.5 and E17.5 was examined (Fig. 3A). Rdh10 expression was high in the proximal and middle Müllerian ducts, and Cyp26a1 was high in the proximal region at E17.5. Dhrs9, Aldh1a1, Aldh1a2, Rara, Rarb,andRarg expression was similar, from the proximal to caudal regions. By immunohistochemistry, slight ALDH1A2 pro- tein expression was detected as brown staining in the stroma of proximal Müllerian duct at E14.5, but not in the middle and caudal Müllerian duct (Fig. 3 B–D). RDH10 protein was not detected in the female reproductive tracts from E14.5 to P90 by immunohis- tochemistry (Fig. S2). These results indicated that RDH10 and ALDH1A2 mRNAs and proteins were expressed in the stroma of proximal Müllerian duct and decreased from the proximal to the caudal Müllerian duct. BIOLOGY

Fig. 1. mRNA and protein expression and localization of the RA-synthesizing DEVELOPMENTAL enzymes, RARs and RA-metabolizing enzymes in female reproductive tracts after birth. The mRNA expression in the uterine epithelia (UtE), uterine stroma (UtS), vaginal epithelia (VgE), and vaginal stroma (VgS) of P2 and P15 mice was analyzed by RT-PCR (A). The expression of RDH10 and ALDH1A2 proteins in the uteri and vaginae of P2 and P15 mice and oil- or E2-treated P90 mice was analyzed by Western blot (B).

postnatal day 2 (P2) and P15, and Dhrs9 was localized in the uterine and vaginal epithelium at P2 and P15, and in the vaginal stroma at P15. Rdh1 and Rdh7 were not detected (Fig. 1A); therefore, the primary producers of retinaldehyde in the uterus and vagina are probably RDH10 and DHRS9. Aldh1a1 was not expressed at P2 and mainly expressed in the uterine epithelium at P15. Aldh1a2 was expressed in the uterine stroma, vaginal epithelium, and vaginal stroma at P2 but only in the uterine stroma at P15. Aldh1a3 was not detected. Rara, Rarg, Rxra, and Rxrb were expressed in the uterus and vagina at P2 and P15, but Rarb and Rxrg were not detected. Cyp26a1 was localized in the uterine epithelium at P15, whereas Cyp26b1 was not detected in the uterus and vagina. RDH10 protein expression in the uterus at P2 was higher than that in the vagina and in the uterus at P15 and P90 (Fig. 1B). ALDH1A2 expression was similar between the uterus and vagina at P2 and P15. A single injection of 17β-estradiol (E2) increased expression of ALDH1A2, but not RDH10 in the uterus and vagina at P90. We used quantitative PCR (qPCR) to characterize temporal changes in gene expres- sion in the uterus and vagina. Rdh10 and Aldh1a2 in the uterus was significantly higher than that in the vagina from P2 to P90 (Fig. 2). In the uterus, Rdh10 was not altered from E17.5 to P90, except for a decrease at P15, and Aldh1a2 decreased at P2 and then increased with age. Thus, Rdh10 and Aldh1a2 were high in the fetal and adult uterus. Dhrs9, Aldh1a1, and Cyp26a1 in the uterus and Dhrs9 in the vagina increased with age. At P15 and Fig. 2. Developmental mRNA expression of the RA-synthesizing enzymes and RA-metabolizing enzymes in female reproductive tracts. The mRNA 90, Aldh1a1 and Cyp26a1 expression in the uterus was signifi- expression in the middle Müllerian duct of E17.5 mice, and uterus and cantly higher than that in the vagina. At P90, E2 treatment sig- vagina of P2 and P15 mice, and P90 mice at diestrus or estrus, or treated with nificantly stimulated Dhrs9 and Aldh1a2 expression in the uterus oil or E2, was analyzed by qPCR. Data were expressed relative to mRNA and Aldh1a2 and Cyp26b1 in the vagina. In contrast, E2 treat- expression of the middle Müllerian duct of E17.5 mice (=1.0). aP < 0.05 ment blunted Aldh1a1, Cyp26a1, and Cyp26b1 in the uterus and compared with the expression in the uterus of E17.5 mice or vagina of Rdh10, Dhrs9, Aldh1a1, and Cyp26a1 in the vagina (Fig. 2). Thus, P2 mice (temporal changes). *P < 0.05 compared with the expression in the expression of a variety of genes encoding RA biosynthetic vagina of age-matched mice (uterus vs. vagina). #P < 0.05 compared with the and catabolic enzymes is regulated by . Rara and Rarg expression in the uterus and vagina of oil-treated P90 mice (effects of E2).

