G C A T T A C G G C A T genes Review Hormonal and Molecular Regulation of Phallus Differentiation in a Marsupial Tammar Wallaby Yu Chen 1,2,* and Marilyn B. Renfree 2,* 1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32603, USA 2 School of BioSciences, The University of Melbourne, Parkville, VIC 3010, Australia * Correspondence: yuchen2@ufl.edu (Y.C.); [email protected] (M.B.R.) Received: 9 December 2019; Accepted: 14 January 2020; Published: 16 January 2020 Abstract: Congenital anomalies in phalluses caused by endocrine disruptors have gained a great deal of attention due to its annual increasing rate in males. However, the endocrine-driven molecular regulatory mechanism of abnormal phallus development is complex and remains largely unknown. Here, we review the direct effect of androgen and oestrogen on molecular regulation in phalluses using the marsupial tammar wallaby, whose phallus differentiation occurs after birth. We summarize and discuss the molecular mechanisms underlying phallus differentiation mediated by sonic hedgehog (SHH) at day 50 pp and phallus elongation mediated by insulin-like growth factor 1 (IGF1) and insulin-like growth factor binding protein 3 (IGFBP3), as well as multiple phallus-regulating genes expressed after day 50 pp. We also identify hormone-responsive long non-coding RNAs (lncRNAs) that are co-expressed with their neighboring coding genes. We show that the activation of SHH and IGF1, mediated by balanced androgen receptor (AR) and estrogen receptor 1 (ESR1) signalling, initiates a complex regulatory network in males to constrain the timing of phallus differentiation and to activate the downstream genes that maintain urethral closure and phallus elongation at later stages. Keywords: lncRNA; WGCNA; marsupial; androstanediol; RNAseq; IGF1; SHH; oestrogen; castration; phallus 1. Introduction The marsupial tammar wallaby has been used as a molecular research model to study sex determination and sexual differentiation for decades. It is a unique model to investigate sex-related molecular regulation due to its extended period of postnatal sexual differentiation. In the tammar, testicular differentiation occurs from two days after birth, while ovarian differentiation does not begin until day eight postpartum (pp) (reviewed in [1]). Although the genital tubercle is detected about two days before birth [2], sexually dimorphic phallus differentiation does not begin until day 50 pp [3] (Figure1). After day 50 pp, the anogenital distance in males becomes longer than that in females [ 2–5]. The male phallus elongates faster and the urethra begins to fuse along the ventral midline, while the female urethra remains unfused [2–5]. By day 150 pp, the urethral meatus has reached the glan penis in males, whereas in females, the urethra remains open [3,5]. Genes 2020, 11, 106; doi:10.3390/genes11010106 www.mdpi.com/journal/genes Genes 2020, 11, 106 2 of 14 Genes 2020, 11, 106 2 of 14 FigureFigure 1. 1.The The timeline timeline of of prostate prostate didifferentiation,fferentiation, phallusphallus development, development, the the androgen androgen imprinting imprinting window,window, the the androgen androgen sensitive sensitive phase, phase, and and the the insulin-like insulin-like growth growth factor factor 11 (IGF1(IGF1)) dependentdependent phasephase in thein tammar the tammar wallaby. wallaby. TheThe development development of of male male tammar tammar phallusphallus is androgenandrogen dependent dependent [5,6], [5,6 ],like like that that of ofeutherian eutherian mammals.mammals. In In males, males, there there is an is increasean increase of testicular of testic testosteroneular testosterone from birthfrom tobirth day to 40 day pp [740]. However,pp [7]. testicularHowever, testosterone testicular testosterone concentration concentration falls sharply falls after sharply day after 40 pp, day but 40 pp, is unmeasurable but is unmeasurable in ovaries, in ovaries, and plasma levels do not differ between the sexes up to day 50 [3,5,7,8]. There is a critical and plasma levels do not differ between the sexes up to day 50 [3,5,7,8]. There is a critical androgen androgen imprinting window (window of androgen sensitivity or androgen programming window) imprinting window (window of androgen sensitivity or androgen programming window) between between days 25 to 30 pp, first described in the tammar in 2004 and then identified in rats and humans days 25 to 30 pp, first described in the tammar in 2004 and then identified in rats and humans [5,9–12]. [5,9–12]. Altering androgen concentrations by castration of male young or treatment of female young Altering androgen concentrations by castration of male young or treatment of female young with the with the potent androgen androstanediol