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Differentiation 91 (2016) 78–89

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Differentiation

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Review article Investigation of sexual dimorphisms through mouse models and /hormone-disruptor treatments

Lerrie Ann Ipulan a,1, Dennis Raga a,1, Kentaro Suzuki a, Aki Murashima a, Daisuke Matsumaru a, Gerald Cunha b, Gen Yamada a,n a Department of Developmental Genetics, Institute of Advanced Medicine, Wakayama Medical University (WMU), Wakayama, Japan b Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, USA article info abstract

Article history: in mouse reproductive tissues is observable in adult, post-natal, and embryonic Received 8 November 2015 stages. The development of sexually dimorphic tissues starts with an ambisexual structure. It is followed Accepted 11 November 2015 by -specific organogenesis as guided by different signaling pathways that occur from late embryonic Available online 2 December 2015 stages. The measurement of the anogenital distance (AGD), and the observation of the external genitalia Keywords: are practical ways to distinguish male and pups at and thereafter. Careful observation of the Reproductive tissue morphological or histological features and the molecular signatures of the external genitalia and peri- Sexual dimorphism neum enable identification of sex or /masculinization of embryos. Aberrations in hormone Mouse Cre lines signaling via castration or treatment with or hormone disruptors result in dysmorphogenesis Hormone of reproductive tissues. Several hormone disruptors have been used to modulate different aspects of Hormone disruption hormone action through competitive inhibition and exogenous hormone treatment. Concomitantly, the Perineum vast advancement of conditional mutant mouse analysis leads to the frequent utilization of Cre re- External genitalia combination technology in the study of reproductive/urogenital tissue development. Mouse Cre-lines that are tissue-specific and cell-specific are also effective tools in identifying the molecular mechanisms during sexually dimorphic development. Cre-lines applicable to different cell populations in the prostate, seminal vesicles, testis and , and mammary glands are currently being utilized. In the external genitalia and perineum, Cre lines that examine the signaling pathways of cells of endodermal, ecto- dermal, and mesenchymal origin reveal the roles of these tissues in the development of the external genitalia. The interaction of hormones and growth factors can be examined further through a variety of techniques available for researchers. Such cumulative information about various technologies is sum- marized. & 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Contents

1. Introduction ...... 79 2. Sexual dimorphism in mouse reproductive tissues ...... 79 2.1. Sexual dimorphism of external reproductive tissues at post-natal stages ...... 80 2.2. Sexual dimorphism of external reproductive tissues at embryonic stages ...... 80 3. Experimental techniques in hormonal modulation and sexual dimorphism ...... 81 3.1. Cessation of androgen signaling by gonadectomy and hormone inhibition experiments ...... 81 3.2. Hormonal modulators are essential tools to determine the mechanisms of in model animals ...... 82 3.3. Critical time window for hormonal modulation and sexual dimorphism...... 82 3.4. Usefulness of animal models and application to studies: variations and limitations ...... 82 4. Transgenic mouse lines for investigating reproductive structures ...... 82 4.1. The use of transgenic mouse lines for the investigation of external genitalia and perineum ...... 84

n Corresponding author at: Department of Developmental Genetics, Institute of Advanced Medicine, Wakayama Medical University (WMU), Wakayama, Japan. E-mail addresses: [email protected], [email protected] (G. Yamada). 1 Equal contributing author. http://dx.doi.org/10.1016/j.diff.2015.11.001 Join the International Society for Differentiation (www.isdifferentiation.org) 0301-4681/& 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved. L.A. Ipulan et al. / Differentiation 91 (2016) 78–89 79

4.1.1. GT outgrowth abnormalities ...... 84 4.1.2. Hypospadias-like phenotype and ventral GT hypoplasia ...... 84 4.1.3. Perineum Defects...... 87 5. Conclusion ...... 87 Disclosure statement...... 87 Acknowledgment ...... 87 References...... 87

1. Introduction Information about different hormone and hormone-disruptors is also discussed together with technical tips in utilizing such che- The is essential for the continuity of spe- micals and their interpretation. cies. The differences and the complimentary of male and female reproductive systems provide optimal , ensure re- production, and facilitate the continuous caring for the offspring. 2. Sexual dimorphism in mouse reproductive tissues The female reproductive system is composed of ovaries, oviducts, uterus, and vagina. Other sexual characteristics include The development of reproductive tissues starts with bipotential mammary glands and deposition in the torso. The male re- primordial organ formation. It is followed by a sexually dimorphic productive system is composed of testes, epididymes, vas de- developmental stage, which is governed by molecular pathways ferens, urethra, seminal vesicles, prostate gland, bulbourethral leading to morphologically different male and female structures. glands, and . Fig. 1 summarizes the timeline of the bipotential stage, the stage of The study of reproductive tract tissues requires a multi- molecular sexual differences, and the stage of observable mor- disciplinary approach. The morphological changes and effects of phological dimorphism of reproductive tract organs. The onset of hormone balance on developing male and female reproductive molecular sexual differences refers to the stage wherein differ- tracts distinguish them from other organ systems. Disruptions in ences in signaling or gene expression in such tissues can be de- hormone signaling affect both the morphology and subsequent tected, which is clearly a moving target subject to continued im- adult physiological functions of reproductive tract tissues. As such, provement in analytical techniques. Some detailed histological factors related with stage-dependent perturbations, tissue-speci- differences and cellular differentiation may occur concomitant ficity, and proper mouse models in the development of such tis- with molecular sexual differences or shortly thereafter. - sues must be considered. logical dimorphism is defined as a stage wherein structural dif- review aims to present technical information for the in- ferences are apparent. vestigation of male and female reproductive tracts such as ob- The and the testis, which originate from the genital ridge, servable sexual dimorphisms, timeline of development, and ap- are the first structures to undergo molecular sexual differentiation propriate mouse Cre lines for conditional mutagenesis (with par- (Koopman et al., 1991; Wilhelm and Koopman, 2006). The testes ticular focus on external genitalia and perineum formation). then secrete hormones (, anti-Mullerian hormone) that

