Gonads are distinct organs in organisms with complicated reproductive systems, and they have specific locations in the body. The gonads of crinoids, however, are scattered in numerous genital pinnules of their arms. We studied seven species of feather stars, Comatella maculata (Carpenter, 1888), Comatella nigra (Carpenter, 1888), Comaster multifidus (M􀎏ller, 1841), Comaster distinctus (Carpenter, 1881), Comanthus parvicirrus (M􀎏ller, 1841), Colobometra perspinosa (Carpenter,1881) and Himerometra magnipinna Clark, 1908 collected at Kenting, southern Taiwan in 2007. The purpose is to explore if the distribution patterns of gonads are adaptative. With bimonthly samplings in a year, it is discovered that the spawning seasons of the species are not synchronized in a particular season. Three models of relationships between maximum arm lengths and numbers of arms exist in the seven species, i.e., constant arm numbers, linear, and quadratic with an asymptote of arm numbers. Therefore, the maximum arm length is more appropriate than arm number as a size index of feather stars. Evidence of bigger mature than immature individuals is found in Comatella maculata, Comaster distinctus and Comaster multifidus, and there is no size difference between males and females. The gonads of feather stars are distributed in the proximal end of arms. The proportion of arms represented by genital pinnules within individuals were constant except in Comanthus parvicirrus where a negative correlation was found. Except the 2 cave-dwelling species, Comatella maculata and Comanthus parvicirrus, most regenerating arms of 5 other species had breakage points occurred near the proximal ends of the arms. Distal breakage may regenerate too fast to recognize. The arms obviously do not grow so fast as to limit gonad development to the proximal ends of arms. Habitat difference may cause variation in genital pinnule widths, but not in the number of genital pinnules as suggested by comparsions of specimens between 2 sites. The cave-dwelling Comanthus parvicirrus has special congregated long arms, which are extended outside. Moreover, these arms have higher probability of breakage, and smaller ranges of genital pinnules than short arms. This phenomenon supports that the limited gonad distribution is adaptive for reducing loss upon arm breakage.

The production of fully functional haematopoietic stem cells (HSCs) for clinical transplantation is a highly sought after goal in the field of regenerative medicine. Given their capacity for extensive self-renewal and differentiation into any cell type, human pluripotent stem cells (hPSCs) provide a potentially limitless source of haematopoietic cells in vitro for clinical application. However, to date, fully functional HSCs have not been produced from hPSCs without the overexpression of transcription factors. In this study I first investigated the production of HSCs and haematopoietic progenitor cells (HPCs) in an established clinical-grade haematopoietic differentiation protocol. I demonstrated the efficient and reproducible production of HPCs but showed that the strategy did not produce fully functional HSCs that could repopulate the haematopoietic system of immune-deficient mice. Modification of the protocol by manipulation of the hedgehog signalling pathway and co-aggregation with OP9 stromal cells did not provide any significant enhancement of HPC production. To gain the required knowledge with which to improve our current protocol, I therefore switched my focus towards studying the development of HSCs in the early human embryo. It has been shown that HSCs first emerge from the ventral wall of the dorsal aorta in the aorta-gonad-mesonephros (AGM) region of the human embryo but the precise location and the mechanisms underpinning this process remain unknown. In this study, I established a culture system to map the spatio-temporal distribution of HSCs and to investigate the presence of HSC precursors. I showed that embryonic HSCs emerge predominantly around and above the vitelline artery entry point in the dorsal aorta and can be maintained in our explant culture system. I then performed RNA-sequencing of cells derived from AGM sub-regions, and this identified molecular signatures which could potentially underlie the ventral polarity of HSC emergence in the AGM. To elucidate the role of the stromal compartment in this unique haematopoietic niche, I derived stromal cell lines from the human AGM region and showed they were capable of supporting haematopoiesis in vitro. This work has provided some important insights into the mechanisms regulating HSC development in the human AGM region and identified interesting candidate molecules for future testing in differentiation protocols. This knowledge brings us a step closer to the successful in vitro production of HSCs for clinical use.

