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The ability of a single genome to produce distinct and often dramatically different male and female forms is one of the wonders of animal development. In Drosophila melanogaster , most sexually dimorphic traits are controlled by sex-specific isoforms of the doublesex ( dsx ) transcription factor, and dsx expression is mostly limited to cells that give rise to sexually dimorphic traits. However, it is unknown how this mosaic of sexually dimorphic and monomorphic organs arises. Here, we characterize the cis -regulatory sequences that control dsx expression in the foreleg, which contains multiple types of sex-specific sensory organs. We find that separate modular enhancers are responsible for dsx expression in each sexually dimorphic organ. Expression of dsx in the sex comb is co-regulated by two enhancers with distinct spatial and temporal specificities that are separated by a genitalia-specific enhancer. The sex comb-specific enhancer from D. willistoni , a species that primitively lacks sex combs, is not active in the foreleg. Thus, the mosaic of sexually dimorphic and monomorphic organs depends on modular regulation of dsx transcription by dedicated cell type-specific enhancers., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Regulatory networks driving morphogenesis of animal genitalia must integrate sexual identity and positional information. Although the genetic hierarchy that controls somatic sexual identity in the fly Drosophila melanogaster is well understood, there are very few cases in which the mechanism by which it controls tissue-specific gene activity is known. In flies, the sex-determination hierarchy terminates in the doublesex (dsx) gene, which produces sex-specific transcription factors via alternative splicing of its transcripts. To identify sex-specifically expressed genes downstream of dsx that drive the sexually dimorphic development of the genitalia, we performed genome-wide transcriptional profiling of dissected genital imaginal discs of each sex at three time points during early morphogenesis. Using a stringent statistical threshold, we identified 23 genes that have sex-differential transcript levels at all three time points, of which 13 encode transcription factors, a significant enrichment. We focus here on three sex-specifically expressed transcription factors encoded by lozenge (lz), Drop (Dr) and AP-2. We show that, in female genital discs, Dsx activates lz and represses Dr and AP-2. We further show that the regulation of Dr by Dsx mediates the previously identified expression of the fibroblast growth factor Branchless in male genital discs. The phenotypes we observe upon loss of lz or Dr function in genital discs explain the presence or absence of particular structures in dsx mutant flies and thereby clarify previously puzzling observations. Our time course of expression data also lays the foundation for elucidating the regulatory networks downstream of the sex-specifically deployed transcription factors.

Steroidogenic factor 1 (SF-1/Nr5a1) is an orphan nuclear receptor encoded by the Ftz-F1 gene and is required for gonad and adrenal development and regulation of hormone production within the reproductive and adrenal axes. To extend our understanding of Ftz-F1 and its role in SF-1 expression, we identified and characterized a yeast artificial chromosome (YAC) containing Ftz-F1. Within this YAC, Ftz-F1 is centrally located and flanked by genes encoding a second orphan nuclear receptor, germ cell nuclear factor, and proteasome (prosome, macropain) subunit beta type 7. Three lines of transgenic mice carrying the YAC were generated and in two lines (lines 7 and 14), RT-PCR and ribonuclease protection analysis showed that expression of transgenic SF-1 mimicked that of endogenous SF-1, both spatially and quantitatively. In the third line (line 15), pituitary and hypothalamic expression were absent. Comparison of the integrated transgenes revealed that line 15 was truncated at the end of intron 4 and revealed a region within the locus that is responsible for SF-1 expression in the pituitary and hypothalamus. The line 14 transgene was introduced into a mouse strain lacking functional SF-1. Examination of SF-1-deficient, transgene-positive mice revealed that the YAC was able to rescue adrenal and gonad development, which normally arrests in the SF-1-null embryos and showed that the 153-kb transgene integrated in line 14 is sufficient to properly direct SF-1 expression and support its biological activity. Thus, the study defines a region of Ftz-F1 that contains the requisite set of regulatory elements to direct SF-1 cell-specific expression and all temporal and quantitative changes need for its biological activity.

