School of BioSciences - Theses
Permanent URI for this collection
Search Results
1 - 10 of 246
-
ItemUnderstanding the role of companion of cellulose synthase1 (CC1) in maintaining cellulose synthesis under salt stress( 2022)Salt stress is one of the most detrimental abiotic stresses for plants, and substantially impacts plant biomass and agricultural productivity. In the last decades, revealing how plants cope with stress conditions and maintain growth under salt stress has been an important focus in plant research and agricultural development. The plant cell wall, which encases plant cells and functions as a cellular exoskeleton, is an important structure to cope with such stresses. One of the main components of the cell wall is cellulose, which is synthesized by cellulose synthase (CESA) complexes (CSC) at the plasma membrane by moving along underlying cortical microtubules. COMPANION OF CELLULOSE SYNTHASE (CC) 1 and 2 are components of the CSCs and links the CSCs to the microtubules. CC1 and CC2 function in maintaining cellulose synthesis under salt stress by supporting microtubules and CESA behaviors. However, the exact regulatory mechanisms of the CC1 and CC2 proteins remain largely unknown. In this thesis, the regulatory mechanisms of CC1 are investigated in more details. In Chapter 2, a comprehensive phylogenetic analysis shows that the CC protein family contains up to seven members in land plants, with six CCs present in the Arabidopsis thaliana (A. thaliana) genome. The chimeric constructs swapping the N-terminus of CC1 with different homologs, i.e., CC2-CC6, were generated and transformed into cc1cc2 double mutants. The phenotypic analyses showed that CC1 to CC4 and CC6 behaved similar to CC1 in supporting plant growth under salt stress, while CC5 did not. These inabilities of CC5 were due to its defects in microtubule-binding, microtubule bundling, and maintenance of microtubules stability under salt stress, as well as other changes in functionalities of the N-terminal part of the protein. CC5 was found to have a dominant negative effect on plant root growth. Furthermore, CC5 was specifically expressed in pollen and inhibited pollen germination under salt stress. These findings reveal functional differences among CC protein family members and improve our understanding of the four microtubule-binding motifs of CC1. In Chapter 3, an immunoprecipitation-mass spectrometry (IP-MS) analysis was performed to identify potential interactors of CC1 under normal conditions and salt stress. Among the candidates, FERONIA (FER) was selected for further study. It was found that FER interacted and phosphorylated CC1, and the phosphorylation of CC1 by FER negatively affected its in vitro microtubule-binding and microtubule-bundling abilities. In Chapter 4, in planta phospho-proteomic analysis was performed and potential phosphorylation sites of CC1 were identified. Here, BRASSINOSTEROID INSENSITIVE 2 (BIN2) was found to phosphorylate CC1, and the relevant phospho-null and phospho-mimetic mutants were generated and briefly characterized. These findings provide further insights into the regulatory mechanisms of CC1, which are important to better understand how plants maintain cellulose synthesis and growth under salt stress.
-
ItemThe metaboloepigenetic regulation of preimplantation embryo development and fetal programming by ketone bodies( 2022)Diet-induced nutritional changes in the maternal reproductive tract elicit embryonic adaptations to maintain growth and survival, changes which nonetheless compromise long-term child and adult health via developmental programming. This is plausibly facilitated by metaboloepigenetic mechanisms, whereby metabolic sensing of nutrient availability coordinates epigenetic regulation of gene expression in a developmentally persistent manner, affecting short- and long-term embryo physiology and health. Despite little evidence for its efficacy and developmental safety, the very high fat, low carbohydrate ketogenic diet (KD) is increasing in popularity as a ‘fertility diet’ worldwide. In line with the hypothesis that metaboloepigenetics links diet with embryo health, KD consumption elevates systemic concentrations of the ketones beta-hydroxybutyrate (BOHB) and acetoacetate (AcAc), which in addition to their role as oxidative fuels, are epigenetic modifiers and may consequently affect embryo development. Therefore, to investigate the safety of a periconceptional KD for offspring health, it is necessary to determine the impact of ketones on the development, physiology, and viability of the preimplantation embryo. Using a mouse model, the impact of in vitro ketone exposure on preimplantation embryo development, physiology and viability was assessed. Exposure to BOHB alone perturbed blastocyst development, decreased glucose metabolism, and increased histone acetylation, predominantly impacting the trophectoderm cell lineage. This was further associated with implantation failure and delayed female fetal development post-transfer, suggesting preimplantation BOHB exposure is detrimental to development in a female-specific manner via a trophectoderm-mediated mechanism. Similarly, in vitro exposure to AcAc alone or AcAc + BOHB in combination did not impair morphological development of the blastocyst, however impacted glucose metabolism and cumulatively increased histone acetylation. Preimplantation AcAc + BOHB exposure also increased miscarriage rates and delayed female fetal development post-transfer. Consistently, RNA-seq analysis identified persistent and sexually dimorphic effects of in vitro preimplantation BOHB and AcAc + BOHB exposure on fetal liver