Nakajima et al. PNAS | December 13, 2016 | vol. 113 | no. 50 | 14355 Downloaded by guest on September 24, 2021 Müllerian duct (Fig. S4 K and N). As expected, RA treatment stim- ulated RA signaling in the stroma of E14.5 and E17.5 Müllerian ducts organ cultured for 8 d (Figs. S5 A–J and S6). In contrast, treatment with the pan-RAR antagonist, AGN193109 (AGN), inhibited RA signaling in the stroma of the Müllerian duct in the presence or absence of RA, whereas AGN treatment alone partially increased RA signaling in the epithelia of caudal Müllerian ducts (Fig. S6P).

Effects of RA or RAR Antagonists on Stromal Differentiation in the Organ-Cultured Müllerian Ducts. Because no marker gene for stromal differentiation in Müllerian ducts has yet been identified, expression of stroma-induced epithelial markers (4, 5) was taken as an indication of stromal differentiation. Organ-cultured E14.5 Müllerian ducts, including proximal and middle regions treated with RA, displayed no overt changes, whereas epithelia treated with AGN differentiated into a vagina-like stratified epithelium, accompanied by expression of the vaginal epithelial marker transformation related protein 63 (TRP63) (Fig. S5 K and L). However, the differentiated vaginal epithelialmarker,keratin14(KRT14),wasnotdetectedintheAGN- treated epithelia, suggesting that differentiation into a vaginal epithe- lium was incomplete (Fig. S5 M–P). At E17.5, the middle Müllerian duct treated with retinaldehyde or RA did not show apparent changes (Fig. 5 B and F and Fig. S7 B and F), whereas caudal ep- ithelia treated with retinaldehyde or RA differentiated into − − TRP63 and KRT14 uterus-like simple columnar epithelia (Fig. 5 + N and R and Fig. S7 N and R). AGN treatment induced TRP63 + and KRT14 vagina-like stratified epithelia in middle Müllerian ducts, and caudal epithelia treated with AGN expressed KRT14

Fig. 3. mRNA and protein expression and localization of the RA-synthesizing enzymes, RARs, and RA-metabolizing enzymes in female reproductive tracts of fetal mice. The mRNA expression in the proximal and middle Müllerian duct of E14.5 mice, and the proximal, middle, and caudal Müllerian duct of E17.5 mice was analyzed by qPCR (A). Data were expressed relative to mRNA expression of whole Müllerian ducts of E14.5 mice (=1.0). *P < 0.05. The protein localization of ALDH1A2 in the proximal (B), middle (C), and caudal (D) Müllerian ducts of E14.5 mice and in the uterus of P90 mice as a positive control (E) was analyzed by immunohistochemistry (n = 5). (Scale bar, 50 μm.) Dashed line, basement membrane; e, epithelium; s, stroma.

Transactivation Activity of Endogenous RA in the Female Reproductive Tracts. Endogenous levels of RA have not previously been mea- sured in female reproductive tracts. Expression of well-known RA-responsive genes was examined in the uterus and vagina (Cyp26a1 in Fig. 2; Rarb in Fig. S1;othersinFig. S3); however, the expression of those genes was inconsistent. Thus, we used a re- porter mouse expressing lacZ under the control of a strong RARE (29) to investigate the temporal and spatial distribution of active RA signaling in the Müllerian duct. The β-galactosidase activity was histochemically detected from E14.5 to P15 (Fig. 4). In the stroma of proximal and middle Müllerian duct, β-galactosidase activity was highly detected from E14.5 to P0, slightly detected at P2, and not detected by P15 (Fig. 4 A–E). From E17.5 to P2, β-galactosidase activity in the proximal Müllerian duct (Fig. 4 G–I) was higher than that in the middle portion (Fig. 4 K–M). In the caudal Müllerian duct and vagina, β-galactosidase activity was not detected (Fig. 4 N–Q). Thus, the levels of biologically active, en- dogenous RA were highest in the stroma of proximal Müllerian ducts and gradually decreased from the proximal to caudal regions.

