Ee Ting Ng

Loss of Wnt5a Disrupts Primordial Germ Cell Migration and Male Sexual Development in Mice

ABSTRACT Disruptions in the regulatory pathways controlling sex determination and differentiation can cause disorders of sex development, often compromising reproductive function. Although extensive efforts have been channeled into elucidating the regulatory mechanisms controlling the many aspects of sexual differentiation, the majority of disorders of sex development phenotypes are still unexplained at the molecular level. In this study, we have analyzed the potential involvement of Wnt5a in sexual development and show in mice that Wnt5a is male-specifically upregulated within testicular interstitial cells at the onset of gonad differentiation. Homozygous deletion of Wnt5a affected sexual development in male mice, causing testicular hypoplasia and bilateral cryptorchidism despite the Leydig cells producing factors such as Hsd3b1 and Insl3. Additionally, Wnt5a-null embryos of both sexes showed a significant reduction in gonadal germ cell numbers, which was caused by aberrant primordial germ cell migration along the hindgut endoderm prior to gonadal colonization. Our results indicate multiple roles for Wnt5a during mammalian reproductive development and help to clarify further the etiology of Robinow syndrome (OMIM 268310), a disease previously linked to the WNT5A pathway.

Female-to-male sex reversal in mice caused by transgenic overexpression of Dmrt1.

Genes related to Dmrt1, which encodes a DNA-binding DM domain transcription factor, act as triggers for primary sex determination in a broad range of metazoan species. However, this role is fulfilled in mammals by Sry, a newly evolved gene on the Y chromosome, such that Dmrt1 has become dispensable for primary sex determination and instead maintains Sertoli cell phenotype in postnatal testes. Here, we report that enforced expression of Dmrt1 in XX mouse fetal gonads using a Wt1-BAC transgene system is sufficient to drive testicular differentiation and male secondary sex development. XX transgenic fetal gonads showed typical testicular size and vasculature. Key ovarian markers, including Wnt4 and Foxl2, were repressed. Sertoli cells expressing the hallmark testis-determining gene Sox9 were formed, although they did not assemble into normal testis cords. Other bipotential lineages differentiated into testicular cell types, including steroidogenic fetal Leydig cells and non-meiotic germ cells. As a consequence, male internal and external reproductive organs developed postnatally, with an absence of female reproductive tissues. These results reveal that Dmrt1 has retained its ability to act as the primary testis-determining trigger in mammals, even though this function is no longer normally required. Thus, Dmrt1 provides a common thread in the evolution of sex determination mechanisms in metazoans. Highlighted article: Even though its function is not normally required for sex determination in mammals, Dmrt1 is able to drive sex reversal in XX mice, suggesting that it has retained sex-determining function.

Structure–function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination

Significance The sex-determining factor SRY is thought to function by up-regulating expression of its key target gene SRY-box 9 (SOX9) in pre-Sertoli cells of the developing gonads, but evidence for a transactivation domain is lacking for human SRY and is limited to in vitro evidence for mouse Sry. The latter is unusual in possessing a polyglutamine tract at its C terminus. We demonstrate, using a combination of biochemical, cell-based, and transgenic mouse assays, that this domain plays essential roles in both protein stabilization and transactivation of Sox9, and is required for male sex determination in mice. Our data indicate that mouse Sry has evolved a novel bifunctional module, revealing an unexpected level of plasticity of sex-determining mechanisms even among mammals. The mammalian sex-determining factor SRY comprises a conserved high-mobility group (HMG) box DNA-binding domain and poorly conserved regions outside the HMG box. Mouse Sry is unusual in that it includes a C-terminal polyglutamine (polyQ) tract that is absent in nonrodent SRY proteins, and yet, paradoxically, is essential for male sex determination. To dissect the molecular functions of this domain, we generated a series of Sry mutants, and studied their biochemical properties in cell lines and transgenic mouse embryos. Sry protein lacking the polyQ domain was unstable, due to proteasomal degradation. Replacing this domain with irrelevant sequences stabilized the protein but failed to restore Sry’s ability to up-regulate its key target gene SRY-box 9 (Sox9) and its sex-determining function in vivo. These functions were restored only when a VP16 transactivation domain was substituted. We conclude that the polyQ domain has important roles in protein stabilization and transcriptional activation, both of which are essential for male sex determination in mice. Our data disprove the hypothesis that the conserved HMG box domain is the only functional domain of Sry, and highlight an evolutionary paradox whereby mouse Sry has evolved a novel bifunctional module to activate Sox9 directly, whereas SRY proteins in other taxa, including humans, seem to lack this ability, presumably making them dependent on partner proteins(s) to provide this function.