Fig. 1. Timeline of development and sexual dimorphisms of reproductive structures. This figure summarizes the approximate timepoints in the development of reproductive structures, which are divided into (i) the bipotential stage, the phase of ambisexual organ formation; (ii) the stage of initiation of molecular sexual differences, the phase of observed differences in signaling pathways/gene expression patterns with subtle histological differences; and (iii) the stage of morphological sexual dimorphisms. (AGD – anogenital distance, BC – bulbocavernosus). 80 L.A. Ipulan et al. / Differentiation 91 (2016) 78–89 initiate or regulate male accessory organ development (Franco and 2.1. Sexual dimorphism of external reproductive tissues at post-natal Yao, 2012). This implies the essential role of hormones in sexually stages dimorphic development. The cloaca divides into the urogenital si- nus (UGS) and rectal/anal compartments. After the differentiation of Mice at early post-natal stages can be sexed according to the the , the Wolffian ducts (WD) and Mullerian ducts (MD) AGD (Fig. 2c–d), inguinal mammary teats, and the small darkened form within the urogenital ridge at embryonic day 13.5 (E13.5) in region in the perineum (Fig. 2c–d), which is applicable for non- both male and female embryos (Kobayashi et al., 2011; Murashima albino mice (Schneider et al., 1978). All other developmental et al., 2015, 2011). In male embryos, the Mullerian ducts degenerate characteristics in the early post-natal stage such as weight gain, as a result of action of anti-Mullerian hormone, while in female lanugo, growth, incisor eruption, and opening of the eyelids embryos the Wolffian ducts degenerates as a consequence of the showed non-sex-biased distinctions (Greenham and Greenham, absence of androgens. After E16.0 the MDs of form the 1977). The AGD of female albino pups have a range between oviduct, uterine horns, cervical canal and upper vagina in female. 1.0 and 2.1 mm with a 1.6 mm mean, while male pups have an The lower vagina or sinus vagina is formed from the urogenital AGD ranging from 1.8 to 2.8 mm with a mean of 2.3 mm (Green- epithelium, which is subsequently replaced by Mullerian epithe- ham and Greenham, 1977). Such measurements apply to pups in the age range of 0–3 days post-natal day (PND). The margin for lium (Kurita, 2010, 2011). In males, the WDs differentiate into the error in using AGD to determine sex of neonatal pups is 14.5% for epididymes, vas deferens, and seminal vesicles (Staack et al., 2003). females and 1.8% for males (Greenham and Greenham, 1977). Si- The UGS forms the prostate and bulbourethral gland during late milarly in non-albino mice, the male AGD is approximately twice embryonic stages. Identification of essential stages in reproductive as long as that of the female when examined at PND1 (Schneider tract development, together with proper skills for distinguishing et al., 1978). Between PND 5 and 10, the presence of inguinal teats such structures is necessary for investigating sexual dimorphisms as can be used as a criterion in segregating male and female pups. A described previously (Staack et al., 2003). more pronounced external genitalia (sometimes referred to as The formation of mammary placodes starts at E10. The sexual genital papilla) is also observed in males in post-natal stages differences in histology and signaling pathways are detected start- (Fig. 2a blue arrow). ing at E14.0. However, the regression of male mammary placodes and formation in females are observable in late embryonic 2.2. Sexual dimorphism of external reproductive tissues at embryo- stages to post-natal stages (PNS). The continued growth in female nic stages mammary glands occurs during (Hennighausen and Ro- binson, 2001; Macias and Hinck, 2012; VanHouten et al., 2003). The protrusion of the bipotential GT is observed at E11.5 (Su- Sexual dimorphism of the external genitalia and perineum are zuki et al., 2002). No sexually dimorphic features in the GT can be observed from mid-embryogenesis (Ipulan et al., 2014a; Suzuki observed between E11.5 and E15.5. Recently, sexing of embryonic et al., 2002; Yamada et al., 2003), which continues into late em- mouse is possible based on the expression of Mafb (v-Maf avian bryonic and post-natal stages. Hence, sexing of the mouse usually musculo aponeurotic fibrosarcoma oncogene homolog B) through relies on these parameters. The adult male mouse is distinguished the Mafb-GFP knock-in mouse line (Suzuki et al., 2014). In the by virtue of a penis, a longer anogenital distance (AGD) in the male mouse embryos at E14.5, GFP signals are prominently ob- perineum and the presence of the scrotal sac (Cunha and Baskin, served at the proximal or lower half of the GT and associated 2004). In the neonatal and adult female mouse, a shorter AGD, and perineum. Thus, it is possible to identify masculinization or fem- vagina are observed. The different parameters for identifying inization of the GT at early stages through gene expression analysis sexual dimorphism of the external reproductive organs at em- before morphological dimorphism is apparent. Other develop- bryonic and post-natal stages are discussed below. mental genes known to be differentially expressed in male and