To date, the majority of studies into sex determination and sexual development have focused on the mammalian system due to the mouse model being an excellent tool for developmental biology as well as obvious implications to human health and development. However, the focus on the mammalian system has caused a large fraction of other vertebrate groups to be overlooked. The last decade has seen an array of studies into various non-mammalian organisms like fishes, birds, reptiles, and amphibians. These studies have unveiled a remarkably conserved molecular background utilized for sexual differentiation amongst all vertebrates, ranging from the alligator which employs temperature dependent sex determination to the mouse, which employs genotypic sex determination In this project, I implemented molecular methods traditionally used to study model organisms, to investigate an amphibian species, the cane toad (Bufo marinus). The cane toad was chosen due to its invasive status in Australia, as well as being a representative of one of the most successful and specious families of vertebrates, Bufonidae. Since, its introduction, this species has rapidly spread across the continent, adversely effecting native species throughout its introduced range. Recent studies have identified the disruption of the sex-determination pathway as a realistic goal in an otherwise, seemingly futile, effort to curtail their expansion. We decided to approach the study of cane toad sex determination and development on three levels, in order to have a broader understanding of the similarities between toads and other groups, and additionally, to better assess the feasibility of utilizing molecular means to control cane toads in non-native habitats. Firstly, I used a candidate gene approach to clone and characterize five genes which were previously known to be involved in both mammalian as well as amphibian sex determination: Sox9, Dmrt1, p450arom, Sf1, and Dax1. I chose Sox9 and Dmrt1 due to their known involvement in the male-specific pathway of mammals. Dmrt1 was also known to be male specific in other groups, including amphibians. Conversely, I also decided to investigate p450arom and Dax1, due to their affiliation with the female pathway, while Sf1 has a role in both sexes. All of these genes were expressed in the gonads of both sexes of cane toads. However, Sox9 exhibited strong transcriptional up-regulation in testes at the time of sexual differentiation, similar to mammals. Thus, using the candidate gene approach, I was successful in identifying a sex-specific marker which could be utilized in the manipulation of the sex determination pathway (e.g. female to male sex reversal). VIIThe second approach involved the study of sex determination on a cytogenetic level. Traditionally, Bufonids have been assumed to utilize a ZZ/ZW (male homogametic/female heterogametic) sex chromosome system, yet only one recent study has been successful in the identification of sex chromosomes in a toad species. Moreover, the authors identified the sex chromosomes in only one population (of 18 studied), highlighting the scarcity of discernible sex chromosomes in toads. When we began to investigate the karyotype of cane toads, we were able to identify a female-specific length polymorphism in the nucleolus organizer region (NOR) of chromosome 7, making this chromosome pair a strong candidates for the Z and W sex chromosomes. In order to verify our hypothesis, we also performed chromomycin A3 staining to reveal a differential signal between the chromosome pair, indicative of heterochromatin accumulation on the brighter NOR. This study lent strong support to the observed difference between the chromosomes, yet we required further investigation in order to identify the W and the Z chromosomes, specifically. By utilizing comparative genomic hybridization (CGH), I was able to identify a female specific region on the chromosome with the larger NOR, identifying it as the W chromosome. This study was successful in identifying the sex chromosomes in the cane toad. Moreover, it allows us to make a more accurate prediction of the possible sex determination method utilized: either a dosage dependent male determination system, or a female-specific gene in a female determination system. The third approach focused on the Bidder’s organ and its role in sexual development of cane toads. The Bidder’s organ is a gonadal structure comprised of ovary-like tissue, found in both males and females, with no known function. Since our study was focused on female to male sex-reversal of cane toads, we decided to investigate the potential of the Bidder’s organ to interrupt this process. By utilizing the candidate genes, which I had previously cloned, I was able to identify the Bidder’s organ as having an entirely distinct transcriptional pattern in comparison to the gonads of either sex. Additionally, the Bidder’s organ showed significantly higher levels of p450arom expression than the gonads, identifying it as a possible key player in the production of aromatase enzyme for oestrogen production. Moreover, gene expression patterns in the Bidder’s organ strongly correlated with the significant developmental time points in the sexual development of the toads, implying a possible function in the development process of toads. In conclusion, I have performed the first study of the molecular, cytogenetic and anatomical aspects of sexual development in a toad. I was able to verify that cane toads utilize a ZZ/ZW chromosome system and furthermore, likely utilize the mammalian male-linked gene, Sox9, in male development. I was also able to show that the Bidder’s organ is transcriptionally active at key time points, likely indicative of a functional role during development.

