No description available.

No description available.

During mammalian development a small number of pluripotent cells proliferate and differentiate to give rise to all the mature cell types of the organism. Among the earliest differentiation events is the process of gastrulation, in which pluripotent primitive ectoderm cells form the three germ lineages, mesoderm, ectoderm and endoderm under the control of complex signalling and environmental cues. This process can be modelled using embryonic stem cells, which have proven to respond to embryologically relevant signals during in vitro differentiation and promise to uncover additional insights into the process of germ lineage specification. This thesis describes the differentiation of mouse ES cells to committed cell types via a second intermediate population of pluripotent cells termed Early Primitive Ectoderm-Like (EPL) cells. The similarity of EPL cells to primitive ectoderm and the rapid acquisition of lineage specific markers and loss of pluripotent characteristics upon differentiation of EPL cells suggest they are an excellent model for the cells in the embryo that undergo germ lineage commitment. EPL cells can be differentiated as EPLEBs, which are highly enriched in mesodermal cell types and contain essentially no ectodermal derivatives and no visceral endoderm. Here it is shown that EPLEBs can be generated from EPL cells grown either adherently or in suspension culture provided the cells are reduced to a single cell suspension before reaggregation as EPLEBs. Since EPLEBs are a rich source of mesoderm and contain less non-mesodermal cell types than traditional ESEBs, they were assayed for definitive blood formation, however none was detected. Alternately, EPL cells can be differentiated in the presence of MEDII in aggregates termed EBMs, which are restricted to ectodermal cell fates. Here it is demonstrated that the switch from mesodermal to ectodermal differentiation observed in ELPEBs and EBMs relies on two variables; a mesoderm suppressing activity within MEDII and the pro-mesodermal activity of cell dissociation as undertaken during EPLEB formation. Evidence has been presented that interventions that modulate the epithelial identity of EPL cells are capable of influencing subsequent differentiation such that protection of the epithelial cell state favours ectoderm while disruption favours mesoderm. Staurosporine (SSP) is a kinase inhibitor that has been shown to induce an epithelial to mesenchymal transition in chick neural tube. Here it was added to EPL cells with the result that mesodermal differentiation was enhanced at the expense of ectoderm. DAPT is a potent inhibitor of ƴ-secretase, which cleaves a number of protein targets including the adherens junction component E-cadherin. Addition of DAPT to differentiating EPL cells has the opposite effect to SSP, with an increase in ectodermal differentiation at the expense of medoderm. It is proposed that DAPT is acting by preventing E-cadherin cleavage and thus stabilising the epithelial state. Modulation of epithelial contacts between pluripotent cells represents a novel way to control lineage induction and as such the incorporation of these findings into methodologies for directed differentiation in defined culture conditions is likely to provide improved outcomes in the production of desired cell types. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

The brain is our most complex organ, with an estimated 1011 neurons. With the spinal cord, it forms the central nervous system which controls our movements and our senses, holds our memories and creates our thoughts. Because of this, neurodegenerative disorders can be extremely distressing and a thorough understanding of how the nervous system develops is essential if progress is to be made in finding ways to treat them. Critically, this includes understanding how the nervous system forms, i.e., the nature of the signals that promote neural identity (neural induction) and determine correct positional information (patterning). The zebrafish (Danio rerio) has become established as a model for embryological studies due to ease of experimental manipulation. Taking advantage of this, the aims of this PhD were to contribute to unravelling the molecular mechanisms of neural induction and patterning, using a variety of embryological and molecular methods. In the first project, functional analyses of the eve1 gene identified a key factor for posterior neural development. Eve1 was found to be a critical posteriorising factor, with an additional role in posterior neural induction. An outstanding question in neural induction is the relative contribution to this process of two key developmentally important signalling pathways, Bmp and Fgf. In the second project, differential analyses of maternal versus zygotic Bmp and Fgf signalling revealed crucial maternal roles for these two pathways in neural development as neural and epidermal capacitators. The results further suggested that Fgf signalling may be the critical neural inducer. Finally, as a third project, a zebrafish ectodermal explant assay was developed using the organiser-deficient ichabod mutant. The aim was to develop a system to analyse how key molecules directly affect ectoderm and neural development, free of mesoderm and endoderm influences, as signalling from these layers can directly or indirectly influence neural development.