and placental gene expression. BOHB downregulated cholesterol synthesis pathways in female placenta, indicating compromised placental function, possibly contributing to the observed impairments in pregnancy establishment and maintenance. Oxidative metabolism was downregulated in ketone-treated female fetal liver, highlighting that ketones also affect the inner cell mass lineage and exert persistent changes in metabolic function. Notably, X-linked genes were overexpressed in ketone-treated female tissues, alluding to a mechanism underlying the sexually dimorphic effects of ketones, plausibly via dysregulation of X-inactivation. Finally, a maternal periconceptional KD was confirmed to elevate BOHB concentrations within mouse oviduct fluid, concomitant with delayed in vivo blastocyst development and altered trophectoderm-specific histone acetylation, corroborating the findings from prior in vitro studies. The data within this thesis provide a comprehensive analysis of the negative impact of ketone exposure on embryonic development and viability, confirming that metaboloepigenetic processes within the preimplantation embryo are sensitive to ketones, and provide mechanistic insight into sex-specific developmental programming. These findings suggest that periconceptional ketogenic diet consumption may be detrimental to long-term health, such that a KD during the periconception period is inadvisable. These findings may provide essential guidance for professional recommendations regarding dietary choices to improve fertility without increasing the time to pregnancy or compromising child health.
-
ItemUsing indigenous microinvertebrates to assess the environmental impacts of soil pollution in Antarctica( 2022)Modern humans have had a continuing presence in Antarctica since at least the 1800s, and this presence has come with an environmental footprint. Since the introduction of the Madrid protocol in the 1990s, the Antarctic Treaty has ensured the protection of the environment from current and future impacts of human activity and enshrines a responsibility to remediate legacy contamination. Ice-free areas of Antarctica only account for a small fraction (<0.5%) of the landmass of the continent but are the location of >94% of current permanent stations. The concentration of human activity in these areas disproportionately exposes them to the impact of our activities. Ecotoxicologically-derived sensitivity estimates for endemic species can be used to assess the impacts of soil contamination on terrestrial Antarctic environments, informing risk assessment and land management for environmental protection. Prior to the work completed herein, there was little ecotoxicological information available for indigenous terrestrial microinvertebrates from Antarctica and none for tardigrades and rotifers. This thesis investigated the use of endemic terrestrial rotifers and tardigrades from Antarctica to assess the impacts of metals (Cu and Ni) present in the environment from historic waste disposal sites on terrestrial Antarctic ecosystems. The thesis was split into two stages: an initial stage in which cultures of terrestrial Antarctic microinvertebrates were established and life history traits recorded, and a second, in which the sensitivity of these microinvertebrates to Cu and Ni was determined in soil-based media. Soil based media were used to model the physiochemical conditions experience by the terrestrially-sourced rotifers and tardigrades in the environment. The bdelloid rotifer 'Habrotrocha' sp. and tardigrade 'Acutuncus' sp. were successfully isolated from moss and soil samples and cultured in two soil-based growth mediums, a soil elutriate, and a balanced salt solution (BSS). Both BSS and soil solution were suitable growth media for 'Habrotrocha' sp. and 'Acutuncus' sp., and moderate dilutions of soil elutriate were optimal for rotifer population growth. This was the first example of a soil-based medium being used to culture tardigrades or rotifers. The life history traits of the rotifer 'Habrotrocha' sp. were also assessed and found that they were long-lived compared to equivalent rotifers from the literature, with similar reproductive output across their lifespan. A meta-analysis conducted on the interaction between bdelloid rotifer life history traits and culturing temperature found that rotifer longevity and time to reproductive maturity were strongly negatively correlated with culturing temperature, expanding our knowledge of these organisms. The sensitivity of three terrestrial Antarctic microinvertebrate species to contaminants was assessed throughout this thesis, two bdelloid rotifers 'Philodina' sp., and 'Habrotrocha' sp, and one or more species of tardigrade, from the Family Hypsibiidae. 'Philodina' sp. directly isolated from environmental samples exposed to Cu in a water medium were highly sensitive compared to other bdelloid rotifers and demonstrated apparent toxicant-induced cryptobiosis as a novel endpoint for sensitivity estimates, a first for these taxa. In contrast, 'Habrotrocha' sp. and 'Hypsibiidae' sp. were exposed to Cu and Ni in a soil elutriate, demonstrating high tolerance to both metals compared to equivalent temperate rotifers, and similar microinvertebrates (e.g. nematodes) from the literature. The procedures and initial response data presented in this thesis will contribute to the establishment of environmental quality guidelines for soil contamination in Australia’s Antarctic Territory. The procedures can also be utilised for terrestrial microinvertebrates from other climates, assisting in incorporating more species into environmental risk assessments.