Abstract Biotechnological strategies aimed at producing male-only offspring have the potential to improve the yield of the Australian beef industry. As a proof-of-concept project, I aimed to target the primary male sex-determining gene Sry to the X chromosome in mice, to produce a transgenic XY male that would transmit Sry – and hence maleness – to both XX and XY offspring. In this project I aimed to target a 14.5 kb DNA fragment containing Sry to an X-chromosome locus that escapes X-inactivation. After considering many potential loci, a targeting strategy and construct were designed for the SMCX locus, which is well conserved between mouse, human and bovine. A targeting vector with 5kb and 3kb arms of homology was also constructed without Sry, to target the locus. Attempts to introduce the 14.5 kb Sry fragment into the construct were unsuccessful, and a smaller construct, containing only the coding sequence of the Sry gene driven by a strong promoter, is currently being made. In order to translate this transgenic approach into cattle, other facets of bovine sex determination required investigation. First, it was important to identify the necessary regulatory regions upstream of bovine SRY needed for the gene to be functional, and secondly to investigate the timing of testis development in male bovine embryos. To enable sequence comparison, I sequenced upstream of the bovine and goat SRY gene and through bioinformatic analysis identified regulatory regions common to several mammals. I identified four regions of high homology upstream of bovine SRY conserved between human, goat, and pig, but not mouse. These regions are likely to be important for the regulation of the gene in these species, as they share unique transcription factor binding sites. From this research I concluded that 9 kb upstream of bovine SRY were likely to be useful in transgenic strategies to produce sex-reversed cattle. Although I attempted to use a 15 kb bovine genomic fragment containing SRY to sex reverse XX mice, this project was unsuccessful. I also investigated the expression pattern of genes known to have a role in sex determination, including SRY, in early bovine embryos. I identified the major time points important for male sex determination, including the first appearance of the gonadal ridge from the mesonephros at day 31, the onset of SRY expression and its peak at day 39, and the appearance of testis cords at day 42, along with the pattern of expression of many other genes downstream of SRY. This information will allow future researchers to check that transgenic SRY expression is occurring at the correct time and place for it to be able to cause XX sex reversal in cattle. I also identified some of the major time points important for female sex determination, including that ovigerous cords form between CRL 37-91 in female bovine embryos. In addition I show the cellular differentiation of the cortex and medulla at this time. I have also predicted the female germ cell entry into meiosis around CRL 40 in bovine embryos through the use of qRT-PCR for STRA8 and SYCP3. This is the first detailed account of gene expression profiles in early female bovine embryos, unfortunately the data is incomplete due to an uneven distribution of embryo ages due to the difficulty of obtaining embryos from timed matings. Hopefully in the future obtaining more female embryos of the missing stages can complete the female data. This project has provided additional basic knowledge about bovine sex-determination events to ensure the possibility of making single-sex livestock a real possibility in the future. The similarity between human and bovine developmental time frames also points to cattle being a good alternative model for human development, and emphasises the need for further research in species other than mouse, with the aim of ultimately understanding our own biology.