Primary cilia function regulates the length of the embryonic trunk axis and urogenital field in mice

The issues of whether and how some organs coordinate their size and shape with the blueprint of the embryo axis, while others appear to regulate their morphogenesis autonomously, remain poorly understood. Mutations in Ift144, encoding a component of the trafficking machinery of primary cilia assembly, result in a range of embryo patterning defects, affecting the limbs, skeleton and neural system. Here, we show that embryos of the mouse mutant Ift144(twt) develop gonads that are larger than wild-type. Investigation of the early patterning of the urogenital ridge revealed that the anterior-posterior domain of the gonad/mesonephros was extended at 10.5 dpc, with no change in the length of the metanephros. In XY embryos, this extension resulted in an increase in testis cord number. Moreover, we observed a concomitant extension of the trunk axis in both sexes, with no change in the length of the tail domain or somite number. Our findings support a model in which: (1) primary cilia regulate embryonic trunk elongation; (2) the length of the trunk axis determines the size of the urogenital ridges; and (3) the gonad domain is partitioned into a number of testis cords that depends on the available space, rather than being divided a predetermined number of times to generate a specific number of cords.

A Site-Specific, Single-Copy Transgenesis Strategy to Identify 5′ Regulatory Sequences of the Mouse Testis-Determining Gene Sry

The Y-chromosomal gene SRY acts as the primary trigger for male sex determination in mammalian embryos. Correct regulation of SRY is critical: aberrant timing or level of Sry expression is known to disrupt testis development in mice and we hypothesize that mutations that affect regulation of human SRY may account for some of the many cases of XY gonadal dysgenesis that currently remain unexplained. However, the cis-sequences involved in regulation of Sry have not been identified, precluding a test of this hypothesis. Here, we used a transgenic mouse approach aimed at identifying mouse Sry 5′ flanking regulatory sequences within 8 kb of the Sry transcription start site (TSS). To avoid problems associated with conventional pronuclear injection of transgenes, we used a published strategy designed to yield single-copy transgene integration at a defined, transcriptionally open, autosomal locus, Col1a1. None of the Sry transgenes tested was expressed at levels compatible with activation of Sox9 or XX sex reversal. Our findings indicate either that the Col1a1 locus does not provide an appropriate context for the correct expression of Sry transgenes, or that the cis-sequences required for Sry expression in the developing gonads lie beyond 8 kb 5′ of the TSS.

A piggyBac transposon- and gateway-enhanced system for efficient BAC transgenesis

Background: Bacterial artificial chromosomes (BACs) have become increasingly popular vectors for making transgenic mice, as they are able to carry large genomic DNA fragments that in many cases are needed to reproduce the endogenous gene expression pattern. However, the efficiency of BAC transgenesis is generally low, and gene transfer to BAC vectors by recombination‐mediated engineering (recombineering) is time‐consuming and technically demanding. Results and Conclusions: We present an enhanced system, comprising a BAC vector retrofitted with piggyBac DNA transposon elements and attL (Gateway) docking sites, that obviates these problems. Using this system, a gene‐of‐interest (such as a reporter gene) is transferred to the vector in a one‐step in vitro reaction, and piggyBac transposition mediates transgene integration at high efficiency when microinjected into mouse zygotes with piggyBac transposase mRNA. We establish proof‐of‐principle for this system using a Wilms tumour‐1 (Wt1) BAC to drive expression of an mCherry‐2A‐EGFP (RG) reporter gene, which yielded transgenic mice at a frequency of 33%, and recapitulated endogenous WT1 expression in developing gonads, kidneys and heart. The system we describe is applicable to any BAC transgenesis strategy. Developmental Dynamics 243:1086–1094, 2014.