Murine genes encoding the forkhead transcription factors Foxf1 and -2 are both expressed in derivatives of the splanchnic mesoderm, i.e., the mesenchyme of organs derived from the primitive gut. In addition, Foxf2 is also expressed in limbs and the central nervous system. Targeted mutagenesis of Foxf1 and -2 suggests that Foxf1 is the more important of the two mammalian FoxF genes with early embryonic lethality of null embryos and a haploinsufficiency phenotype affecting foregut-derived organs. In contrast, the only reported defect in Foxf2 null embryos is cleft palate. To investigate if the differences in mutant phenotype can be attributed to nonoverlapping expression patterns or if distinct functions of the encoded proteins have to be inferred, we analyzed the early embryonic expression of Foxf2 and compared it with that of the better investigated Foxf1. We find that in the early embryo, Foxf1 is completely dominating-in terms of expression-in extraembryonic and lateral plate mesoderm, consistent with the malformations and early lethality of Foxf1 null mutants. Along the developing gut, Foxf1 is highly expressed throughout, whereas Foxf2 expression is concentrated to the posterior part-fitting the foregut haploinsufficiency phenotypes of Foxf1 mutants. Foxf2, on the other hand, is more prominent than Foxf1 in mesenchyme around the oral cavity, as would be predicted from the cleft palate phenotype. The differences in expression pattern also highlight areas where defects should be sought for in the Foxf2 mutant, for example limbs, the posterior gut, genitalia, and derivatives of the neural crest mesenchyme.

Fushi tarazu factor-1 (FTZ-F1) genes belong to the nuclear receptor family 5A (NR5A). The distribution pattern of NR5A genes in teleosts suggests that they control functions separate to, or in addition to, those of other vertebrates. In mammals NR5A1 genes, including steroidogenic factor-1 (SF-1), are primarily involved in steroidogenesis. NR5A2 contain the alpha-fetoprotein transcription factor (FTF) genes, which protect mammalian embryos against maternal estrogens, and are involved in cholesterol transfer and metabolism. In this study we have analysed the expression of two Arctic char FTZ-F1 forms belonging to the NR5A2 group. The expression starts during early development and the transcripts are present in embryonic liver/pancreas and gonadal regions. The genes are up-regulated during embryogenesis as the embryo develops towards hatch, as shown by increased mRNA and protein levels. In adult Arctic char the FTZ-F1 forms are primarily located to tissues involved in steroidogenesis as well as cholesterol metabolism. Thus, a division of NR5A into SF-1 (NR5A1) and FTF (NR5A2) specific functions does not appear to have occurred in teleosts.

To study the process of mammalian sex determination and in particular to further understand the mechanisms of transcriptional regulation of the SRY gene, we have isolated a 4.5-kilobase (kb) pig SRY 5' flanking sequence. To facilitate the in vitro analysis of these sequences, we have generated a porcine genital ridge (PGR) cell line (9E11) that expresses SRY as well as SOX9, steroidogenic factor-1 (SF-1), and DAX1. Via primer extension analysis on RNA from this cell line, a transcription start site for porcine SRY was identified at -661 base pairs (bps) 5' from the translation initiation site. Deletion studies of the SRY 5' flanking sequences in PGR 9E11 cells demonstrated that -1.4 kb of 5' flanking sequences retained full transcriptional activity compared with the -4.5 kb fragment, but that transcriptional activity fell when further deletions were made. Sequences downstream of the transcriptional start site are important for promoter activity, because deleting transcribed but not translated sequences eliminated promoter activity. Sequence analysis of the -1.4 kb fragment identified two potential binding sites for SF-1, at -1369 and at -290 from the ATG. To address the role of SF-1 transactivation in SRY promoter activity, mutagenesis studies of the potential SF-1 binding sites were performed and revealed that these sites were indeed important for SRY promoter activity. Cotransfection studies in a heterologous cell system (mouse CV-1 cells) demonstrated that pig SF-1 was able to transactivate the pig SRY promoter. Gel shift assays confirmed that the upstream site was recognized by mouse SF-1 protein. We conclude that two sites for SF-1 transactivation exist within the pig SRY promoter, at -1369 bp and at -290 bp, and that the site at -1369 bp is quantitatively the most important.