Despite the general advice of avoiding medication during pregnancy, the majority of pregnant woman use one or more ‘over the counter’ analgesics. During the last few years there has been growing evidence that analgesic exposure, such as paracetamol, ibuprofen or indomethacin, during pregnancy can have detrimental effects on rodent and human fetal gonads. The majority of previous studies have focused in alterations in testosterone production and male reproductive disorders. However, few studies have analysed the effect of these analgesics on fetal germ cells and possible consequences on fertility. During my thesis, I first focused on the effect of paracetamol and indomethacin exposure during pregnancy on rat fetal gonads. These showed that both paracetamol and indomethacin are able to alter the expression of genes important for fetal gonad and germ cell development. Previous studies on germ cells and analgesics have focused on rat models, but there is a lack of similar studies performed in human models. Therefore, I investigated the consequences of exposure of therapeutically relevant doses of paracetamol and ibuprofen on human gonads, with a special attention to the germ cells. Fetal gonads from the 1st and 2nd trimester were used in two different models: hanging drop cultures for 1st trimester testes and ovaries and a xenograft system for 2nd trimester fetal testes. Fetal gonad culture in the presence of paracetamol or ibuprofen reduced AP2γ+ (gonocyte) GC number in both 1st trimester fetal testes (22-28% reduction) and ovaries (43-49% reduction). 2nd trimester fetal testes were exposed to three different regimes, 1 or 7 days paracetamol and 7 days ibuprofen, which led to reductions of 17% and 30%, respectively in AP2γ+ GC number for paracetamol and a 53% reduction in total germ cell number for ibuprofen.

In the mouse embryo, the first definitive haematopoietic stem cells (HSCs), capable of repopulating adult irradiated mice, emerge at mid-gestation by embryonic day E11. At this stage, the aorta-gonad-mesonephros (AGM) region is able to initiate and expand HSCs. Recently, it has been shown that the development of HSC in the AGM region results from the maturation of haematopoietic precursors called pre-HSCs. Mounting evidence points at an endothelial origin for these cells, the haematogenic endothelium. Analysis of VEGFs mutants, a critical pathway for endothelial developement, suggested that it also plays a role during early haematopoiesis. The main receptor of the pathway, FLK-1 (also known as VEGRR2 or KDR), is expressed in early hematopoietic and endothelial cells in the mouse embryo. Knock-out mutants for Flk-1 showed a decrease of endothelial and intra-embryonic haematopoietic progenitors. Although Flk-1 has been identified as an essential gene for HSC emergence, its exact point of action in HSC development remains unknown. In this thesis, I investigated the role of FLK-1 signalling in haematopoietic development and defined precise stages and cell types during HSC emergence in which FLK-1 is critically involved. by using a reporter line and antibody staining, I demonstrated that FLK-1 is expressed in the pre-HSCs/HSC lineage. Germ-line Flk-1 knockout results in embryonic lethality at around E9.0, before HSC emergence, mainly due to defects in vasculogenesis. Since arterial specification precedes HSC formation, it has never been elucidated whether the haematopoietic defects found in the knockouts are a secondary effect of the loss of vasculature or it FLK-1 is directly involved in haematopoietic specification. Therefore, to determine the role of the receptor in HSC development, I used a conditional inducible mutagenesis approach that allowed the deletion of Flk-1 precisely when pre-HSCs mature into HSCs at E10.5 and E11.5. My data showed that Flk-1 deletion at these stages affects both endothelial and haematopoietic progenitors, as well as HSCs. This suggests that the VEGF pathway is not only essential in early stages of haematopoietic development, as previously demonstrated, but it may be also involved in the maturation of pre HSC into HSCs at later stages.

Environmental and anthropogenic stressors can affect wildlife populations in a number of ways. For marine mammals (e.g. the Florida manatee Trichechus manatus latirostris), certain stressors or conservation risk factors have been identified, but sublethal effects have been very difficult to assess using traditional methods. The development of 'biomarkers' allows us to correlate effects, such as impaired reproduction, with possible causes. A recently developed biomarker (anti-Müllerian hormone, AMH) provides an enzyme-linked immunosorbent assay of gonadal function. The study objective was to determine AMH levels in wild manatees. In total, 28 male and 17 female manatee serum samples were assayed. Animal demographics included collection date, body weight (kg) and total length (cm). In certain cases, age of individuals was also known. AMH levels ranged from 160 to 2451.85 ng ml -1 (mean = 844.65 ng ml -1) in males and 0.00 to 0.38 ng ml -1 (mean = 0.10 ng ml -1) in females. Linear regression analyses revealed a significant relationship between male AMH levels and body weight (R 2 = 0.452; p < 0.001) and length (R 2 = 0.338; p < 0.001). Due to the small sample size, regression analyses for female AMH and body weight and length were not significant. This represents the first report of AMH detection in a marine mammal. AMH levels in male manatees are the highest of any species observed to date, whereas levels in females are within reported ranges. Further studies will promote improved conservation decision by assessing AMH levels in the manatee as a function of various stressors including, but not limited to, nutritional status, serious injuries (e.g. watercraft collisions), exposure to biotoxins or contaminants, or disease.