-
ItemBuilding a wall: Developing small molecule biosensors to visualize cell wall biosynthesis and untangling mechanisms underlying nucleotide sugar transport( 2022)The cell wall is one of the main energy sinks in plants constituted of many polysaccharides and glycoproteins. The synthesis of most polysaccharides and the proteins glycosylation occur in the Golgi apparatus. The nucleotide sugars are the precursors of the cell wall building blocks. These molecules are biosynthesized in the cytosol from sugars of the primary metabolism and are transported from the cytosol to the Golgi by the nucleotide sugar transporters (NSTs). The glycosyltransferases (GTs) then consume these nucleotide sugars to produce polysaccharides and the glycoproteins. Although we have a generalized overview, our knowledge of the exact roles of NSTs and nucleotide sugars in regulating the cell wall synthesis is still sparse. The recent determination of the crystal structures of two NSTs, the GDP-D-mannose transporter VRG4 from yeast and the orthologue of CMP-sialic acid transporter from maize indicate that conformational changes occur during the transport process. Therefore, NSTs are excellent candidates to develop fluorescent proteins-based sensors to study the flow of nucleotide sugars. In chapter II, our aim was to generate fluorescent protein- biosensors based on the UDP-XYLOSE TRANSPORTER 1 (UXT1) to follow the import of UDP-D-xylose into the Golgi in vivo as a proxy for xylan and xyloglucan biosynthesis. We show that the UXT1-based FRET sensors maintain the physiological localization of UXT1 in planta, while transporting UDP-D-Xyl in vitro. We also designed ratiometric sensors based on the sfGOMatryoshka. However, these sensors were found to disrupt the Golgi localization of UXT1. In chapter III, we aimed to probe the existence of protein complexes involved in arabinosylation and study the regulation of the arabinosylation pathway. Investigating higher order mutants of the UDP-ARABINOFURANOSE TRANSPORTERs (UAfTs), allowed us to decipher the relative contribution of each of the UAfTs to arabinosylation. These newly generated mutants in combination with other mutants of the arabinosylation pathway led us to propose a mechanistic model to explain a glucose hypersensitivity phenotype in the dark. Phenotypic assessment of nucleotide sugar levels and hypocotyl elongation allowed us to make progress towards dissecting the role of cytosolic UDP-GLUCOSE EPIMERASE 1 and 3 in regulating nucleotide sugar metabolism. Finally, using affinity purification, split-ubiquitin assays and in silico co-expression approaches, we unravel putative NST-GT complexes and suggest that these complexes also involve nucleotide sugar interconverting enzymes.