Fig. 2. Sexual dimorphism of post-natal and embryonic mouse external genitalia and perineum. A larger genital papilla (arrow in a,b) and longer AGD (blue line in c,d) are observed in PND3 males. Male perineum exhibits a striation pattern (yellow arrow in e,f) indicative of midline fusion at E15.5. A well-developed prepuce (blue lines) (g,h) and S-shaped urethra (I,j) are observed in male at E16.5. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) L.A. Ipulan et al. / Differentiation 91 (2016) 78–89 81 female GT are Dkk2 (Dickkopf 2), Sfrp1 (secreted Frizzled-related conditions such as reduced AGD, persistent cloaca, cleft phallus, protein 1), B-cat (B-catenin), Cyp1b1 (Cytochrome P450,Family 1, hypospadias, as well as epididymal hypoplasia (Murashima et al., Subfamily B,Polypeptide 1), and Fkbp51 (FK506-binding protein 2015; Virtanen and Adamson, 2012; Hsieh et al., 2008; Baskin, 51) (Miyagawa et al., 2009a; Nishida et al., 2008). 2007; Hutson et al., 2010). The development of urogenital struc- The AGD is sexually dimorphic starting at E15.5 (Ipulan et al., tures such as the prostate, Wolffian duct, perineum and the genital 2014b). By scanning electron microscope (SEM), the male exhibits a tubercle are androgen-dependent (reviewed in Murashima et al. longer perineum with observable medial striations indicating (2015)). “midline fusion” (Fig. 2e–f), being more prominent at later stages Experimental parameters need to be defined to clearly evaluate (E16.5) (Fig. 2g). The use of the AGD measurement as a method to the effects of hormonal induction or ablation on the development of discriminate male from female is widely known in adult stage with reproductive/urogenital organs. AGD, malformation of the phallus, different methods of measurement. The striation pattern of the testicular descent, prostate bud number, seminal formation and perineum is one possible method of assessing dimorphism in em- epididymal coiling are among the published parameters used in bryos . evaluating the effects of hormonal regulation. AGD is the distance The male GT is morphologically distinguishable from the female from the center of the external genitalia (genital papilla) to the GT starting at E16.5 (Suzuki et al., 2002). The male ventral GT un- proximal end of the anus (Hotchkiss and Vandenbergh, 2005; An- dergoes a rapid growth leading to a well-developed prepuce thony and Manno, 2008)(Fig. 2c and d). AGD is a sexually di- (Fig. 2g). Moreover, the male GT exhibits midline fusion of the pre- morphic index that reflects in utero exposure to androgens result- putial/urethral folds leading to the formation of the tubular urethra. ing to a longer distance in males as compared to females (Bornehag The urethra of the male also exhibits an S-shaped structure within et al., 2015, Salazar-Martinez et al., 2004, Swan et al., 2015, Mitchell the (Fig. 2i–j). This may be due to the differential growth of et al., 2015). Prostate bud distribution and patterning are responsive the perineal region between males and females. The bulboca- to androgen signaling (Allegeier et al., 2010; Freestone et al., 2003). vernosus (BC) muscle is more evident in males at E16.5 (Ipulan et al., The degree of epididymal coiling and seminal vesicle formation is 2014b). The male BC muscles continue to increase in size while this also influenced by androgens thus reflecting the degree of mascu- muscle remains under-developed until birth in female. linization in male reproductive/urogenital organs (Welsh et al., Overall, sexual dimorphism is observable from E15.5 onwards 2007, 2006; Shima et al., 1990). Androgenic, estrogenic, and anti- by examining the perineum and from E16.5, through examination androgenic compounds have been suggested to regulate testicular of the prepuce and urethral formation. The continuous growth of descent (reviewed in Biason-Lauber (2010), Virtanen and Adamson the male GT and BC muscle is another marker of masculinization. (2012) and Kaftanovskaya et al. (2012)).

3.1. Cessation of androgen signaling by gonadectomy and hormone 3. Experimental techniques in hormonal modulation and inhibition experiments sexual dimorphism Hormonal ablation is an essential tool for investigating the Experimental systems have been employed to investigate hor- roles of sex hormones in development of reproductive/urogenital monal involvement during sexually dimorphic development of organs. Reproductive tracts can be investigated in castrated ani- male and female reproductive tracts. These techniques are generally mals treated with oil vehicle or androgens (Sugimura et al., 1986; classified into ablation of endogenous hormones (gonadectomy Donjacour and Cunha, 1988). An alternate approach is to culture such as castration or ovariectomy and in vitro organ culture) and reproductive/urogenital organs in vitro in medium with or without ligand activity modulation by administration of exogenous hor- androgens, or to transplant organ rudiments beneath the renal mone modulators (Fig. 3). Disruption of hormonal signaling can capsule or subcutaneously into castrated or -intact host alter sexual differentiation resulting to specificpathologic animals treated with hormone modulators (Ma et al., 2009;

Fig. 3. Experimental systems used in the investigation of hormonal modulation and sexual dimorphism of the reproductive/urogenital organ formation. 82 L.A. Ipulan et al. / Differentiation 91 (2016) 78–89

