Germ Cells Are Not Required to Establish the Female Pathway in Mouse Fetal Gonads

The fetal gonad is composed of a mixture of somatic cell lineages and germ cells. The fate of the gonad, male or female, is determined by a population of somatic cells that differentiate into Sertoli or granulosa cells and direct testis or ovary development. It is well established that germ cells are not required for the establishment or maintenance of Sertoli cells or testis cords in the male gonad. However, in the agametic ovary, follicles do not form suggesting that germ cells may influence granulosa cell development. Prior investigations of ovaries in which pre-meiotic germ cells were ablated during fetal life reported no histological changes during stages prior to birth. However, whether granulosa cells underwent normal molecular differentiation was not investigated. In cases where germ cell loss occurred secondary to other mutations, transdifferentiation of granulosa cells towards a Sertoli cell fate was observed, raising questions about whether germ cells play an active role in establishing or maintaining the fate of granulosa cells. We developed a group of molecular markers associated with ovarian development, and show here that the loss of pre-meiotic germ cells does not disrupt the somatic ovarian differentiation program during fetal life, or cause transdifferentiation as defined by expression of Sertoli markers. Since we do not find defects in the ovarian somatic program, the subsequent failure to form follicles at perinatal stages is likely attributable to the absence of germ cells rather than to defects in the somatic cells.Stem Cell and Regenerative Biolog

Identification of Novel Interactors of SEC6 via Genetic Suppressor Screen Using a Saccharomyces cerevisiae Genomic DNA Library

Polarized protein secretion is a fundamental process for all organisms, from yeast to higher eukaryotes. The secretory pathway in eukaryotes includes many steps mediated by hundreds of essential proteins. Our interest lies on Sec6, which is an 88kDa protein subunit of the tethering complex named exocyst, which is known to play diverse roles in recognition, tethering, and SNARE-mediated fusion of secretory vesicles. Previous work on Sec6 done by Munson and Songer (2009) explored phenotypes of two novel SEC6 mutant proteins, whose conserved surface amino acids had been altered. Both sec6-49 and sec6-54 mutant alleles displayed severe temperature sensitive growth and secretion defects. Interestingly, at non-permissive conditions trafficking of secretory vesicles to the plasma membrane was unimpaired, but none of the exocyst subunits were correctly polarized at sites of secretion. Biochemical analyses examining the state of exocyst assembly in both mutant backgrounds showed that the complex is intact at non-permissive conditions. We hypothesize that Sec6 has an important anchoring function for the exocyst, and that mislocalization of exocyst stems from compromising Sec6’s interaction with an unknown factor(s). Our research aims to identify and validate novel interactors of Sec6 that are critical for anchoring the exocyst complex at the plasma membrane. We utilized a classic genetic high copy suppressor screen to identify genes that suppress the temperature sensitive (ts) phenotype of sec6-49 cells upon overexpression. The plasmids from cells that survived temperature shift after transformation were purified, and genomic inserts were confirmed via restriction digests. Currently, sequencing via primers flanking the genomic insert has been initiated to identify the location of the genomic inserts and candidate genes. Overall, the isolation and characterization of novel interactors will shed light on mechanistic details of Sec6 function, which is critical for understanding mechanistic details of quality control in the secretory pathway

Temporal Transcriptional Profiling of Somatic and Germ Cells Reveals Biased Lineage Priming of Sexual Fate in the Fetal Mouse Gonad

The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process, because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates

Oestrogen shuts the door on SOX9

Oestrogen exerts a robust yet imperfectly understood effect on sexual development in vertebrate embryos. New work by Pask and colleagues in BMC Biology indicates that it may interfere with male development by preventing nuclear localization of SOX9, a master regulator of the testis differentiation pathway

The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage

Background: We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.Results: Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.Conclusions: Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders

