<p>Pathologies of sexual development are common in humans and reflect the precarious processes of sex determination and sexual differentiation. The gonad forms as a bipotential organ, and recent results from the Capel lab revealed that it is initially balanced between testis and ovarian fates by opposing and antagonistic signaling networks. In XY embryos, this balance is disrupted by the transient expression of the Y-linked gene, Sry, which activates genes that promote the testis pathway and oppose the ovarian pathway. While the roles of a few genes have been defined by mutation, current evidence suggests that the interactions of many genes and signaling pathways are involved in the establishment of sexual fate. For example, most cases of disorders of sexual development (DSDs) are unexplained by mutations in known sex determination genes. In addition, recent microarray studies in the mouse revealed that nearly half the transcriptome is expressed in the gonad at the time of sex determination (Embryonic day 11.5, or E11.5), and as many as 1,500 genes are expressed in a sexually dimorphic pattern at this early stage. Thus the sexual fate decision in the developing gonad likely depends on a complex network of interacting factors that converge on a critical threshold. </p><p>To begin to elucidate the transcription network topology underlying sex determination, we exploited two inbred mouse strains with well-characterized differences in sex reversal. The common inbred strain C57BL/6J (B6) is uniquely sensitive to XY male-to-female sex reversal in response to a number of genetic perturbations, while other strains, including 129S1/SvImJ (129S1) and DBA/2J (D2) are resistant to sex reversal. We hypothesized that these strain differences in gonad phenotype likely result from underlying expression differences in the gonad at the critical timepoint of E11.5. Using microarrays, we identified significant, reproducible differences in the transcriptome of the E11.5 XY gonad between B6 and 129S1 indicating that the reported sensitivity of B6 to sex reversal is consistent with a higher expression of a female-like transcriptome in B6 XY gonads. Surprisingly, a well-characterized master regulator of the testis pathway, Sox9, was found to be upregulated in the sensitive B6 background, which may serve as a compensatory mechanism to counteract the female-leaning transcriptome and activate the testis pathway in wild type B6 XY gonads.</p><p>We extended our expression analysis to a large set of F2 XY gonads from B6 and 129S1 intercrosses. From each pair of gonads, we analyzed the expression of 56 sex-associated genes by nanoliter-scale quantitative RT-PCR (qRT-PCR). The expression levels of most genes were highly variable across the F2 population, yet strong correlations among genes emerged. We employed a First-Order Conditional Independence (FOCI) algorithm to estimate the F2 coexpression network. From this unbiased analysis of XY expression data, we uncovered two subnetworks consisting of primarily male and female genes. Furthermore, we predicted roles for genes of unknown function based on their connectivity and position within the network. </p><p>To identify the genes responsible for these strain expression differences, we genotyped each F2 embryo at 128 single nucleotide polymorphisms (SNPs) located evenly throughout the 19 autosomes and X chromosome. We then employed linkage analysis to detect autosomal regions that control the expression of one or more of the 56 genes in the F2 population. These regions are termed expression quantitative trait loci, or eQTLs. We identified eQTLs for many sex-related genes, including Sry and Sox9, the key regulators of male sex determination. In addition, we identified multiple prominent trans-band eQTLs that controlled the expression of many genes. My work represents the first eQTL analysis of a developing vertebrate organ, the mouse gonad. This systems-level approach revealed the complex transcription architecture underlying sex determination, and provides a mechanistic explanation for sensitivity to sex reversal seen in some individuals.</p> / Dissertation

In Drosophila melanogaster, sex is determined at the preblastoderm stage via an Xchromosome counting mechanism. During this process embryos that carry two X chromosomes begin to develop as females while embryos with one X start the male developmental program. The Xlinked genes involved in sex determination, also called Xsignal elements (XSEs), are: sisterlessA (sisA), sisterlessB (sisB), unpaired (upd), and runt. These genes are responsible for the transcriptional activation of the master regulatory gene Sexlethal (Sxl). Expression of Sxl is initially accomplished only in females through activation of the establishment promoter SxlPe. Later in development, Sxl is transcribed in both sexes through a maintenance promoter, SxlPm, but functional Sxl protein is only produced in female flies. Since Sxl is at the top of the sex determination cascade, understanding its regulation is key to comprehend the process of sex determination. The experiments in this dissertation were designed to better understand two aspects of the sex determination mechanism: How the protein encoded by XSE element sisA interacts with SxlPe, and how the transition from regulation by SxlPe to regulation by SxlPm occurs. The sisA protein (SisA), as part of the bZIP protein family, is thought to bind to its target as a dimer, but a dimerization partner has not yet been found. This work uses knockouts and germline clones to examine interaction between sisA and three SisA partner candidates, atf4, CG16813, and CG16815. Although the evidence described here suggest that none of the three SisA partner candidates genetically interact with Sis, we cannot rule out the possibility of redundancy between the different candidate proteins. This research unravels the timing and regulation of SxlPm expression. I have shown, contrary to previous thought, that expression of SxlPe and SxlPm overlaps for a brief period. Several of the same proteins that are involved in the regulation of SxlPe, including the XSE sisB, also regulate SxlPm. This sex specific regulation leads to a sexually dimorphic pattern of activation and early expression of SxlPm. A common enhancer region was found to regulate SxlPe as well as SxlPm. These results highlight the importance of the transition between SxlPe and SxlPm for the proper establishment of sex determination and have implications for how the sex determination mechanism evolved.