Effects of RA or an RAR Antagonist on Activation of RA Signaling in Fig. 4. Transactivation activity of endogenous RA in the female reproductive Organ-Cultured Müllerian Ducts. To investigate the role of RA sig- tracts. β-Galactosidase activity in whole female reproductive tracts (A–E)and naling in the Müllerian duct stroma, RA and/or RAR antagonists the sagittal section of female reproductive tracts (F–Q)ofE14.5(A, F, J,andN), were added to the medium of organ-cultured Müllerian duct of E17.5 (B, G, K,andO), P0 (C, H, L,andP), P2 (D, J, M and Q), and P15 (E)RARE-lacZ RARE-lacZ mice at E14.5 and E17.5. Absence of mice was analyzed by X-gal staining (n = 3). [Scale bar, 1 mm (A and B), (AR)-expressing Wolffian ducts (Fig. S4 A and B) and paired box μ – − 2mm(C and D), 3 mm (E), and 50 m(F Q). Solid arrows, predicted area of 2 (PAX2) epithelium of the urogenital sinus in the organ-cultured oviducts at E14.5 and E17.5 or oviducts from P0 to P15; dashed arrows, pre- Müllerian ducts (Fig. S4 C–N) were confirmed by immunohisto- dicted area of uteri at E14.5 and E17.5 or uteri from P0 to P15; arrowheads, − chemistry, except for PAX2 partially stratified epithelium of caudal vaginae; dashed line, basement membrane; e, epithelium; s, stroma.

14356 | www.pnas.org/cgi/doi/10.1073/pnas.1608808113 Nakajima et al. Downloaded by guest on September 24, 2021 Müllerian ducts had oviduct-like epithelia consisting of β-tubulin + + (TUBβ) ciliated cells or oviductal glycoprotein 1 (OVGP1) secretory cells (Fig. 6 E–G and Fig. S10 I and J) and uterus-like + epithelia (Fig. 6G). AGN treatment irreversibly induced TRP63 vagina-like epithelia in host mice, irrespective of E2 treatment (Fig. 6 C and D). KRT14 was expressed in the vagina-like epithelium in E2-treated host mice (Fig. S10B), but not in oil-treated host mice (Fig. S10A). PGR was not detected in the vagina-like epithelium in the oil-treated host mice (Fig. S10E), indicating that the PGR expres- sion pattern was similar to vaginal epithelium (2). These data sug- gested that AGN treatment irreversibly induced vaginal epithelium. In E2-treated host mice, grafted AGN-treated Müllerian ducts did not have oviduct-like epithelia (Fig. 6F and Fig. S10J). Cotreatment with RA + AGN canceled the effects of AGN on induction of vaginal epithelium (Fig. 6 C and D and Fig. S10B) and inhibition of oviductal epithelium (Fig. 6F and Fig. S10J). RA treatment appeared to decrease the size of the smooth muscle actin (SMA) expression domain in both oil- and E2-treated animals and this effect was blocked by AGN (Fig. S10 K and L). Grafted, organ- cultured E17.5 middle Müllerian ducts at day 30 postgrafting had only uterus-like epithelia (Fig. 6L). AGN treatment irreversibly + induced TRP63 vagina-likeepitheliainthepresenceorabsenceof E2 (Fig. 6 J and K). In the vagina-like epithelia, E2 treatment BIOLOGY DEVELOPMENTAL

Fig. 5. Effects of RA or RAR antagonists on stromal differentiation in the organ-cultured Müllerian duct. The immunohistochemistry of TRP63 (A–D and M–P) and KRT14 (E–L and Q–X) in the organ-cultured middle (A–L)or caudal (M–X) Müllerian duct of E17.5 mice with or without 1 μMRA(B, F, J, N, R, and V), 1 μMRA+ 2(C, G, and K), or 10 μM(O, S, and W) AGN or 10 μM AGN (D, H, L, P, T, and X)for8d.n = 5–8. [Scale bar, 50 μm(A–H and M–T) and 200 μm(I–L and U–X). e, epithelium; s, stroma.

strongly (Fig. 5 D, H,andT). In middle Müllerian ducts, BMS195614 (BMS; RARα antagonist) treatments induced only a few TRP63- expressed epithelial cells (Fig. S7C, arrows). In contrast, treatments with LE135 (LE; RARβ antagonist) or MM11253 (MM; RARγ antagonist) did not appear to affect morphology (Fig. S8). Co- treatment with RA + AGN (Fig. 5 C and G)orRA+ BMS (Fig. S7D) canceled the effects of AGN or BMS, and AGN canceled the effects of RA (Fig. 5 O, S,andW). The organ-cultured P15 uterus treated with RA or AGN showed no morphological changes (Fig. S9 B and G). RA treatment decreased KRT14 expression in the vaginal epithelium, but it did not affect TRP63 expression (Fig. S9 D and I). AGN treatment increased the number of epithelial layers in the vagina, but it did not affect TRP63 and KRT14 expression (Fig. S9 E and J). These data suggested that retinaldehyde or RA treatment induced uterine epithelium in the Müllerian duct via activation of RA signaling in the stroma at the developing stage, whereas inhi- bition of RAR signaling induced vaginal epithelium.