-
ItemRibosome Heterogeneity and Specialization during Temperature Acclimation in Plants( 2022)Ribosomes decode mRNA to synthesize proteins. Ribosomes, once considered static, executing machines, are now viewed as dynamic modulators of translation. Increasingly detailed analyses of structural ribosome heterogeneity led to a paradigm shift toward ribosome specialization for selective translation. As sessile organisms, plants cannot escape harmful environments and evolved strategies to withstand. Plant cytosolic ribosomes are in some respects more diverse than those of other metazoans. This diversity may contribute to plant stress acclimation. The goal of this thesis was to determine whether plants use ribosome heterogeneity to regulate protein synthesis through specialized translation. I focused on temperature acclimation, specifically on shifts to low temperatures. During cold acclimation, Arabidopsis ceases growth for seven days while establishing the responses required to resume growth. Earlier results indicate that ribosome biogenesis is essential for cold acclimation. REIL mutants (reil-dkos) lacking a 60S maturation factor do not acclimate successfully and do not resume growth. Using these genotypes, I ascribed cold-induced defects of ribosome biogenesis to the assembly of the polypeptide exit tunnel (PET) by performing spatial statistics of rProtein changes mapped onto the plant 80S structure. I discovered that growth cessation and PET remodeling also occurs in barley, suggesting a general cold response in plants. Cold triggered PET remodeling is consistent with the function of Rei-1, a REIL homolog of yeast, which performs PET quality control. Using seminal data of ribosome specialization, I show that yeast remodels the tRNA entry site of ribosomes upon change of carbon sources and demonstrate that spatially constrained remodeling of ribosomes in metazoans may modulate protein synthesis. I argue that regional remodeling may be a form of ribosome specialization and show that heterogeneous cytosolic polysomes accumulate after cold acclimation, leading to shifts in the translational output that differs between wild-type and reil-dkos. I found that heterogeneous complexes consist of newly synthesized and reused proteins. I propose that tailored ribosome complexes enable free 60S subunits to select specific 48S initiation complexes for translation. Cold acclimated ribosomes through ribosome remodeling synthesize a novel proteome consistent with known mechanisms of cold acclimation. The main hypothesis arising from my thesis is that heterogeneous/ specialized ribosomes alter translation preferences, adjust the proteome and thereby activate plant programs for successful cold acclimation.
-
ItemSystematics and biogeography of Spyridium with a focus on Spyridium parvifolium and its hybrids( 2022)Spyridium is a genus of c. 45 species endemic to south-western and south-eastern Australia, with a disjunct distribution across the Nullarbor Plain and Bass Strait. The genus also includes several morphologically distinct phrase name taxa. Spyridium parvifolium is a widespread and morphologically variable shrub from south-eastern Australia. Several varieties and forms of this species have been recognised, but there is disagreement on the accepted taxonomy between Australian states. Spyridium parvifolium is known to hybridise with S. daltonii in the Grampians and is thought to hybridise with S. vexilliferum in locations where these taxa co-occur in western Victoria and south-eastern South Australia. The aim of this research project was to develop a comprehensive molecular systematic understanding of Spyridium, and S. parvifolium and its hybrids, to inform the treatment of Rhamnaceae in the Flora of Australia (Kellermann et al. 2022-). The objectives were to investigate: the species circumscription and biogeographic history of Spyridium, the infraspecific taxa and phylogeographic patterns of S. parvifolium and introgression associated with this species (i.e. S. xramosissimum and S. parvifolium x S. vexilliferum). Entire chloroplast genomes (c. 160k base pairs) and the nuclear ribosomal array (18S–5.8S–26S; c. 6k base pairs) were analysed using both Bayesian and Maximum Likelihood phylogenetic methods. In total sequences from 230 samples were analysed across these phylogenies, including representatives of all recognised species of Spyridium, six phrase name taxa, seventy-two accessions of S. parvifolium, eight putative hybrids and four outgroup taxa. This study provides the most comprehensive phylogenies of Spyridium and S. parvifolium to date. For Spyridium, several biogeographic patterns were identified, including deep diverging clades of taxa endemic to Western Australia, New South Wales and Tasmania. Several taxa were identified as polyphyletic (e.g. S. eriocephalum and S. phylicoides), warranting taxonomical review. For S. parvifolium, early divergence of individuals from west of the Murray Darling Depression, isolation on the inland side of the Great Dividing Range and recent seed-mediated gene-flow across Bass Strait were identified in the chloroplast genome phylogeny. The variants of S. parvifolium were not supported as genetically distinct suggesting the infraspecific recognition of var. parvifolium and var. molle in Tasmania is not warranted. Molecular evidence of introgression between S. parvifolium and both S. daltonii and S. vexilliferum was identified, providing molecular support for hybrids also inferred from intermediate morphology. Other findings include inferred parentage, unidirectional introgression and recombination of the nuclear ribosomal array for some hybrid accessions.