The Lake Malawi cichlid fishes are a pre-eminent example of adaptive evolutionary radiation. The diversity of species (nearly 1000 extant) is mirrored by an array of variation in dozens of phenotypes (e.g. trophic morphology, tooth shape, color patterns, behavior, development). The unique characteristics of this system have produced unparalleled diversity with very little genetic differentiation between species. This dissertation is composed of four studies addressing different aspects of the variation in the LM cichlids and the mechanisms generating and maintaining this level of diversity at multiple biological levels. Community-level diversity is investigated using null model analysis of species co-occurrence data. We detect signals of non-random community assembly at only the broadest and finest spatial scales. Based on the unique ecological and evolutionary characteristics of this assemblage we suggest that different mechanisms are responsible for these patterns. A core‟ group of species is posited to act as a foundation on which these diverse communities are created as a result of fine-scale species interactions. We identify both positive and negative depth-based correlations between species and suggest these interactions play an important role in species diversity in these fish. The Lake Malawi cichlids exhibit an array of trophic morphologies which may play a role in the fine-scale species interactions described in chapter one. In the second chapter we build a genetic model to predict the evolution of jaw morphology and a complex functional jaw trait. We use a complex biomechanical system, the 4-bar jaw linkage, to simulate trait evolution during interspecific hybridizations. We find rampant transgression (trait values beyond parental distributions) in jaw function in a large proportion of potential crosses. This result is characterized by a lack of novel morphological components but rather is the result of recombinations of existing component traits thus producing functional novelty. In the third chapter we create a laboratory cross of one of the parental combinations suggested from the genetic model. The results of this study serve as a proof of principle to the simulations as we observe a large proportion of transgressive 4-bar function in the F2. As predicted this diversity is produced in the absence of transgressive morphology. We contrast these results between this complex system and data generated from several simple jaw lever traits and report differences in the patterns. Using quantitative trait locus (QTL) mapping approaches we examine the genetic basis for complex and simple jaw traits and discuss correlative patterns within and between systems. Finally we examine the genetic architecture of sex-determination and color morphs in this hybrid cross. We find both ZW and XY sex systems segregating as well as linkage to sex-specific color patterns. Several loci and epistatic interactions are associated with sex-determination and color morphs in this cross. The orange-blotch (OB) color is found associated with ZW as predicted from previous work but a previously undescribed (in these species) male nuptial color (blue) is found associated with both ZW and XY genetic systems as well as other loci segregating for sex-determination. These results are discussed in the context of models of sex chromosome evolution as a result of sexual conflict and the potential importance of sexual selection in the diversification of Lake Malawi cichlids. Overall we observe various mechanisms generating and maintaining diversity at different levels of biological organization. We use community co-occurrence analyses, genetic simulation, and QTL analysis of an F2 hybrid population to examine these mechanisms in this rapidly radiating assemblage. These results bolster our understanding of the origins of diversity and the interplay between variation and aspects of evolution in all biological systems.

Components of the molecular pathway underlying gonadogenesis in organisms with temperature-dependent sex determination (TSD) have been retained from genetic sex determination. Furthermore, although much of this network has been conserved, new functions for these genes have evolved in this different mode of sex determination. We find that the transcription factors Sox9 and Dmrt1 and the hormone Mis are involved in the formation of a testis and/or the repression of an ovary at a male-producing temperature. While Mis expression may be maintained by Sox9, the initial upregulation of Mis in the developing testis is most likely modulated by some other upstream factor. Dmrt1 appears to play an upstream role in testis sex determination. We provide evidence that the transcription factor Dax1 and the signaling molecule Wnt4, cloned for the first time in an organism with TSD, play roles in gonadogenesis in both sexes. Finally, we show that the transcription factor FoxL2 and the signaling molecule Rspo1 are involved in the formation of an ovary and/or the repression of a testis at a female-producing temperature. In the first investigation of Rspo1 in any organism exhibiting TSD, we demonstrate it is involved upstream in ovarian sex determination. Complementary to descriptive studies, we optimize a whole organ culture system in which gonad explants develop in vitro for up to three weeks. We show that expression of the sex-determining network in isolated gonads mimics in ovo patterns, revealing an endogenous temperature-sensing mechanism that does not require other embryonic tissues. Ectopic expression of Sox9 reveals a possible positive feedback regulation of Dmrt1. The use of this culture system opens the door to functional manipulation of the gonad at the molecular level and is suitable for a myriad of future studies. This work makes strides in elucidating the molecular network underlying gonadogenesis in an organism exhibiting TSD, and invites investigation of the evolution of gene function. The data lend insight into the changing roles of molecules in sex determination across diverse taxa, and into the evolution of developmental pathways in general. / text