Murakami, 1986; Uemura et al., in press). Tissues grown in vitro in embryonic rats, flutamide exposure at E17–19 reduced seminal hormone-regulated environments allow controlled investigations vesicle size and branching relative to control. Similarly, flutamide of a specific ligand's action. treatment at E17 induced a high incidence of hypospadias, vaginal pouch and cleft prepuce indicating a critical window of sensitivity 3.2. Hormonal modulators are essential tools to determine the me- to androgen action on reproductive/urogenital organ development chanisms of sexual differentiation in model animals (Foster and Harris, 2005). A critical time window (E15.5 to E17.5) has been identified in rats exposed to flutamide that relates to Exposure to exogenous hormones may disrupt normal devel- AGD, prostate, Wolffian duct and phallic development (Welsh opment of reproductive/urogenital organ formation and differ- et al., 2007, 2008). The timing of administration of hormone entiation. Well-studied hormonal modulators are generally clas- modulators is critical suggesting the temporal susceptibility of the sified as estrogenic, anti-androgenic or compounds with de- reproductive/urogenital organs to different hormonal modulators. masculinizing effects. The phenotypes observed in laboratory an- imals exposed to exogenous hormones or hormone modulators 3.4. Usefulness of animal models and application to human studies: can be compared to the phenotypes of controls and certain variations and limitations spontaneous urogenital disorders. Anti-androgens administered perinetally cause changes in reproductive organ weight, altered Animal models have advantages and limitations as experi- prostate morphology and dysgenesis and hypoplasia of seminal mental systems. Animal models that mimic human diseases allow vesicles, epididymis, and external genitalia including hypospadias investigations that are potentially applicable to human cases. (Metzdorff et al., 2007). Flutamide is a non-steroidal synthetic Teratological societies have suggested a set of parameters to anti-androgen, and prenatal flutamide treatment of male rats re- evaluate the applicability of animal models to study human dis- duced AGD to the female size range (Yamada et al., 2000; McIntyre eases (Wise et al., 1997; Makris et al., 2009). Although animal et al., 2001, Suzuki et al., 2002, Welsh et al., 2008). Hypospadias, models are commonly utilized to study reproductive/urogenital cleft prepuce, epispadias, abnormal seminal vesicles, and reduced tissue abnormalities, these models do not always reflect human prostate size and UGS bud number are frequently reported in conditions (reviewed in Cunha et al. (2015)). Reproductive/ur- prenatally flutamide-treated rats (Foster and Harris, 2005, Im- ogenital anomalies observed in hormone modulated animal perato-McGinley et al., 1992, Allegeier et al., 2010). Finasteride is models may aid in elucidating the mechanisms and incidences of an inhibitor of 5α-reductase (Sudduth and Koronkowski, 1993; pathologic phenotypes observed in (Hsieh et al., 2008; Dallob et al.,2013; Mahendroo et al., 2001), the enzyme that con- Baskin, 2007; Virtanen and Adamson, 2012). Animal var- verts to the more potent androgen, dihy- iation and sensitivity to hormone modulators are important fac- drotestosterone (Pham and Ziboh, 2002). Finasteride treatment tors affecting the outcome of the reproductive/urogenital tract reduced AGD (Bowman et al., 2003) and induced hypospadias malformations. Rats are commonly utilized as animal models in (Clark et al., 1990,). Prenatal and neonatal exposure to diethyl- the investigations of the effects of flutamide and other anti-an- stilbestrol (DES) resulted in feminized external genitalia, hypos- drogens (McIntyre et al., 2001; Foster and Harris, 2005; Bowman padias, reduced phallus weight and length, preputial abnormality, et al., 2003; Welsh et al., 2008). Different strains of mice are used and smaller BC (Mahawong et al., 2014a, b; Blaschko et al., 2013; specifically in the investigation of effect of the , DES. Goyal et al., 2005; Kim et al., 2004). Administration of DES to C57BL/6J and CD1 mice have been utilized to investigate on the neonatal mice altered the expression of genes that mediate cell effects of DES (Mahawong et al., 2014a, b; Blaschko et al., 2013; proliferation and differentiation (Miyagawa et al., 2004; Maha- Rodriguez et al., 2012; Miyagawa et al., 2004; Laronda et al., 2013). wong et al., 2014a, b; Rodriguez et al., 2012; Kurita et al., 2004; Yin CD-1 mice exhibit a lower rate of DES-induced urethral anomalies et al., 2012). Wnt and Hox genes are known targets of DES during (Mahawong et al., 2014a, b) consistent with its reduced sensitivity embryonic stages inducing reproductive organ malformations to estrogen (Spearow et al., 1999). However, exogenous estrogen (Miyagawa et al., 2011). Hormonal treatment can be used to ana- and testosterone treatment experiments commonly utilize differ- lyze hormone dependent growth factor signaling (Miyagawa et al., ent animal species and strains (Yucel et al., 2003; He et al., 2015; 2009a, b; Welsh et al., 2008) in developing reproductive/ur- Allegeier et al., 2010). ogenital organs. Despite restrictions in acquiring human data, the tacit (but in many cases unproven assumption) is that actions of exogenous hormone modulators in animal models are predictive 4. Transgenic mouse lines for investigating reproductive of pathological processes in humans. structures

3.3. Critical time window for hormonal modulation and sexual Application of genetic manipulation in animal models has been dimorphism an effective tool for the study of developmental processes. The discovery of the inducible Cre loxP system enables spatial and The expression of mesenchymal and epithelial androgen re- temporal control of gene modification (Feil et al., 1996, 1997). This ceptors (AR) plays crucial roles in regulating cell proliferation and greatly aids in clarifying the specific roles of different genes and migration contributing to development of sexual dimorphism of signaling pathways during development. Additionally, the sig- the urogenital tract (Suzuki et al., 2002; Murashima et al., 2015; nificance of different cell populations in the developmental pro- Cunha et al., 2005). Mice exposed to dihydrotestosterone (DHT) as cess was elucidated through the use of cell-specific Cre lines. early as E12.5 to PND1 (Lin et al., 2003, Allegeier et al., 2010, Ro- Table 1 lists representative examples of tissue-specific Cre lines driguez et al., 2012) or as early as E13.5 utilized for the study of different reproductive tissues. (Suzuki et al., 2014, Miyagawa et al., 2009a, b) exhibit a masculi- Normal testicular function relies on the proper development of nized female urethra. Such treatment also induced developmental the Sertoli cells, Leydig cells, and cells. Leydig cells produce regulators, which are necessary to establish a sexually dimorphic testosterone, while Sertoli cells provide a structural barrier to reproductive/urogenital tract. Flutamide treatment at E13.5 or protect and secrete factors that nourish developing sperm. The E14.5 results in normal male phenotypes, while a later treatment development of the testes can be manipulated at an early devel- period (E15.5 or E16.5) results in demasculinized male genital opmental stage through Sertoli- and Leydig cell-specific Cre lines tubercle (Miyagawa et al., 2009a, b, Welsh et al., 2008). In by utilizing the AMH-Cre (anti-Mullerian hormone) and AMHR2- Table 1 Summary of mouse Cre lines for the study of reproductive tissues development.