Transcription factor GATA-4 has been suggested to have a role in mammalian gonadogenesis, e.g., through activation of the Müllerian-inhibiting substance (MIS) gene expression. Although the expression of GATA-4 during gonadogenesis has been elucidated in detail, very little is known about FOG-2, an essential cofactor for GATA-4, in ovarian development. We explored in detail the expression of FOG-2 and GATA-4 in the fetal and postnatal mouse ovary and in the fetal testis using Northern blotting, RNA in situ hybridization, and immunohistochemistry. GATA-4 and FOG-2 are evident in the bipotential urogenital ridge, and their expression persists in the fetal mouse ovary; this result is different from earlier reports of GATA-4 downregulation in the fetal ovary. In contrast to ovary, FOG-2 expression is lost in the fetal Sertoli cells along with the formation of the testicular cords, leading to the hypothesis that FOG-2 has a specific role in the fetal ovaries counteracting the transactivation of the MIS gene by GATA-4. In vitro transfection assays verified that FOG-2 is able to repress the effect of GATA-4 on MIS transactivation in granulosa cells. In postnatal ovary, granulosa cells of growing follicles express FOG-2, partially overlapping with the expression of MIS. These data suggest an important role for FOG-2 and the GATA transcription factors in the developing ovary.

Each Drosophila genital imaginal disc contains primordia for both male and female genitalia and analia. The sexually dimorphic development of this disc is governed by the sex-specific expression of doublesex (dsx). We present data that substantially revises our understanding of how dsx controls growth and differentiation in the genital disc. The classical view of genital disc development is that in each sex, dsx autonomously "represses" the development of the inappropriate genital primordium while allowing the development of the appropriate primordium. Instead, we show that dsx regulates the A/P organizer to control growth of each genital primordium, and then directs each genital primordium to differentiate defined adult structures in both sexes.

We have studied the spatial dynamics of Sry transcription in the genital ridges of mouse embryos. We find that Sry is expressed in a dynamic wave that emanates from the central and/or anterior regions, extends subsequently to both poles, and ends in the caudal pole. This dynamism may explain the relative positioning of ovarian and testicular tissue seen in ovotestes in mice. Since direct regulatory targets of SRY ought to be expressed in a corresponding or complimentary wave, our observations pave the way for identification of target genes. Sry is expressed in internal cells but not in coelomic surface epithelial cells, indicating that its effect on proliferation of surface cells is achieved non-cell-autonomously. The cellular dynamism of Sry expression revealed in this study thus provides important insights into both the cellular and molecular mode of action of SRY, and how perturbations in Sry expression can lead to anomalies of sexual development., (Copyright 2001 Wiley-Liss, Inc.)

Pax2 and Pax5 arose by gene duplication at the onset of vertebrate evolution and have since diverged in their developmental expression patterns. They are expressed in different organs of the mouse embryo except for their coexpression at the midbrain-hindbrain boundary (MHB), which functions as an organizing center to control midbrain and cerebellum development. During MHB development, Pax2 expression is initiated prior to Pax5 transcription, and Pax2(-/-) embryos fail to generate the posterior midbrain and cerebellum, whereas Pax5(-/-) mice exhibit only minor patterning defects in the same brain regions. To investigate whether these contrasting phenotypes are caused by differences in the temporal expression or biochemical activity of these two transcription factors, we have generated a knock-in (ki) mouse, which expresses a Pax5 minigene under the control of the Pax2 locus. Midbrain and cerebellum development was entirely rescued in Pax2(5ki/5ki) embryos. Pax5 could furthermore completely substitute for the Pax2 function during morphogenesis of the inner ear and genital tracts, despite the fact that the Pax5 transcript of the Pax2(5ki )allele was expressed only at a fivefold lower level than the wild-type Pax2 mRNA. As a consequence, the Pax2(5ki )allele was able to rescue most but not all Pax2 mutant defects in the developing eye and kidney, both of which are known to be highly sensitive to Pax2 protein dosage. Together these data demonstrate that the transcription factors Pax2 and Pax5 have maintained equivalent biochemical functions since their divergence early in vertebrate evolution.