Morphogenesis and Female Fate Determination in Vertebrates

<p>A unique feature of the fetal gonad is its ability to form two distinct organs, the testis and the ovary, from a single bipotential primordium. The outcome of this decision, which is made by a population of somatic cells known as the bipotential supporting cell precursors, determines whether an embryo will develop as a phenotypic male or female. Though several molecular pathways have been shown to be required for female fate determination in vertebrates, the intricacies of ovarian morphogenesis are not well understood. A key event in ovarian development occurs around birth, when meiotic germ cells and somatic granulosa cells organize into primordial follicles, the structures that generate mature oocytes for ovulation in adult females. We investigated the embryonic origins and proliferative properties of granulosa cells in the fetal mouse ovary and found that the precursors emerge from the ovarian surface epithelium and then enter mitotic arrest in a specification process that extends from the bipotential stage to the end of the postnatal follicle assembly period. Maintenance of cell cycle arrest in granulosa cell precursors appears to be regulated by Wnt signaling. The first granulosa cells to be specified were exclusively incorporated into the subset of follicles that begin to grow immediately upon assembly. We show that this first group of granulosa progenitors derives from the supporting cell precursors present in the bipotential gonad. Interestingly, both XX and XY supporting cell precursors were mitotically arrested towards the end of the bipotential period, indicating that adoption of supporting cell fate might be regulated by the cell cycle. We also show that antagonism of Notch signaling may be required for these precursor cells to exit the cell cycle and differentiate.</p><p>In Witschi's classic model of vertebrate gonad development, the cortex and medulla of the undifferentiated gonad expand and differentiate in a mutually exclusive manner to yield the mature ovary and testis (Witschi 1951). Estrogen acts on both the cortex and medulla to promote female fate determination and ovary development in non-mammalian vertebrates. However, the downstream receptors and targets through which estrogen exerts its effects on the gonad have not yet been elucidated. We selected the red-eared slider turtle Trachemys scripta as a model with which to address this question. We first characterized the cellular composition of the turtle gonad before and after sex determination, identifying four populations of somatic cells distinguishable by their location within the gonad as well as the complement of transcription factors expressed. This information was then applied to an investigation of estrogen signaling pathways in the turtle ovary. We show that i) estrogen likely acts through its canonical receptors rather than a non-canonical pathway involving ERK signaling; ii) early exposure to estrogen resulted in the premature downregulation of a testis-specific gene, SOX9, in the medulla; iii) less estrogen is needed to promote ovarian differentiation in the cortex of the gonad than to repress testicular differentiation of the medulla, consistent with the localized production of estrogen in the medulla; and iv) estrogen's repressive effect on SOX9 expression may be mediated by Wnt signaling. </p><p>Our findings add complexity to the standard model of how the male and female supporting cell lineages are established in mice, reveal evolutionary conservation between mice and turtles in the timing of granulosa cell specification relative to sex determination., and refine our understanding of how estrogen acts to promote ovarian development in non-mammalian species.</p>Dissertatio

Germ cells are not required to establish the female pathway in mouse fetal gonads.

The fetal gonad is composed of a mixture of somatic cell lineages and germ cells. The fate of the gonad, male or female, is determined by a population of somatic cells that differentiate into Sertoli or granulosa cells and direct testis or ovary development. It is well established that germ cells are not required for the establishment or maintenance of Sertoli cells or testis cords in the male gonad. However, in the agametic ovary, follicles do not form suggesting that germ cells may influence granulosa cell development. Prior investigations of ovaries in which pre-meiotic germ cells were ablated during fetal life reported no histological changes during stages prior to birth. However, whether granulosa cells underwent normal molecular differentiation was not investigated. In cases where germ cell loss occurred secondary to other mutations, transdifferentiation of granulosa cells towards a Sertoli cell fate was observed, raising questions about whether germ cells play an active role in establishing or maintaining the fate of granulosa cells. We developed a group of molecular markers associated with ovarian development, and show here that the loss of pre-meiotic germ cells does not disrupt the somatic ovarian differentiation program during fetal life, or cause transdifferentiation as defined by expression of Sertoli markers. Since we do not find defects in the ovarian somatic program, the subsequent failure to form follicles at perinatal stages is likely attributable to the absence of germ cells rather than to defects in the somatic cells