Reproductive organ Cre mouse lines Target cell types Reported Cre activity Ref.

Testis AMH-Cre Sertoli cells E15.0 Initial Cre recombination Lecureuil et al., 2002 AMHR2-Cre Leydig cells E11.5 Initial Cre recombination Xu et al., 2007; Jamin et al., 2002 SM22a-Cre Smooth muscles/peritubularmyoid cells E17.5 Initial Cre recombination Welsh et al., 2009; Regan et al., 2000 Wolffian duct (WD) AP2a-Cre WD epithelial cells (some also in mesenchyme) E14.5 Observed Cre/LacZ expression Murashima et al., 2011; Macatee et al., 2003 Hoxb7-Cre WD epithelial cells E9.5 Initial Cre recombination Yu et al., 2002; Okazawa et al., 2015 ..Iua ta./Dfeetain9 21)78 (2016) 91 Differentiation / al. et Ipulan L.A. Mullerian duct (MD) Amhr2-Cre Mesenchyme surrounding MD E12.5 Initial Cre recombination Jamin et al., 2002 Wnt7a-Cre MD epithelial cells E12.0 Initial Cre recombination Huang et al., 2014; Winuthanayanon et al., 2010 Prostate Probasin-Cre Prostate epithelial cells PND1 Observed Cre/LacZ expression Maddison et al., 2000 ARR2PB-Cre/ARR2PBi-Cre Prostate epithelial cells PND1 Observed Cre/LacZ expression Wu et al., 2001; Jin et al., 2003 Nkx3.1-Cre Prostate epithelial cells E17.5 Initial Cre recombination Wu et al., 2011; Stanfel et al., 2006 FSP1-Cre Fibroblast PNS (post natal stage) Observed Cre/LacZ expression (Yu et al., 2012); Bhowmick et al., 2004 Seminal Vesicles ARR2PB-Cre Fibromuscularstroma 8-week old Observed Cre/LacZ expression Wu et al., 2001 ARR2PBi-Cre Epithelial cells 8-week old Observed Cre/LacZ expression Jin et al., 2003 MMTV-Cre Epithelial cells 3 month old Observed Cre/LacZ expression Wagner et al., 2001 Bulbourethral gland NKX3.1-Cre Epithelial cells E17.5 Initial Cre recombination Wu et al., 2011 Mammary gland BLG-Cre Mammary epithelial cells and Observed Cre/LacZ expression VanHouten et al., 2003 WAP-Cre Mammary epithelial cells Pregnancy and lactation Observed Cre/LacZ expression Wagner et al., 1997 MMTV-Cre Mammary epithelial cells PND 6 Initial Cre recombination Wagner et al., 2001 Fsp-Cre Mammary stroma 8 week-old Observed Cre/LacZ expression Trimboli et al., 2009

AP2-Cre Mammary adipose tissues PNS (virgin mouse) Observed Cre/LacZ expression Morroni et al., 2004 – Mammary epithelial cells During pregnancy 89

Ovary Amhr2-Cre Granulosa cells E17.5 Initial Cre recombination Jorgez et al., 2004 Gdf9i-Cre Primordial follicles and of all follicular stages PND 3 Initial Cre recombination Lan et al., 2004 Zp3-Cre of all follicular stages expect for primordial follicles PND 5 Initial Cre recombination de Vries et al., 2000 Msx2-Cre Secondary follicles and late follicular stage PND 12 (late PNS) Initial Cre recombination Lan et al., 2004 Vagina Pax2-Cre Mullerian vagina epithelial cells P0 Observed Cre/LacZ expression Kurita, 2010 Osr1-Cre Sinus vagina epithelial cells E17.5 Initial Cre recombination Grieshammer et al., 2008; Kurita, 2010 83 84 L.A. Ipulan et al. / Differentiation 91 (2016) 78–89