The role of mammalian Hox genes in regulating segmental patterning of axial structures and the limb is well established. A similar role in development of soft tissue organ systems has recently been suggested by observations linking several 5' members of the HoxA and HoxD clusters to segmentation events and morphogenesis in the gastrointestinal and genitourinary systems. We have specifically examined the role of Hoxa-10 in development of the male accessory sex organs by characterizing expression of Hoxa-10 in the developing male reproductive tract and correlating expression to morphologic abnormalities in knockout mice deficient for Hoxa-10 function. We report that Hoxa-10 expression in the Wolffian duct and urogenital sinus is regionally restricted and temporally regulated. The domain of expression is defined anteriorly by the caudal epididymis and extends posteriorly to the prostatic anlagen of the urogenital sinus. Expression was maximal at E18 and down-regulated postnatally, well before accessory sex organ morphogenesis is completed. Expression in the prostatic anlagen of the urogenital sinus cultured in vitro does not depend upon the presence of testosterone. Loss of Hoxa-10 function is associated with diminished stromal clefting of the seminal vesicles and decreased size and branching of the coagulating gland. The ductal architecture of the coagulating gland was altered in approximately 30% of mutants examined and suggests a partial posterior morphologic transformation of the coagulating gland. We interpret these data to indicate that Hoxa-10 is expressed in a region specific manner during late gestation and into the perinatal period and that Hoxa-10 is required for normal accessory sex organ development.

We have isolated mammalian homologues of the Drosophila dachshund gene. Two domains of high conservation, one of which contains an alpha-helical, coiled-coil motif, show similarity to the Ski family of genes. We therefore propose that Dachshund belongs to a superfamily including these genes. Mouse Dachshund (Dach) is expressed in the eye and limb, structures affected by the Drosophila loss-of-function mutant, and rib primordia, CNS and genital eminence. Pax6 and Dach show overlapping but non-identical expression patterns. Dach expression is unaffected in smalleye mouse brain, indicating that Pax6 is not directly activating Dach. In Drosophila eye development dachshund is a component of an interacting network of proteins. Genes homologous to many of these exist in mammals; Dach joins this expanding group., (Copyright 1998 Elsevier Science Ireland Ltd. All rights reserved)

Wilms' tumour is a childhood kidney cancer, and a classic example of cancer arising through disrupted development (Armstrong et al., 1992). It is one of the most common solid paediatric malignancies, affecting one in 10000 children. The genetics of Wilms' tumour is complicated, with several different genes or chromosomal regions being implicated (Armstrong et al., 1992). However, the gene we know most about is the Wilms' tumour predisposition gene, WT1 (Bickmore et al., 1992; Bruening and Pelletier, 1996). It is now clear that mutations in this gene in humans can lead to abnormalities of the kidneys and gonads, as well as to the eponymous tumour. Also, as discussed below, WT1 is essential for kidney, testis and ovary development, as revealed in knockout mice.

GATA-3 is expressed in a temporally dynamic manner and fulfills vital functions during vertebrate fetal development. Homozygous mGATA-3 mutant embryos die at midgestation, thus complicating the analysis of its contribution to the development of specific cell fates in the many tissues where it is expressed during embryogenesis. We show here that the elements controlling GATA-3 regulation can be precisely refined, using transgenic mice, to discrete cis-acting domains: within 6 kb surrounding the transcriptional initiation site, separate sequences were found to control the expression of mGATA-3 in early muscle masses, in a subset of PNS neurons, in the genital tubercle, and in the branchial arches. The branchial arch regulatory element is particularly robust and was refined to a discrete enhancer sequence lying between nt -2832 and -2462 from the transcription initiation site. The enhancer contains potential binding sites for many well-characterized transcription factors, suggesting that mGATA-3 transcriptional activity may be regulated by these proteins (or related family members) in the mesenchyme of the arches that contribute to formation of the jaw. These studies show that discrete regulatory elements required for the elaboration of complex developmental programs can be individually localized, suggesting that the developmentally transient expression of individual transcription factors collaboratively contributes to the temporal and spatial pattern of cellular differentiation leading to the formation of adult anatomy.

A reverse transcription-polymerase chain reaction approach was used in an attempt to identify a chick homologue to Sry. Gene transcripts from the genital ridge were analysed at the stage of development at which the expression of such a gene might be expected. A number of Sry-related genes expressed in the developing genital ridge of the chick were identified.