Modulating RA Signaling Irreversibly Alters Stromal Differentiation in the Female Reproductive Tracts. To investigate the role of RA signaling in the differentiation of female reproductive tracts, RA and/or RAR antagonists were added to the medium in organ- Fig. 6. Modulating RA signaling irreversibly alters stromal differentiation in cultured Müllerian ducts at E14.5 or E17.5, and those tissues were the female reproductive tracts. HE staining (A, B, H, I, M,andN)andimmu- then grafted under the renal capsules of host mice. Epithelia of the nohistochemistry of TRP63 (C, D, J, K, O,andP)andOVGP1(E and F)inthe α organ-cultured Müllerian duct including proximal and middle regions in grafted Müllerian ducts expressed (ESR1) and – – – – – E14.5 mice (A G) or organ-cultured middle (H L) or caudal (M Q)Müllerianduct receptor (PGR) (Figs. S10 C F and S11 C F and of E17.5 mice with or without RA and AGN at 30 d postgrafting treated with oil – + M P) and showed cell proliferation in response to E2 treatment or E2. (Scale bar, 50 μm.) Arrows, TRP63 cells induced by AGN treatment − (Figs. S10 G and H and S11 G, H, Q,andR), suggesting that the in vitro; arrowheads, TRP63 area induced by RA treatment in vitro. The per- grafted Müllerian ducts retained characteristics of the female centage of oviduct-like (Ov), uterus-like (Ut), and vagina-like ducts (Vg) per all reproductive tracts. At day 30 postgrafting, the grafted E14.5 grafted ducts in the sections is shown (G, L,andQ). e, epithelium; s, stroma.