Cre (anti-Mullerian hormone receptor), respectively (Lécureuil (Wagner et al., 2001). BLG-Cre (B-lactoglobulin) (VanHouten et al., et al., 2002; Xu et al., 2007). A population of cells known as 2003) and WAP-Cre (Whey acidic protein) (Wagner et al., 1997) peritubular myoid cells that surround the seminiferous tubules is target luminal secretory mammary epithelial cells and are suitable also essential for . These cells are marked through for investigating gene functions in mammary gland during lacta- SM22a-Cre (smooth muscles actin) at late embryonic stage to tion and late pregnancy. It is important to note the MMTV-Cre adulthood (Regan et al., 2000; Welsh et al., 2009). targets all mammary epithelial cells (myoepithelial cells and lu- The epithelial cells of the Wolffian ducts develop into the epi- minal epithelial cells), while BLG-Cre and WAP-Cre strategies tar- didymes. The function of genes expressed in epididymal epithelial get luminal mammary epithelial only. The stroma of the mammary cells can be analyzed through epididymal-specific Cre such as gland is labeled using the FSP-Cre at PNS (8 week old), while the AP2a (activating enhancer binding protein 2 alpha)-Cre (Macatee adipocytes are marked using aP2-Cre(adipocyte binding protein 2). et al., 2003; Murashima et al., 2011) and Hoxb7-Cre (Okazawa Interestingly, the aP2-Cre also marks mammary epithelial cells et al., 2015; Yu et al., 2002). In the female, investigations on the during pregnancy indicating possible transdifferentiation from development of the Mullerian duct (MD) can be achieved through adipocyte to epithelia occurring in the mammary gland (Morroni analyzing the MD epithelia or surrounding mesenchyme by uti- et al., 2004). lizing Wnt7a-Cre (Wingless-Type MMTV Integration Site Family, Member 7A) (Huang et al., 2014; Huang et al., 2013; Winuthaya- 4.1. The use of transgenic mouse lines for the investigation of ex- non et al., 2010) and Amhr2-Cre (Jamin et al., 2002), respectively. ternal genitalia and perineum During vaginal development, the epithelium of the Mullerian va- gina is marked through different MD markers, while the sinus The GT is composed of different tissues derived from several vagina can be targetted through Osr1-Cre at E17.5 (Grieshammer lineages. The urethra originates from the endoderm. The me- et al., 2008; Kurita, 2010). senchyme of the GT is of lateral plate mesodermal origin and the Investigation of other male accessory organs such as the outer skin is derived from the ectoderm (Ipulan et al., 2014a; Ya- prostate, seminal vesicles, and bulbourethral glands utilize epi- mada et al., 2003). Hence, investigations on the growth and dif- thelial or mesenchymal Cre lines (Cooke et al., 1987a, b; Table 2). ferentiation of the GT rely on the morphogenesis of these tissues Among all the accessory organs, the prostate is one of the most with corresponding molecular pathways. Tissue-specific gene- studied structures. The study of the prostate requires the dissec- targeting in the GT is necessary to identify the roles of each sig- tion of the role of epithelial, fibroblasts, and smooth muscle cells naling pathway. Table 2 shows a summary of mouse Cre lines used in its development. Nkx3.1 (NK3 homeobox 1) and Fsp1 (fibroblast in the previous studies of GT development. The use of these Cre specific protein 1) Cre lines are utilized for investigating relevant lines is discussed based on the prevalent phenotypes for the signaling pathways in epithelia and fibroblast cells, respectively, at analysis of GT and perineum development. embryonic stages (Bhowmick et al., 2004; Prins and Putz, 2008; Stanfel et al., 2006; Wu et al., 2011). Probasin-Cre (PB-Cre) and 4.1.1. GT outgrowth abnormalities different forms of this the Cre line (ARR2PB-/ARR2PBi-Cre line) can The outgrowth of the bipotential GT starts at E11.5. Perturba- be utilized for post-natal signaling events in the prostatic epithe- tions in signaling derived from the GT endoderm and mesenchyme lium. Some slight variations of Cre activity are detected in these before this stage usually results in defective GT outgrowth. The three Probasin Cre lines. PB-Cre utilizes the basic Probasin pro- tamoxifen-inducible Shh (Sonic hedgehog) mutation through the moter which exhibits moderate expression in the ventral prostate Shh-Cre leads to defective GT outgrowth when tamoxifen treat- (Maddison et al., 2000). The ARR2PB-Cre, composed of a compo- ment was performed at E9.5. Furthermore, tamoxifen treatment at site Probasin promoter, with two androgen responsive elements, E11.5–E12.5 results in ventral GT hypoplasia (Lin et al., 2009; expresses higher levels of Cre especially in the lateral prostate and Miyagawa et al., 2009a). ShhCreERT2;AR (androgen receptor)flox/Y some Cre expression in fibroblasts of the seminal vesicles (Wu (Miyagawa et al., 2009b) and ShhCreERT2;Fgfr1/2 (fibroblast growth et al., 2001). The ARR2PBi-Cre, which includes an insulator ele- factor 1/2)flox/flox (Harada et al., 2015) mutants treated with ta- ment, exhibits higher and uniform Cre activity in all prostate lobes moxifen at E9.5 exhibit reduced AR and Fgf signaling in the en- and epithelia of the seminal vesicles (Jin et al., 2003). Of note doderm with no observable outgrowth defects. This further sug- about Probasin-based Cre lines is that the expression of the gests that Shh-Cre could be utilized to ablate endoderm specific transgene can only be induced postnatally. signals and introduction of mutation at E9.5 enables analyzing During ovarian development, differentiation of granulosa cells signals for GT outgrowth. and their function can be assessed at late embryonic stages using Aberration in the mesenchyme contributing to the GT also re- the Amhr2-Cre line (Jorgez et al., 2004). The granulosa cells secrete sults in outgrowth defects as is the case for Dermo1(or Tiwst2, estrogen and other factors that support oocyte development. Twist family factor 2)Cre;B-catflox/flox (Lin et al., 2008) Using Amhr2-Cre, granulosa cells can be targeted for genetic ma- and Isl1(islet 1)Cre;Fgfr1/2flox/fox (Harada et al., 2015). The expres- nipulation as early as E17.5. Oocytes at various stages of follicular sion of Isl1 is widely observed in the posterior part of the embryo development can be manipulated using appropriate Cre lines such as early as E9.5. The Isl1-expressing cells contribute to the bladder, as Gdf9i-Cre (growth differentiation factor 9), Zp3-Cre (Zone pel- rectum, hindlimb and GT. Early tamoxifen treatment (E8.5–9.5) of lucida protein 3), and homeobox gene, Msx2-Cre. The Gdf9i-Cre Isl1(islet 1)Cre;Fgfr1/2flox/fox mice results in Isl1 contribution to the marks all oocytes at the primordial follicular stage to late follicular hindlimb, bladder and dorsal GT. Later tamoxifen treatment stage (Lan et al., 2004). Zp3 (de Vries et al., 2000) and Msx2 (Lan (E10.5–11.5) reduced the contribution of such cells to the bladder et al., 2004) marks oocytes at different follicular stages but not and limb, while increased contribution is observed in the GT primordial germ cells. (Suzuki et al., 2012). Hence, the Isl1-Cre line could be utilized for The development of mammary gland is frequently studied after analyzing caudal embryo morphogenesis and GT. birth during early postnatal and late pregnancy stages. It is com- posed of epithelial cells, fibroblasts (stroma) and adipose tissues in 4.1.2. Hypospadias-like phenotype and ventral GT hypoplasia varying proportions depending on the stage of development. The Hypospadias is a human birth defect characterized by an ec- MMTV-Cre (mouse mammary tumor virus) is utilized to mark topic urethral meatus located in proximal regions of the mammary epithelial cells which enable identification of essential penis (Cunha et al., 2015). Associated phenotypes include under- signaling in such cells during the pubertal growth of developed foreskin, bending of the penis (also known as chordee), Table 2 Summary of mouse Cre lines and mutant lines for investigating the development of the mouse GT and perineum.

