Sex in mammals is genetically determined and is defined at the cellular level by sex chromosome complement (XY males and XX females). The Y chromosome-linked gene sex-determining region Y (SRY) is believed to be the master initiator of male sex determination in almost all eutherian and metatherian mammals, functioning to upregulate expression of its direct target gene Sry-related HMG box-containing gene 9 (SOX9). Data suggest that SRY evolved from SOX3, although there is no direct functional evidence to support this hypothesis. Indeed, loss-of-function mutations in SOX3 do not affect sex determination in mice or humans. To further investigate Sox3 function in vivo, we generated transgenic mice overexpressing Sox3. Here, we report that in one of these transgenic lines, Sox3 was ectopically expressed in the bipotential gonad and that this led to frequent complete XX male sex reversal. Further analysis indicated that Sox3 induced testis differentiation in this particular line of mice by upregulating expression of Sox9 via a similar mechanism to Sry. Importantly, we also identified genomic rearrangements within the SOX3 regulatory region in three patients with XX male sex reversal. Together, these data suggest that SOX3 and SRY are functionally interchangeable in sex determination and support the notion that SRY evolved from SOX3 via a regulatory mutation that led to its de novo expression in the early gonad.

DEPDC5 mutations have recently been shown to cause epilepsy in humans. Evidence from in vitro studies has implicated DEPDC5 as a negative regulator of mTORC1 during amino acid insufficiency as part of the GATOR1 complex. To investigate the role of DEPDC5 in vivo we generated a null mouse model using targeted CRISPR mutagenesis. Depdc5 homozygotes display severe phenotypic defects between 12.5-15.5 dpc, including hypotrophy, anaemia, oedema, and cranial dysmorphology as well as blood and lymphatic vascular defects. mTORC1 hyperactivity was observed in the brain of knockout embryos and in fibroblasts and neurospheres isolated from knockout embryos and cultured in nutrient deprived conditions. Heterozygous mice appeared to be normal and we found no evidence of increased susceptibility to seizures or tumorigenesis. Together, these data support mTORC1 hyperactivation as the likely pathogenic mechanism that underpins DEPDC5 loss of function in humans and highlights the potential utility of mTORC1 inhibitors in the treatment of DEPDC5-associated epilepsy.

Long non-coding RNAs (lncRNAs) are classified as RNAs greater than 200 nucleotides in length that do not produce a protein product. lncRNAs are expressed with cellular and temporal specificity and have been shown to play a role in many cellular events, including the regulation of gene expression, post-transcriptional modifications and epigenetic modifications. Since lncRNAs were first discovered, there has been increasing evidence that they play important roles in the development and function of most organs, including the placenta. The placenta is an essential transient organ that facilitates communication and nutrient exchange between the mother and foetus. The placenta is of foetal origin and begins to form shortly after the embryo implants into the uterine wall. The placenta relies heavily on the successful differentiation and function of trophoblast cells, including invasion as well as the formation of the maternal/foetal interface. Here, we review the current literature surrounding the involvement of lncRNAs in the development and function of trophoblasts and the human placenta.

Single nucleotide polymorphisms and pre-and peri-conception folic acid (FA) supplementation and dietary data were used to identify one-carbon metabolic factors associated with pregnancy outcomes in 3196 nulliparous women. In 325 participants, we also measured circulating folate, vitamin B12 and homocysteine. Pregnancy outcomes included preeclampsia (PE), gestational hypertension (GHT), small for gestational age (SGA), spontaneous preterm birth (sPTB) and gestational diabetes mellitus (GDM). Study findings show that maternal genotype MTHFR A1298C(CC) was associated with increased risk for PE, whereas TCN2 C766G(GG) had a reduced risk for sPTB. Paternal MTHFR A1298C(CC) and MTHFD1 G1958A(AA) genotypes were associated with reduced risk for sPTB, whereas MTHFR C677T(CT) genotype had an increased risk for GHT. FA supplementation was associated with higher serum folate and vitamin B12 concentrations, reduced uterine artery resistance index and increased birth weight. Women who supplemented with <800 μg daily FA at 15-week gestation had a higher incidence of PE (10.3%) compared with women who did not supplement (6.1%) or who supplemented with ≥800 μg (5.4%) (P < .0001). Higher serum folate levels were found in women who later developed GDM compared with women with uncomplicated pregnancies (Mean ± SD: 37.6 ± 8 nmol L −1 vs. 31.9 ± 11.2, P = .007). Fast food consumption was associated with increased risk for developing GDM, whereas low consumption of green leafy vegetables and fruit were independent risk factors for SGA and GDM and sPTB and SGA, respectively. In conclusion, maternal and paternal genotypes, together with maternal circulating folate and homocysteine concentrations, and pre-and early-pregnancy dietary factors, are independent risk factors for pregnancy complications.