lin-28 was first characterized as a developmental timing regulator in Caenorhabditis elegans. Loss of lin-28 function (lin-28(lf)) mutants skip the hypodermal cell fates specific to the 2nd larval stage. Here, we studied two aspects of lin-28 which had not yet been investigated. First, we show that lin-28(lf) mutants exhibit reduced fertility associated with abnormal somatic gonadal morphology. In particular, the abnormal spermatheca-uterine valve morphology of lin-28(lf) hermaphrodites traps embryos in the spermatheca, which disrupts ovulation and causes embryonic lethality. The same genes downstream of lin-28 in the regulation of hypodermal developmental timing also act downstream of lin-28 in somatic gonadal morphogenesis and fertility. Importantly, we find that hypodermal expression, but not somatic gonadal expression, of lin-28 is sufficient for restoring normal somatic gonadal morphology in lin-28(lf) mutants. We propose that the abnormal somatic gonadal morphogenesis of lin-28(lf) hermaphrodites results from temporal discoordination between the accelerated hypodermal development and normally timed somatic gonadal development. Thus, our findings exemplify how a cell-intrinsic developmental timing program can also control proper development of other interacting tissues, cell non-autonomously. We also investigated the expression patterns and functions of two lin-28 isoforms in C. elegans. Our analysis of spatial expression patterns suggests that lin-28a and lin-28b are co-expressed in diverse tissues. Consistently, neither of isoform specific knock-out mutant, lin-28a(lf) or lin-28b(lf), exhibits defects in hypodermal development, somatic gonad, or fertility, indicating functional redundancy of two isoforms. Our study will contribute to further investigation of lin-28 isoforms by providing the mutants of each isoform as well as the primary analysis of their phenotypes.

<p>Understanding transcriptional regulation in development and disease is one of the central questions in modern biology. The current working model is that Transcription Factors (TFs) combinatorially bind to specific regions of the genome and drive the expression of groups of genes in a cell-type specific fashion. In organisms with large genomes, particularly mammals, TFs bind to enhancer regions that are often several kilobases away from the genes they regulate, which makes identifying the regulators of gene expression difficult. In order to overcome these obstacles and uncover transcriptional regulatory networks, we used an approach combining expression profiling and genome-wide identification of enhancers followed by motif analysis. Further, we applied these approaches to uncover the TFs important in mammalian sex determination.</p><p>Using expression data from a panel of 19 human cell lines we identified genes showing patterns of cell-type specific up-regulation, down-regulation and constitutive expression. We then utilized matched DNase-seq data to assign DNase Hypersensitivity Sites (DHSs) to each gene based on proximity. These DHSs were scanned for matches to motifs and compiled to generate scores reflecting the presence of TF binding sites (TFBSs) in each gene's putative regulatory regions. We used a sparse logistic regression classifier to classify differentially regulated groups of genes. Comparing our approach to proximal promoter regions, we discovered that using sequence features in regions of open chromatin provided significant performance improvement. Crucially, we discovered both known and novel regulators of gene expression in different cell types. For some of these TFs, we found cell-type specific footprints indicating direct binding to their cognate motifs.</p><p>The mammalian gonad is an excellent system to study cell fate determination processes and the dynamic regulation orchestrated by TFs in development. At embryonic day (E) 10.5, the bipotential gonad initiates either testis development in XY embryos, or ovarian development in XX embryos. Genetic studies over the last 3 decades have revealed about 30 genes important in this process, but there are still significant gaps in our understanding. Specifically, we do not know the network of TFs and their specific combinations that cause the rapid changes in gene expression observed during gonadal fate commitment. Further, more than half the cases of human sex reversal are as yet unexplained. </p><p>To apply the methods we developed to identify regulators of gene expression to the gonad, we took two approaches. First, we carried out a careful dissection of the transcriptional dynamics during gonad differentiation in the critical window between E11.0 and E12.0. We profiled the transcriptome at 6 equally spaced time points and developed a Hidden Markov Model to