Mouse Cre line/ Mutant Mutant phenotype Important timepoints Lineage/Expression pattern Ref. n Reporter

Cre flox/flox GT ectoderm Wnt7a-Cre Wnt7a ; Bcat Urethral cleft E10.5 – detected Cre- Marks the penile skin Mazahery et al. (2013) recombination E13.5 – detected Cre- GT ectoderm recombination E14.5 – phenotype observed Cre flox/flox Wnt7a ;Fgfr1/2 Prepuce hyplopasia E16.5 – phenotype observed Harada et al. (2015) Disrupted ectodermal urethral E14.5 – phenotype observed epithelium Cre flox/flox Msx2-Cre Msx2 ;Bcat2 GT outgrowth defects and proximal E10.5 – detected Cre GT ectoderm Lin et al. (2008) hypospadias recombination fl K5-Cre K5Cre ;AR ox/Y No obvious defect E15.5 – loss of AR detected Miyagawa et al. (2009b) n Bmp7 Bmp7 LacZ/LacZ Cloacal epithelial abnormality E10.5 – LacZ detected Marks dorsal mesenchyme and ventral Xu et al. (2012) endoderm of GT and URS mesenchyme Wu et al. (2009) ..Iua ta./Dfeetain9 21)78 (2016) 91 Differentiation / al. et Ipulan L.A. CreERT2 flox/flox GT endoderm Shh-Cre Shh ;Shh Defective GT outgrowth E9.75 – tamoxifen treatment for Marks the penile urethra and cloacal Miyagawa et al. (2009b) and Lin Cre-recombination endoderm et al. (2009) GT ventral hypoplasia E11.5–E12.5 – tamoxifen treat- ment for Cre-recombination No obvious phenotype E13.5 – tamoxifen treatment for Cre-recombination CreERT2 flox/Y – Shh ;AR No obvious phenotype E9.5 tamoxifen treatment for Miyagawa et al. (2009a) Cre-recombination fl fl ShhCreERT2 ;Fgfr1/2 ox/ ox differentiation abnormalities in the E9.5 – tamoxifen treatment for Harada et al. (2015) endodermal epithelium Cre-recombination þ n Ephrin-B2LacZ/ Reduced perineum, hypospadias, and Adult – observed phenotype Dravis et al. (2004) incomplete cloacal septation E16–E17 – LacZ observation Marks urethral endoderm (and sur- rounding mesenchyme) n ki/ki / – EphB2-Bgal EphB2 ;EphB3 Slightly open cloaca and un- E17 phenotype observed – tubularized urethra 89 Ki-kinase inactive E13 – LacZ detected Marks anorectal endoderm (and sur- rounding mesenchyme) CreERT2 Foxa2-Cre Foxa2 E7.5–9.5 – tamoxifen treatment Marks GT and perineum endoderm Ipulan (unpublished) for Cre-recombination E10.5–E11.5 – tamoxifen treat- Marks GT endoderm only ment for Cre-recombination CreERT2 Foxa2 ;dNBcat hypoplastic URS and hypospadias E15.5 – phenotype observed

GT mesenchyme Gli1-Cre Gli1CreERT2 E8.75–9.0 – tamoxifen treat- Marks dorsal GT mesenchyme Haraguchi et al. (2007) ment for Cre-recombination fl Gli1CreERT2 ;AR ox/Y hypoplastic external genitalia E10.5 – tamoxifen treatment for Marks mesenchyme around the Miyagawa et al. (2009a, b) Cre-recombination urethra underdeveloped prepuce CreERT2 flox/Y Sall1-Cre Sall1 AR2 hypospadias E11.5 – tamoxifen treatment for Marks mesenchyme around the Suzuki et al. (2014) Cre-recombination urethra MafB-GFP MafB GFP-knockin mutants (KO) hypospadias E14.5 – observed sexually di- Marks mesenchyme around the Suzuki et al. (2014) morphic expression of MafB urethra E16.5 – phenotype observed fl fl

Cre ox/ ox 85 Isl1-Cre Isl1 ;Bmp42 Sirenomelia, urogenital dysgenesis, E9.5 – detected Cre- Marks caudal embryo Suzuki et al. (2012) hypoplastic external genitalia recombination nnn Isl1Cre-MCM E8.5–9.5 – tamoxifen treatment Marks hindlimb, dorsal GT and bladder Table 2 (continued ) 86