Urogenital system development in mammals requires the coordinated differentiation of two distinct tissues, the ductal epithelium and the nephrogenic mesenchyme, both derived from the intermediate mesoderm of the early embryo. The former give rise to the genital tracts, ureters and kidney collecting duct system, whereas mesenchymal components undergo epithelial transformation to form nephrons in both the mesonephric (embryonic) and metanephric (definitive) kidney. Pax-2 is a transcriptional regulator of the paired-box family and is widely expressed during the development of both ductal and mesenchymal components of the urogenital system. We report here that Pax-2 homozygous mutant newborn mice lack kidneys, ureters and genital tracts. We attribute these defects to dysgenesis of both ductal and mesenchymal components of the developing urogenital system. The Wolffian and Müllerian ducts, precursors of male and female genital tracts, respectively, develop only partially and degenerate during embryogenesis. The ureters, inducers of the metanephros are absent and therefore kidney development does not take place. Mesenchyme of the nephrogenic cord fails to undergo epithelial transformation and is not able to form tubules in the mesonephros. In addition, we show that the expression of specific markers for each of these components is de-regulated in Pax-2 mutants. These data show that Pax-2 is required for multiple steps during the differentiation of intermediate mesoderm. In addition, Pax-2 mouse mutants provide an animal model for human hereditary kidney diseases.

We investigated the expression of Ad4BP (also known as SF-1), a transcription factor regulating steroidogenic P-450 genes, in the steroidogenic tissues such as adrenal glands, testes and ovaries through the prenatal and postnatal life of rats. Ad4BP was detected in the primordial adrenal glands and gonads of the 13.5 day postcoitum (d.p.c.) fetus. After the appearance of Ad4BP, a steroidogenic P-450 (P-450(SCC)) was also detected in the adrenal glands and its amount increased gradually. In the fetal gonads of 14.5 d.p.c., a significant amount of Ad4BP was detected in the somatic cells of the testes, whereas only a trace amount was present in the ovaries. The sexually dimorphic expression of Ad4BP continued throughout the neonatal age. Drastic alterations occurred during the first to third week of postnatal age accompanied by functional and structural changes of the gonads. The expression of Ad4BP in the testes attained a maximal level one week after birth and decreased markedly thereafter. By contrast, increase of Ad4BP in the ovary was detected after the first postnatal week. Expression of P-450c17 showed a good correlation with the proliferation of Leydig cells in the testes and theca cells in the ovaries. Immunohistochemical studies revealed the presence of Ad4BP in Sertoli cells as well as Leydig cells up to the pubertal age. In the adult rat testis, however, staining of Sertoli cells decreased significantly. Ad4BP was detected in granulosa, theca, corpus luteum and interstitial gland cells in the ovary although the expression levels in granulosa cells varied among follicles. It is suggested that the Müllerian inhibitory substance gene may be a target of Ad4BP since this gene has a conserved Ad4-binding site within the promoter, which is recognized by Ad4BP expressed in the fetal testes.

The mouse Evx-2 gene is located in the immediate vicinity of the Hoxd-13 gene, the most posteriorly expressed gene of the HOXD complex. While the Evx-1 gene is also physically linked to the HOXA complex, it is more distantly located from the corresponding Hoxa-13 gene. We have analysed the expression of Evx-2 during development and compared it to that of Evx-1 and Hoxd-13. We show that, even though Evx-2 is expressed in the developing CNS in a pattern resembling that of other Evx-related genes, the overall expression profile is similar to that of the neighbouring limbs and genitalia. We propose that the acquisition of expression features typical of Hox genes, together with the disappearance of some expression traits common to Evx genes, is due to the close physical linkage of Evx-2 to the HOXD complex, which results in Evx-2 expression being partly controlled by mechanisms acting in the HOX complex. This transposition of the Evx-2 gene next to the Hoxd-13 gene may have occurred soon after the large scale duplications of the HOX complexes. A scheme is proposed to account for the functional evolution of eve-related genes in the context of their linkage to the HOM/Hox complexes.