-
ItemVisual, thermal, and behavioural adaptions of jewel beetles( 2022)Living organisms have evolved a myriad of traits to adapt to different environments. Understanding the relationship between trait, function, and adaptive value is fundamental to understanding the evolution of this diversity. Insects are one of the most diverse animal groups that have adapted to almost every terrestrial and freshwater environment on earth. Among them, jewel beetles (Coleoptera: Buprestidae) are reported to have a relatively uncommon ability to see longer wavelengths, exploit sunlight beyond the human visible range, and employ a unique predator escape behaviour. In this dissertation, I examine these fascinating visual, thermal, and behavioural adaptations and their ecological significance. I first use visual modelling and a behavioural assay to provide evidence that having visual sensitivity to longer ‘red’ wavelengths enhances ability to discriminate resources in insects (Chapter 2). I next demonstrate that near-infrared reflectance has a stronger influence on heat transfer than ultraviolet-visible reflectance using empirical experiments (Chapter 3). However, phylogenetic comparative analyses I conducted show that near-infrared reflectance is not driven by climate but rather by body size in jewel beetles (Chapter 4). Finally, I document the kinematics of the unexplored flicking behaviour in Astraeus jewel beetles with high-speed videography and reveal its potential importance for predator escape at low ambient temperatures with a series of behavioural experiments (Chapter 5). To understand these diverse adaptations in jewel beetles, I used interdisciplinary approaches and integrated knowledge from different fields. The results of this dissertation advance our understanding of the evolution and ecology of exploitation of light beyond the human visible range. It also provides new knowledge of animal ultrafast movement with promising potential for bioinspired applications, such as robotic design in energy-efficient 3D locomotion. Taken together, this dissertation highlights the importance of research on the relationship between traits, mechanism, and adaptive value for biological understanding as well as broader applications.
-
ItemAn investigation into monogenic causes of male infertility( 2022)Infertility affects 7% of men in developed societies, and the aetiology of approximately 70% of cases remains unexplained. Genetics plays a role in severe forms of male infertility, however, using current clinical approaches, it only explains 4% of cases. The overarching objective of this thesis was to identify novel genetic causes of male infertility. The work carried out here can be split into two branches – the first is an unbiased method of gene discovery using exome sequencing data from infertile men, and the second is a gene-targeted approach conducted using model organisms. In the first approach, I performed exome sequencing in 186 men with low sperm counts and their fertile parents. Potentially pathogenic variants were identified by filtering for rare variants (gnomAD AF < 0.01) classified as pathogenic or unclear by the guidelines of the American College of Medical Genetics. Genes affected by these variants were prioritized according to their likely role in male fertility. A replication analysis of candidate genes was performed in a cohort of infertile men (n=1,580) and fertile men (n=5,784). I identified 50 potential pathogenic variants (20 homozygous, 30 compound heterozygous) affecting 49 protein-encoding genes with a potential role in male fertility. Our replication analysis revealed a significant enrichment of likely pathogenic homozygous mutations in 5 of these 49 genes (AATF, FKBP6, M1AP, TEX14, and TOPAZ1, burden test p<0.05) in infertile compared to fertile men. In the second approach, model organisms were used to test and characterise the role of microtubule-associated proteins in male fertility. Microtubules are an essential component of eukaryotic cells that during spermatogenesis are required for multiple processes including cell division, sperm head shaping, and tail formation. Microtubules are regulated via microtubule-associated proteins, and while the function of many of these proteins in spermatogenesis remains poorly understood, mutations that result in male infertility in humans and model species are increasingly being identified in genes that encode microtubule-associated proteins. As part of this approach, 37 microtubule-associated genes identified from literature searches were knocked down in the testes of D. melanogaster to determine their requirement for spermatogenesis. In doing so, 25 genes that were required to maintain normal male fertility were identified, as measured by the number of offspring produced when mated to wild-type females. The genes that caused a fertility phenotype were enriched for those in the kinesin family of microtubule severing enzymes. In addition, a mouse model containing a loss of function mutation in the microtubule-severing enzyme spastin was characterised. Data revealed that spastin plays a critical role in the assembly and function of the meiotic spindle. Consistent with meiotic failure, round spermatid nuclei were enlarged, indicating aneuploidy, but were still able to enter spermiogenesis. During spermiogenesis, extreme abnormalities in manchette structure, supernumerary acrosome formation, and, commonly, loss of nuclear integrity were observed. Collectively, this thesis identified multiple genes with high-confidence roles in male infertility, as well as novel mechanisms required for germ cell development. These data demonstrate the highly heterogeneous nature of male infertility and underscore the importance and complexity of regulation of microtubules during spermatogenesis.