during this programming window contributes to abnormal potent androgen androstanediol during this programming window contributes to abnormal phallus phallus development, including hypospadias or phallus sex reversal [5,6]. Interestingly, although development,androgen controls including both hypospadias urethral closure or phallus and phal sexlus reversal elongation, [5,6]. the Interestingly, molecular regulation although androgenbehind controlsthese two both phases urethral can differ. closure When and males phallus are elongation,castrated at day the 25 molecular pp, their phalluses regulation are behind feminized these and two phasesthe urethra can di ffremainser. When unfused males [5]. are When castrated males at are day castrated 25 pp, theirat day phalluses 40 pp or are at feminizedday 80 pp, andtheir the urethraphalluses remains become unfused shorter [5 but]. When the treatment males are has castrated no effect on at dayurethral 40 pp closure or at [5]. day These 80 pp, results their indicate phalluses becomethat urethral shorter closure but the is treatment regulated hasby the no eandrogffect onen urethral priming, closure whereas [5 ].phallus These elongation results indicate requires that urethralconstant closure or increasing is regulated levels by of the androgen. androgen Although priming, several whereas phallus phallus regulating elongation genes requires in the constant tammar or increasinghave been levels reported, of androgen. there has Although been less several attent phallusion on regulatingthe signalling genes pathways in the tammar during havephallus been reported,development. there has been less attention on the signalling pathways during phallus development. InIn this this paper, paper, we we review review the the molecular molecular regulationregulation of of androgen androgen priming priming on on sonicsonic hedgehog hedgehog (SHH(SHH), ), insulin-likeinsulin-like growth growth factor factor 1 1(IGF1 (IGF1)) and and longlong non-codingnon-coding RNA RNA (lncRNAs), (lncRNAs), during during phallus phallus differentiation differentiation inin the the tammar. tammar. First, First, we we presentpresent the molecular molecular mechanism mechanism that that initiates initiates the thephallus phallus differentiation differentiation at atday 50 50 pp pp and, and, then, then, the theregulatory regulatory mechanism mechanism of phallus of phallus elongation elongation at day 90 at pp. day We 90 also pp. identify We also identifyhormonal hormonal responsive responsive lncRNAs lncRNAs during during phallus phallus development development in the in thetammar tammar and and describe describe their their relationshiprelationship to to their their neighboring neighboring coding coding genes. genes. 2. A2. A Unique Unique Androgen-Sensitive Androgen-Sensitive RegulationRegulation Network of of SonicSonic Hedgehog Hedgehog (SHH) (SHH) InIn the the tammar, tammar,SHH SHHexpression expressionremains remains lowlow in males when when testicular testicular testosterone testosterone is ishigh, high, but but increases after the content of testosterone (ng/mg protein) in the testes drops [7,9]. Similarly, in increases after the content of testosterone (ng/mg protein) in the testes drops [7,9]. Similarly, in phallus phallus transcriptome data (Figure 2), SHH expression increases after removing the testes, but transcriptome data (Figure2), SHH expression increases after removing the testes, but decreases in decreases in female phalluses when given androgen [13]. The negative association between SHH female phalluses when given androgen [13]. The negative association between SHH expression and expression and androgen is also seen in a lymph node carcinoma of the prostate (LNCaP) cell line androgen is also seen in a lymph node carcinoma of the prostate (LNCaP) cell line [14]. Through steroid [14]. Through steroid treatment and RNA-sequencing (RNA-seq) data analysis in the tammar, a treatmentnumber andof genes RNA-sequencing are shown to (RNA-seq) have a similar data expression analysis in pattern the tammar, to that a numberof SHH. ofSHH genes, Wnt are family shown to havemember a similar5A (WNT5A expression), and MAF pattern BZIP to thattranscription of SHH .factorSHH B, Wnt (MAFB family) are member all downregulated 5A (WNT5A in), andfemaleMAF BZIPphalluses transcription by androgen factor B treatment(MAFB) are at day all downregulated 50 pp, but are upregulated in female phalluses after castration by androgen in males treatment [13,15], at daywhile 50 pp, fibroblast but are growth upregulated factor 10 after (FGF10 castration) is upregulated in males[ 13by, 15androgen], while fibroblasttreatment, growth but downregulated factor 10 (FGF10 ) is upregulatedafter castration by in androgen males [15].