De novo and inherited mutations of X-chromosome cell adhesion molecule protocadherin 19 (PCDH19) cause frequent, highly variable epilepsy, autism, cognitive decline and behavioural problems syndrome. Intriguingly, hemizygous null males are not affected while heterozygous females are, contradicting established X-chromosome inheritance. The disease mechanism is not known. Cellular mosaicism is the likely driver. We have identified p54nrb/NONO, a multifunctional nuclear paraspeckle protein with known roles in nuclear hormone receptor gene regulation, as a PCDH19 protein interacting partner. Using breast cancer cells we show that PCDH19-NONO complex is a positive co-regulator of ERα-mediated gene expression. Expression of mutant PCDH19 affects at least a subset of known ERα-regulated genes. These data are consistent with our findings that genes regulated by nuclear hormone receptors and those involved in the metabolism of neurosteroids in particular are dysregulated in PCDH19-epilepsy girls and affected mosaic males. Overall we define and characterize a novel mechanism of gene regulation driven by PCDH19, which is mediated by paraspeckle constituent NONO and is ERα-dependent. This PCDH19-NONO-ERα axis is of relevance not only to PCDH19-epilepsy and its comorbidities but likely also to ERα and generally nuclear hormone receptor-associated cancers.

The transcription factor SOX3 is expressed within most neural progenitor (NP) cells of the vertebrate central nervous system (CNS) and is essential for normal brain development in mice and humans. However, despite the widespread expression of Sox3, CNS defects in null mice are relatively mild due to functional redundancy with the other SOXB1 sub-group members Sox1 and Sox2. To further understand the molecular function of SOX3, we investigated the genome-wide binding profile of endogenous SOX3 in NP cells using ChIP-seq. SOX3 binding was identified at over 8,000 sites, most of which were intronic or intergeneic and were significantly associated with neurodevelopmental genes. The majority of binding sites were moderately or highly conserved (phastCons scores >0.1 and 0.5, respectively) and included the previously characterised, SOXB1-binding Nestin NP cell enhancer. Comparison of SOX3 and published ChIP-Seq data for the co-activator P300 in embryonic brain identified hundreds of highly conserved putative enhancer elements. In addition, we identified a subset of highly conserved putative enhancers for CNS development genes common to SOXB1 members in NP cells, all of which contained the SOX consensus motif (ACAAWR). Together these data implicate SOX3 in the direct regulation of hundreds of NP genes and provide molecular insight into the overlapping roles of SOXB1 proteins in CNS development.

Polyalanine expansions in transcription factors have been associated with eight distinct congenital human diseases. It is thought that in each case the polyalanine expansion causes misfolding of the protein that abrogates protein function. Misfolded proteins form aggregates when expressed in vitro; however, it is less clear whether aggregation is of relevance to these diseases in vivo. To investigate this issue, we used targeted mutagenesis of embryonic stem (ES) cells to generate mice with a polyalanine expansion mutation in Sox3 (Sox3-26ala) that is associated with X-linked Hypopituitarism (XH) in humans. By investigating both ES cells and chimeric mice, we show that endogenous polyalanine expanded SOX3 does not form protein aggregates in vivo but rather is present at dramatically reduced levels within the nucleus of mutant cells. Importantly, the residual mutant protein of chimeric embryos is able to rescue a block in gastrulation but is not sufficient for normal development of the hypothalamus, a region that is functionally compromised in Sox3 null embryos and individuals with XH. Together, these data provide the first definitive example of a disease-relevant PA mutant protein that is both nuclear and functional, thereby manifesting as a partial loss-of-function allele.

Gene duplication provides spare genetic material that evolution can craft into new functions. and are evolutionarily related genes with overlapping and unique sites of expression during embryogenesis. It is currently unclear whether SOX2 and SOX3 have identical or different functions. Here, we use CRISPR/Cas9-assisted mutagenesis to perform a gene-swap, replacing the ORF with the ORF to investigate their functional equivalence in the brain and testes. We show that increased expression of SOX2 can functionally replace SOX3 in the development of the infundibular recess/ventral diencephalon, and largely rescues pituitary gland defects that occur in null mice. We also show that ectopic expression of SOX2 in the testes functionally rescues the spermatogenic defect of null mice, and restores gene expression to near normal levels. Together, these data provide strong evidence that SOX2 and SOX3 proteins are functionally equivalent.