-
ItemMolecular evolution of algal genomes( 2022)Patterns of molecular evolution vary strikingly among diverse organisms across the tree of life. Molecular evolution is speculated to be associated with various factors such as endosymbiosis and organismal traits like body architecture and lifecycles. These factors are hypothesised to impact selection-drift balance that underlies molecular evolution. Moreover, transition to a new intracellular environment during endosymbiosis is speculated to be associated with relaxed selection due to reduced functional constrains and decreased effective population size. Increased body architecture is speculated to decrease effective population size, which will increase genetic drift. Likewise increased ploidy levels associated with lifecycles will increase genetic drift due to masking mutations in less dominant alleles. Most of these speculations have been studied only on limited taxon groups or model systems and they remain largely unexplored on diverse group of organisms. Algae are my model to study these hypotheses. Algae have long evolutionary history and exhibit wide range of diversity in their origin, body architecture and lifecycles. In my second chapter, I studied the molecular evolution of plastid genomes through secondary endosymbiosis and proposed a general model for molecular dynamics during secondary plastid endosymbiosis that underlined the role of population bottleneck in the endosymbiont establishment. This provides further insights to the molecular dynamics during the origin of the secondary plastids. My third and fourth chapter aims to study the impacts of body architecture and lifecycles on green algal and brown algal genomes respectively. My studies highlight the prominent impacts of body architectures including the features of cellularity and multiple nuclei on the evolution of green algal genomes. In addition to lifecycle and body architecture, brown algal studies highlighted the impacts of lineage specific selection pattern in a few brown algal lineages.
-
ItemDeveloping the Dunnart Model: iPSCs and ART as tools for marsupial conservation and genetic manipulation( 2022)With many marsupials at risk of extinction, we need powerful marsupial laboratory models and innovative new tools to buffer conservation efforts for endangered species. However, improved resources for marsupial models are also essential for deepening our understanding of mammalian development and evolution through the power of comparative genetics. We are developing technologies that will establish the fat-tailed dunnart (Sminthopsis crassicaudata) as a robust marsupial model, capable of targeted genetic manipulation. This goal requires stem cell advances, as well as in vitro derived embryos, however marsupial models lack protocols for these. Since the derivation of embryonic stem cells from marsupials is challenging, I reprogrammed dunnart fibroblasts to induced pluripotent stem cells (iPSCs). These iPSCs express core pluripotency genes and are capable of differentiation to all three embryonic germ layers. I, further, used single-cell RNA-sequencing to explore the transcriptome for these cells and discovered enrichment for networks implicated in pluripotency and early development. Dunnart iPSCs offer a route to loss and gain-of-function experimentation via the integration of modified iPSCs into embryos. However, a simpler method would necessarily involve editing zygotes, enabling the direct generation of genetically modified lines. Timing ovulation in marsupials is difficult, however, the use of IVF would allow the generation of many embryos at the right stage, facilitating repeatable high throughput genetic manipulation. Unfortunately, IVF has not yet been successfully applied to Australian marsupials. In vitro, sperm from these animals fail to undergo the maturational changes that enable fertilisation, known as capacitation. The only exception to this, is when they undergo a complex system of co-culture with oviductal secretions. In pursuit of the optimal media conditions for this process, I used liquid chromatography mass spectrometry (LC/MS) to characterise the in vivo environment in the dunnart oviduct - the site of fertilisation. I then tested candidate metabolites and additional factors on dunnart sperm, with the aim of inducing capacitation. This had limited success, however, in the process, I discovered that incubation in bicarbonate buffered media at atmospheric oxygen is sufficient to produce the physiological outcomes associated with sperm capacitation. In these conditions, dunnart sperm bind to oocytes, undergo the acrosome reaction, and display tyrosine phosphorylation. Finally, sperm exhibit the characteristic head realignment that indicates capacitation for marsupials. This is the first time simple in vitro capacitation has been achieved for an Australian marsupial, and paves the way for progress in IVF for these iconic mammals. Importantly, both IVF and iPSC technology will greatly enhance the utility of the dunnart model. Advances in these areas are critical if we are to generate the first genetically modified Australian marsupial-lines. Furthermore, these resources will also have a vast impact on our ability to conserve vulnerable marsupial species, enabling banking of stem cells and embryos, use of in vitro derived embryos and iPSCs to reintroduce genetic diversity to inbred populations, as well as targeted intervention in disease and pathogen susceptibility. Finally, this thesis presents research that can be used to gain a better understanding of conserved developmental pathways in mammalian biology, with a novel role suggested for the oviduct as the signaling niche that regulates the earliest and most fundamental events for mammalian gametes and embryos.