Nakajima et al. PNAS | December 13, 2016 | vol. 113 | no. 50 | 14357 Downloaded by guest on September 24, 2021 Rdh10 and Aldh1a2 were highly expressed in the proximal and middle Müllerian duct in fetal mice. Furthermore, RDH10 or ALDH1A2 protein expression in the uterus or the proximal Mül- lerian duct was also higher than that in the vagina or caudal Müllerian duct. The phenotypes of Adh or Rdh1 knockout mice are nonlethal, whereas a point mutation in Rdh10 impairs RA synthesis and causes embryonic lethality by E13.0 (31). RDH10 oxidizes retinol on the , but ADHs do not, suggesting that RDH10 is the primary enzyme responsible for the first step of RA synthesis in embryonic mice (31). Our results are consistent with a model in which RA is synthesized from retinol via RDH10 and ALDH1A2 in the stroma of Müllerian ducts. RARs and RXRs were expressed in the fetal and neonatal female reproductive tracts. Endogenous RA activated an RAR-dependent reporter gene in the stroma of proximal and middle Müllerian ducts but it was not de- tected by P15. Therefore, RA was synthesized, and transcription by RAR was activated in the stroma of proximal and middle Müllerian Fig. 7. Schematic model for determination of the borderline between ducts. Uterine CYP26A1 activity is important for im- uterine and vaginal stroma by RA–RAR signaling in the Müllerian duct. plantation in pregnant mice (32); however, Cyp26a1 expression was low in the Müllerian duct, suggesting that RA was not catabolized in the Müllerian duct. Expression of Dhrs9, Aldh1a1,andCyp26b1 induced KRT14 expression (Fig. S11B), whereas PGR was not de- was confirmed in the adult uterus and/or vagina, and it was regu- tected in the oil-treated host mice similar to those in vaginal epithelia lated during the or by E2 treatment (20–22). Thus, (Fig. S11E). Grafted, organ-cultured E17.5 caudal Müllerian ducts these enzymes might act in the adult uterus and/or vagina. + + had TRP63 and KRT14 vagina-like epithelia (Fig. 6 O and P RA treatment induced uterine epithelial differentiation in the and Fig. S11 K and L). RA treatment permanently induced uterus- Müllerian duct, whereas inhibition of RAR signaling induced vaginal + + – like epithelia, but it did not induce TUBβ or OVGP1 oviduct-like epithelial differentiation, suggesting that presence of RA RAR sig- epithelia (Fig. 6Q). These results indicated that RA–RAR signaling naling in the Müllerian duct determined the fate of the uterine and vaginal stroma. RA or RAR antagonist treatment at P0 (Fig. S14)or permanently induced the uterine epithelium in the Müllerian E17.5 affected TRP63 and KRT14 expression, whereas RA or RAR ducts. Because RA treatment did not induce oviductal differ- antagonist treatment at P15 did not alter TRP63 expression. There- entiation in the organ-cultured caudal Müllerian duct, even fore, RA–RAR signaling is able to regulate the uterine and vaginal after grafting, we hypothesized that the fate of oviductal stroma differentiation by P0 (Fig. S15). Retinaldehyde treatment also in- had been already determined by E14.5. To investigate this hy- duced uterine differentiation, butretinoltreatmentdidnot(Fig. S14), pothesis, the intact proximal and middle E14.5 Müllerian ducts indicating that the stroma of the caudal Müllerian duct or neonatal were grafted. At day 30 postgrafting, the proximal duct grafts + + vagina could synthesize RA from retinaldehyde, but not from retinol. had only TUBβ or OVGP1 oviductal epithelia, whereas Thus, RDH10 is a key enzyme for uterine stromal differentiation. middle Müllerian ducts had only uterine epithelia (Fig. S12 A– Compared with robust effects of AGN (an inverse agonist that both E). Furthermore, (Hoxa10) expression in the antagonizes the effects of RA and promotes RAR-mediated re- middle Müllerian duct at E14.5 was already higher than that in pression of target genes) (33), BMS slightly stimulated vaginal dif- the proximal Müllerian duct (Fig. S12F). ferentiation, suggesting that it may act as a partial agonist or neutral RARα antagonist (which does not promote target gene repression) Indirect Transcriptional Regulation of Hoxa Genes by RA Signaling in the (34).However,RARβ or RARγ antagonists might affect morphol- − − − − − − − − Müllerian Duct. Expression of Hoxa genes as candidate downstream ogy. In Rara / /Rarb2 / or Rara / /Rxra / mice, the Müllerian duct effectors of RA signaling was investigated in organ-cultured E14.5 is absent at E12.5 and E13.5 (24, 25). Taken together, RARα may be Müllerian duct. Hoxa10 and Hoxa11 expression in RA-treated the most important RAR for the uterine differentiation. Müllerian ducts was slightly higher than that in vehicle-treated ducts Grafting experiments showed that characteristics of the uterine (Fig. S13A). AGN cotreatment abrogated the effects of RA treat- epithelium induced by RA and of the vaginal epithelium induced ment. Hoxa9 and Hoxa13 expression was not changed by RA by RAR antagonist in the Müllerian duct were retained at 30 d treatment. The Hoxa10 promoter region from −2,000 to 1,000 bp postgrafting, irrespective of E2 treatment. Therefore, RA–RAR contains a putative RARE by bioinformatical search (RARE score signaling irreversibly determined the fate of uterine and vaginal matrix based on ref. 30). To investigate the ability of this putative stroma. The induced epithelia in the organ-cultured P0 vagina – were sloughed after E2 treatment at 30 d postgrafting (Fig. S16), Hoxa10 RARE to recruit RAR, we used ChIP qPCR. RNA poly- – merase II bound to Hoxa10 transcription start site (TSS) both in the indicating that a critical window of RA RAR signaling for uterine proximal or middle Müllerian ducts at E14.5 (Fig. S13B). We did not differentiation is up to E17.5. Although RAR antagonist treatment inhibited oviductal differentiation at E14.5, RA treatment might detect binding of RAR to the putative Hoxa10 RARE, the Hoxa10 not affect the oviductal differentiation. At E16.5, Hoxa10 is expressed TSS, or a nonspecific region located at −3,000 kb from putative in the Müllerian duct at the boundary between future uterine and Hoxa10 RARE (Fig. S13C). Thus, we infer that RAR did not bind oviductal stroma (35). In this study, we found that Hoxa10 expression to TSS or the predicted RARE of Hoxa10 in the Müllerian duct. is already high in the middle at E14.5 and that the E14.5 proximal or Discussion middle Müllerian duct differentiated into oviducts or uterus after grafting, respectively. These results suggest that the border between Results from ontogenic mRNA and protein expression of genes the future uterine and oviductal stroma is already determined at in the RA signaling pathway together with the activity of RA E14.5. Therefore, the role of RA–RAR signaling in the oviductal signaling in the female reproductive tracts showed that RA was differentiation must be investigated before E14.5. In addition, RA synthesized and acted in the stroma of proximal and middle treatment appeared to decrease SMA protein in the Müllerian duct. Müllerian duct. Addition of RA or RAR antagonists in organ- SMA protein expression is inhibited by RA in cultured proepicardium cultured Müllerian ducts and the grafting experiment revealed derived from mesoderm (36); therefore, RA signaling might inhibit that RA–RAR signaling determines the fate of the uterine the differentiation of smooth muscle in the Müllerian duct. stroma and that inhibition of this signaling is essential for the Hoxa10 expression at E14.5 was increased by RA treatment; fate determination of the vaginal stroma. however, RAR did not bind to the 5′ Hoxa10 promoter. RA