SOX4 regulates gonad morphogenesis and promotes male germ cell differentiation in mice.

The group C SOX transcription factors SOX4, -11 and -12 play important and mutually overlapping roles in development of a number of organs. Here, we examined the role of SoxC genes during gonadal development in mice. All three genes were expressed in developing gonads of both sexes, predominantly in somatic cells, with Sox4 being most strongly expressed. Sox4 deficiency resulted in elongation of both ovaries and testes, and an increased number of testis cords. While female germ cells entered meiosis normally, male germ cells showed reduced levels of differentiation markers Nanos2 and Dnmt3l and increased levels of pluripotency genes Cripto and Nanog, suggesting that SOX4 may normally act to restrict the pluripotency period of male germ cells and ensure their proper differentiation. Finally, our data reveal that SOX4 (and, to a lesser extent, SOX11 and -12) repressed transcription of the sex-determining gene Sox9 via an upstream testis-specific enhancer core (TESCO) element in fetal gonads, raising the possibility that SOXC proteins may function as transcriptional repressors in a context-dependent manner.

Reduced activity of SRY and its target enhancer Sox9-TESCO in a mouse species with X∗Y sex reversal

In most eutherian mammals, sex determination is governed by the Y-linked gene Sry, but in African pygmy mice Mus minutoides, Sry action is overridden by a variant X chromosome (X*), yielding X*Y females. We hypothesized that X*Y sex reversal may be underpinned not only by neomorphic X chromosome functionality, but also by a compromised Sry pathway. Here, we show that neither M. minutoides SRY nor its target, the Sox9-TESCO enhancer, had appreciable transcriptional activity in in vitro assays, correlating with sequence degradation compared to Mus musculus counterparts. However, M. minutoides SRY activated its cognate TESCO to a moderate degree, and can clearly engage the male pathway in M. minutoides in the wild, indicating that SRY and TESCO may have co-evolved in M. minutoides to retain function above a threshold level. We suggest that weakening of the SRY/TESCO nexus may have facilitated the rise and spread of a variant X* chromosome carrying female-inducing modifier gene(s).

Agarose/gelatin immobilisation of tissues or embryo segments for orientated paraffin embedding and sectioning.

The technique described in this protocol allows the user to position small tissues in the optimal orientation for paraffin embedding and sectioning by first immobilising the tissue in an agarose/gelatin cube. This method is an adaptation of methods used for early embryos and can be used for any small tissues or embryo segments. Processing of larger tissue sections using molds to create agarose/gelatin blocks has been described previously; this detailed protocol provides a method for dealing with much smaller tissues or embryos (≤5mm). The tissue is briefly fixed then an agarose/gelatin drop is created to surround the tissue. The tissue can be orientated as per the users preference in the drop before it sets as is carved into a cube with a domed top. The cube is then dehydrated and goes through the embedding and sectioning process. The domed cube is easy to orientate when embedding the tissue in a wax block giving the user assured orientation of the small tissue for sectioning. Additionally, the agarose/gelatin cube is easy to see in the unmolded wax once embedded, making the region of interest easy to identify.

Transcriptomic analysis of mRNA expression and alternative splicing during mouse sex determination.

Mammalian sex determination hinges on sexually dimorphic transcriptional programs in developing fetal gonads. A comprehensive view of these programs is crucial for understanding the normal development of fetal testes and ovaries and the etiology of human disorders of sex development (DSDs), many of which remain unexplained. Using strand-specific RNA-sequencing, we characterized the mouse fetal gonadal transcriptome from 10.5 to 13.5 days post coitum, a key time window in sex determination and gonad development. Our dataset benefits from a greater sensitivity, accuracy and dynamic range compared to microarray studies, allows global dynamics and sex-specificity of gene expression to be assessed, and provides a window to non-transcriptional events such as alternative splicing. Spliceomic analysis uncovered female-specific regulation of Lef1 splicing, which may contribute to the enhanced WNT signaling activity in XX gonads. We provide a user-friendly visualization tool for the complete transcriptomic and spliceomic dataset as a resource for the field.