Mouse Cre line/ Mutant Mutant phenotype Important timepoints Lineage/Expression pattern Ref. n Reporter

for Cre-recombination E10.5–11.5 – tamoxifen treat- Marks GT mesenchyme and bladder ment for Cre-recombination fl fl Cre ;Fgfr1/2 ox/ ox GT outgrowth defects E12.5 – phenotype observed Harada et al. (2015) Isl1 fl fl Hoxa3-Cre Hoxa3Cre ;Isl1 ox/ ox Hypoplastic cloacal mesenchyme E8.5 – detected Cre Marks caudal embryo Suzuki et al., (2012) recombination Defective hindlimb formation Cre flox/flox Dermo1-Cre Dermo1 ;Bcat2 Hypoplastic GT E10.5 – detected Cre GT mesenchyme Lin et al. (2008) recombination Six1-Cre E10.5 – observed Six1 Wang et al. (2011) expression nn Six1-Cre hpAP/LacZ E10.5 – E11.5 detected Cre Cloacal mesenchyme recombination E13.5–E15.5 – detected Cre GT mesenchyme recombination ..Iua ta./Dfeetain9 21)78 (2016) 91 Differentiation / al. et Ipulan L.A. Cre flox/Y – Perineum Mck-Cre MCK;AR2 No observable phenotype E13.5 detected Cre Marks BC muscles Ipulan et al. (2014b) recombination fl Sall1-Cre Sall1CreERT2 ;AR ox/Y Defective BC formation E11.5 – tamoxifen treatment for Marks non-myocytic cells Ipulan et al. (2014b) Cre-recombination Reduced AGD Cre flox/Y Rarb-Cre Rarb ;AR Cryptorchidism E14.5 – detected Cre Marks the gubernaculum Kaftanovskaya et al. (2012) and recombination Kobayashi et al. (2005)

n Reporter mouse line. nn hpAP – human placental Alkaline phosphatase. nnn Cre-MCM – mer-Cre-mer, Cre recombinase and two mutated estrogen receptor domains. – 89 L.A. Ipulan et al. / Differentiation 91 (2016) 78–89 87 and undescended testis in varying degrees and frequency (Baskin reproductive organs in males have an equivalent structure in fe- et al., 1998). The commonly observed hypospadias-like phenotype males such as the uteri as female and the vagina as female in mouse is the lack of tubular urethra accompanied by ventral GT prepuce. It is now possible to discuss the development of male and hypoplasia. A severe form of hypospadias exhibits a persistent female reproductive organs with greater depth and complexity. cloaca due to the incomplete cloacal separation (Matsumaru et al., Although these structures are derived from common anlage, the 2015). Hypospadias-like phenotype is a common outcome of critical stages of their development and the essential events aberrations in the endoderm, the mesenchyme surrounding the leading to the sexual dimorphisms are now being clarified. This is endoderm, and/or the ectoderm of the GT. Severe hypospadias-like in part due to the availability of new technology, such as Cre re- phenotypes are observed with early aberrations of signaling in the combination, that enables the dissection of several tissue- and fl endoderm as observed in ShhCreERT2;Shh ox/fox (Miyagawa et al., stage-specific molecular pathways. Similarly, castration studies 2009a) and Foxa2 (forkhead box a2)CreERT2;dNBcat (constitutively and treatment with hormones or hormonal disruptors are useful active B-catenin mutant). Aberrations in ectoderm signaling in the for identifying the role of hormone signaling during specific stages fl Msx2Cre;Bcat ox/fox (Lin et al., 2009), Bmp7 (bone morphogenetic of development. Continuous researches on the hormone-de- protein 7), LacZ/LacZ (Wu et al., 2009; Xu et al., 2012), pendency/independency, molecular signaling and cellular inter- fl Wnt7aCre;Bcat ox/fox (Mazahery et al., 2013) and actions, with the help of the above-integrated techniques, will fl Wnt7aCre;Fgfr1/2 ox/fox (Harada et al., 2015) mutants results in a facilitate researchers in uncovering more aspects of reproductive relatively milder hypospadias-like phenotype. tissue development. Hypospadias-like phenotype is also observed in mutants with defective signaling in the mesenchyme surrounding the en- doderm. Mutants of Gli2 (Gli family zinc finger 2)–/–, Gli1(Gli fa- Disclosure statement mily zinc finger 1)CreERT2;ARflox/Y, Sall1 (Spalt-like transcription CreERT2 flox/fox factor) AR , and Mafb KO exhibit defective tubular ure- The authors have nothing to declare. thra formation with unclosed prepuce (Miyagawa et al., 2011, 2009b; Suzuki et al., 2014). Gli1-Cre;ARflox/Y shows hypoplastic ventral GT and unclosed prepuce. MafB is expressed in a popula- Acknowledgment tion of cells surrounding the urethra in male which is reported to be regulated by androgen signaling (Suzuki et al., 2014). Mutants We would like to extend our gratitude to Drs. Larry Baskin, CreERT2 flox/Y of MafB and Sall1 Cre AR , which both result in loss of Ryuichi Nishinakamura, and Richard Behringer for their support signaling in the mesenchyme surrounding the urethra, exhibit and expertize. We would also like to acknowledge the contribu- hypospadias. Loss of signaling in both the endoderm and its sur- tions of Drs. Shinichi Miyagawa, Ryuma Haraguchi, Akiko Omori, rounding mesenchyme results to hypospadias which is observed Ahmad Mazahery and Masayo Harada. We also thank Iola Iba, and in the case of Ephrin mutants (Table 2; Dravis et al., 2004; Yucel all laboratory colleagues for their assistance. This work is sup- et al., 2007). ported by Challenging Exploratory Research 15K15403 and Sci- Overall, these observations suggest a possible signaling inter- entific Research (B) 15H04300 from the Ministry of Education, action between the endoderm and its surrounding mesenchyme. It Culture, Sports, Science, and Technology of Japan. is also likely that formation of the urethra depends on the growth of the mesenchyme surrounding it. In summary, hypospadias-like phenotype is observed when perturbations in endoderm and the References surrounding mesenchyme occur.

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