14358 | www.pnas.org/cgi/doi/10.1073/pnas.1608808113 Nakajima et al. Downloaded by guest on September 24, 2021 activates a heterodimer of RARα/RXRα to stimulate Hoxa10 collected at the diestrus or estrus as determined by vaginal smear. All experi- promoter activity (37). Therefore, in the middle Müllerian ducts, ments were approved by the animal care committee in Yokohama City RAR may bind to the upstream 5′ promoter region of Hoxa10 University. because RA also increased Hoxa11 (located in the 5′ region of Hoxa10) expression or to the 3′ region of Hoxa10 because the Organ Culture System and Grafting. Müllerian duct organ culture was per- downstream DNA sequence is also important for Hoxa genes (38), formed as described previously (42). E14.5 Müllerian duct including proximal and and a predicted RARE is located at +2,869. On the other hand, middle regions (caudal regions could not be separated), middle and caudal E17.5 Müllerian duct, and P0 or P15 uterus and vagina were cultured in a collagen gel Hoxa10 expression has already been associated with determining μ the border between oviduct and uterus (39); therefore, other fac- for 8 or 5 d, respectively with daily medium changes. A total of 10 Mall transretinol (Sigma), 10 μM all transretinaldehyde (Sigma), 1 μM all trans-RA tors can also mediate the effects of RA signaling on determination (Sigma), 2 or 10 μM AGN (Santa Cruz), 10 μMBMS(Tocris),10μMLE(SantaCruz), of the borderline between uterine and vaginal stroma. μ – or 10 M MM (Tocris) were added to the medium. Four to six organ-cultured In conclusion, the presence of RA RAR signaling in the Müllerian ducts or intact proximal or middle E14.5 Müllerian ducts were grafted Müllerian duct determined the border between uterine and under the renal capsules of C57BL/6J from P50 to P90 as described previously (4). vaginal stroma in prenatal stages (Fig. 7). The fate-determined At the time of grafting, all host mice were OVX. At 30 d postgrafting, host mice stroma then secreted factors to induce their respective epithelia. were given s.c. injections of 0.1 μg/25 g BW E2 or sesame oil for 3 d, euthanized, In female mouse embryos, RA inhibits inhibin βA expression in and tissues harvested. Two independent grafting experiments were carried out. the urogenital sinus (40), and activin A formed by the inhibin βA Assay for treatment, β-galactosidase activity, RNA isolation, RT-PCR, qPCR, is important for vaginal epithelial differentiation (7, 8). These Western blot, immunohistochemistry, ChIP assay, and statistical analysis were data support a model in which key factors regulated by RA– performed as described in SI Materials and Methods. Primers were listed in RAR signaling can play critical roles in fate determination in Table S1. Negative control for immunohistochemistry was shown in Fig. S17. epithelium and stroma of the female reproductive tracts. ACKNOWLEDGMENTS. We thank Dr. Bruce Blumberg (Department of Materials and Methods Developmental and Cell Biology, University of California, Irvine) for critical reading of this manuscript. This work was supported by the Ministry of Animals. C57BL/6J and transgenic mice, which have three RAREs linked to the Education, Culture, Sports, Science, and Technology of Japan [Grant-in-Aid Hspa1b promoter and lacZ gene (RARE-lacZ mice purchased by The Jackson for Scientific Research (B) to T.I., Grant-in-Aid for Scientific Research (C) to Laboratory) (29) were maintained in accordance with the NIH’s Guide for the T.S.] and Yokohama City University (Grants for Support of the Promotion of Care and Use of Laboratory Animals (41). At P90, uteri and vaginae were Research W18005, K2109, G2314, G2401, and IR2502 to T.S.). BIOLOGY

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