reveal the cascades of transcription that drive the differentiation of the gonad. Further, we discovered that while the ovary maintains its transcriptional state at this early stage, concurrent up- and down-regulation of hundreds of genes are orchestrated by the testis pathway. Further, we compared two different strains of mice with differential susceptibility to XY male-to-female sex reversal. This analysis revealed that in the C57BL/6J strain, the male pathway is delayed by ~5 hours, likely explaining the increased susceptibility to sex reversal in this strain. Finally, we validated the function of Lmo4, a transcriptional co-factor up-regulated in XY gonads at E11.6 in both strains. RNAi mediated knockdown of Lmo4 in primary gonadal cells led to the down-regulation of male pathway genes including key regulators such as Sox9 and Fgf9. </p><p>To find the enhancers in the XY gonad, we conducted DNase-seq in E13.5 XY supporting cells. In addition, we conducted ChIP-seq for H3K27ac, a mark correlated with active enhancer activity. Further, we conducted motif analysis to reveal novel regulators of sex determination. Our work is an important step towards combining expression and chromatin profiling data to assemble transcriptional networks and is applicable to several systems.</p> / Dissertation

<p>The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly <italic>in vivo</italic>. 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. The early gonad is composed of four lineages: supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. Each lineage in the early gonad consists of bipotential progenitors capable of adopting either a male or female fate, which they do in a coordinated manner to form a functional testis or ovary. The supporting cell lineage is of particular interest because the decision of these cells to adopt the male or female fate dictates the fate of the gonad as a whole. </p><p><p>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 of the four lineages 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. Our analysis identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We also determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. Multipotent progenitors that show lineage priming express markers of the various fates into which they can differentiate and subsequently silence genes associated with the fate not adopted as they differentiate. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cell progenitors were primed with a female bias. This yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates. </p><p><p>We also used a genetic approach to investigate how individual factors contribute to the adoption of the male supporting cell fate. We previously demonstrated that <italic>Fgf9</italic> and <italic>Wnt4</italic> act as mutually antagonistic factors to promote male or female development of the bipotential mammalian gonad. <italic>Fgf9</italic> is necessary to maintain <italic>Sox9</italic> expression, which drives male development. However, whether FGF9 acted directly on <italic>Sox9</italic> or indirectly through repression of <italic>Wnt4</italic>, was unknown. <italic>Wnt4</italic> is a female-primed gene, and is therefore repressed during male development. To determine how <italic>Fgf9</italic> functioned, we generated double <italic>Fgf9/Wnt4</italic> and <italic>Fgfr2/Wnt4</italic> mutants. While single XY <italic>Fgf9</italic> and <italic>Fgfr2</italic> mutants showed partial or complete male-to-female sex reversal, loss of <italic>Wnt4</italic> in an <italic>Fgf9</italic> or <italic>Fgfr2</italic> mutant background rescued normal testis development. We also found that <italic>Wnt4</italic> and another female-associated gene (<italic>Rspo1</italic>) were derepressed in <italic>Fgf9</italic> mutants prior to the down-regulation of <italic>Sox9</italic>. Thus, the primary function of <italic>Fgf9</italic> is the repression of female genes, including <italic>Wnt4</italic>. We also tested the reciprocal possibility: that de-repression of <italic>Fgf9</italic> was responsible for the aspects of male development observed in XX <italic>Wnt4</italic> mutants. However, we show that loss of <italic>Fgf9</italic> in XX <italic>Wnt4<super>-/-</super></italic> gonads does not rescue the partial female-to-male sex reversal. </p><p><p>Based on the <italic>Fgf9/Wnt4</italic> double mutant studies, we propose a two part model of male sex determination in which both the activation of male genes and repression of female genes is required. Also, this work demonstrates that the repression of the female-primed gene <italic>Wnt4</italic> is required for male development, and <italic>Fgf9</italic> is one factor that leads to the repression of female-primed genes.</p> / Dissertation