In marine flatfish of the genus Paralichthys, temperature plays a large role in sex determination. Thus, global climate change could have significant effects on southern flounder (Paralichthys lethostigma), a commercially and recreationally important flatfish whose populations have steadily declined in Texas in the last 25 years. The most susceptible areas to global climate change are shallow water environments, particularly estuaries, which serve as essential nursery habitats for juvenile southern flounder. While in the estuaries, juveniles develop, and sex is determined. Juvenile southern flounder possess genotypic sex determination; however, the sex of females is highly influenced by temperature and can result in sex reversal. The temperature-sensitive enzyme complex responsible for estrogen biosynthesis in vertebrates is aromatase cytochrome P450 (P450arom), a critical component in ovarian differentiation that can be used to measure presumptive males and females exposed to a gradient of temperatures. This research identifies that sex is influenced by temperature between 35 and 65 mm total length (TL) and establishes that increases in temperature from 18 °C during this size range produce increasingly male skewed sex ratios in southern flounder from Texas. The findings presented here are critical for optimizing production of females in culture and for developing stock enhancement programs of southern flounder in Texas. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

In the B6.YTIR mouse strain, half of the XY progeny develop bilateral ovaries and the female phenotype. These XY females are infertile mainly due to the death of their embryos. This developmental failure has been attributed to a defect intrinsic to the XY oocyte. / The present study examined the transcriptional activity of the X and Y chromosomes in these oocytes. RT-PCR results show that the Ube1y gene is transcribed in the XY ovary at all stages examined and also in growing XY oocytes. The Sry gene was transcribed only at the onset of ovarian differentiation whereas the Zfy gene was undetectable at all stages during fetal life. The Xist gene, which is involved in X inactivation, was not expressed in XY oocytes. We speculate that expression of Y-encoded genes may have a deleterious effect on the quality of the oocytes and thus renders them incompetent for post-fertilization development.

HOAP was originally identified as a component of an ORC-containing multi-protein complex of Heterochromatin Protein 1 (HP1) from early Drosophila embryos. HOAP immunostaining showed prominent association of it with telomeres, and mutants for HOAP (cav1) showed it functions along with HP1 in forming a telomere capping complex that prevents telomeric fusions. Weaker HOAP immunostaining is also observed in regions of pericentric heterochromatin and euchromatin. To examine the role of HOAP at these non-telomeric sites, we applied Affymetric Drosophila Genome Arrays to undertake a microarray expression profiling study of genes that are mis-expressed in cav1 mutant larvae. The data from four publicly available databases were used to assess the normal expression patterns of the affected genes. We found that the majority (67%) of genes with decreased expression levels in cav1 mutants (log2R< -2.0, pvalue≤ 0.01) have normally testis-specific expression. These results could indicate a role of HOAP in testis-specific gene expression. Alternatively they could reflect reduced male viability due to the loss of HOAP, which resulted in the under-representation of males in the cav1 larval sample. The latter hypothesis is supported by the observation of 2.8-fold under-representation of males in cav1 larvae when I used a yellow+-marked X chromosome to differentially mark male and female cav1 larvae. Thus, this project is focused on determining and characterizing the cause of the reduced male viability. Here I report a role for both HOAP and HP1 in regulating the establishment promoter, SxlPe, of the sex determination masterswitch, Sex lethal (Sxl). Female-specific activation of SxlPe is essential to females as it provides SXL protein to initiate productive female-specific splicing of the late Sxl transcripts which are transcribed in both sexes. We find inappropriate firing of SxlPe and splicing of Sxl transcripts in male cav mutants, whereas mutants for HP1 display Sxl splicing defects in both sexes. Both proteins are associated with SxlPe sequences. In embryos from HP1 mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to SxlPe is severely compromised. Our genetic and biochemical assays suggest a repressing activity for HOAP and both activating and repressing roles for HP1 at SxlPe.

Molecular techniques, particularly noninvasive genetic sampling (NGS) and nondestructive sampling (NDS), are increasingly being used as tools to study the ecology of free-ranging mammals. A specific application of these methods is the molecular sexing of species for which external sex differentiation is challenging. Star-nosed moles (Condylura cristata) are a little-studied species in which females possess a peniform clitoris making them externally indistinguishable from males. To my knowledge, no studies have employed NDS to study any aspect of their ecology. I therefore sequenced fragments of one X-chromosome (Zfx) and two Y-chromosome (Sry and Zfy) genes from known-sex specimens, and designed species-specific primers to co-amplify these loci from hair, claw and fecal samples of 16 star-nosed moles. I found all tissue types were highly (90-100%) reliable for sex determination. I envision that this NDS method will facilitate future capture-and-release studies on the natural history and social structure of this fascinating, semi-aquatic mammal.

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