Preplacodal ectoderm (PPE) is a contigous horse-shoe shaped domain that enwraps the anterior neural plate towards the end of gastrulation and eventally resolves into a number of focal epithelial thickenings called placodes. These placodes together with Neural Crest (NC), contributes to the peripheral nervous system in vertebrates. PPE and NC arise at the neural-non neural interface by distinct mechanisms during development. However, a general idea in the field was that a Bmp signaling gradient specifies different ectodermal fates: high Bmp levels specify epidermis, intermediate levels PPE and NC and no Bmp signaling is required for neural fate specification. We showed that while NC responds to intermediate levels of Bmp signals, PPE is specified by a distinct mechanism that involves a two step model for PPE specification. In the first step, Bmp is positively required to activate four competence factors, tfap2a, tfap2c, foxi1 and gata3 throughout the ventral ectoderm and renders this domain competent to respond to inductive factors. In the second step, inductive factors Fgf and Bmp antagonists act to completely block all Bmp signaling to specify PPE at neural-non neural interface. These Bmp-activated competence factors do not need Bmp for subsequent maintenance because they positively cross-regulate and autoregulate each other’s expression forming a gene regulatory network. This network is sufficient to rescue both PPE and NC in the complete absence of Bmp. The subsequent resolution of PPE into discerte placodal thickenings was hypothesized to involve localized migration of placodal progenitors and one of the molecules that could play an important role during cell migration was extracellular matrix binding molecule, integrin alpha 5 (itga5) because it was expressed at the right time and place. Knockdown of itga5 results in disorganised trigeminal, epibranchial ganglia and smaller otic placodes. Tracing the cell trajectories of placodal progenitors revealed that cells failed to migrate directionally. Additionally, we observed elevated levels of cell death in itga5 morphants which could be rescued by overexpression of Fgf ligands suggesting that Itga5 and Fgf pathways cooperate during placodal development. All together, this dissertation reveals novel genetic mechanisms that regulate placodal development from late-blastula to mid-somitogenesis stages.

Title page, table of contents and summary only. The complete thesis in print form is available from the University of Adelaide Library. / During early embryogenesis pluripotent cells of the inner cell mass (ICM) give rise to a second pluripotent cell population known as the primitive ectoderm an obligate developmental intermediate and the substrate for gastrulation. The ICM and primitive ectoderm are distinguished on the basis of morphology, gene expression and differentiation potential. However, the signals and mechanisms involved in the transition form ICM to primitive ectoderm are not understood. Culture of ES cells in the presence of a conditioned medium MEDII leads to a transition of ES cells to a population of pluripotent primitive ectoderm-like (EPL) cells that are the in vitro equivalent of the primitive ectoderm. In terms of EPL cell formation the bioactive component of MEDII was identified as L-proline. In this thesis the molecular mechanism by which L-proline induces EPL-cell formation was elucidated. As well as L-proline, short L-proline containing peptides were also shown to induce EPL-cell formation but different peptides displayed different abilities to induce the transition with some inducing the complete transition and others inducing morphology changes only. The mechanism of L-proline induced EPL-cell formation was shown to be independent of NK receptors. The mechanism of L-proline induced EPL-cell formation, as deduced from the results presented in this thesis, was suggested to involve the internalisation of L-proline via the SAT2 amino acid transporter into ES cells as competitive inhibitors of SAT2 prevented EPL-cell formation. MAPK signalling via the action of MEK1 was implicated in L-proline induced EPL-cell formation as inhibitors of MEK1 prevented EPL-cell morphology, gene expression and differentiation potential in the presence of Lproline. PI3K signalling was implicated in L-proline-induced EPL-cell morphology since PI3K inhibitor L Y294002 maintained domed colonies in the presence of L-proline but failed to maintain an ES-cell gene expression profile and differentiation potential. Both MAPK and PI3K signalling were suggested to lie down-stream of L-proline action since treatment of ES cells with L-proline induced the activation of ERK1/2 and Akt down-stream effectors of MAPK and PI3K signalling respectively. A gene potentially involved in the Pl3K-rnediated rnorphology change was Lefty2. Therefore, the mechanism of L-proline induced EPL-cell formation appears to involve internalisation of L-proline and at least two signalling pathways down-stream of L-proline, which regulate different components of the transition. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1281009 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2007

No description available.

Elongation of the vertebrate rostrocaudal axis depends on localised populations of axial progenitors. Previous work has demonstrated the presence of Neuromesodermal (NM) progenitors that behave as multipotent stem cells, which contribute to the neurectoderm and mesoderm throughout axis elongation. They have been localised to the Node-­‐Streak Border (NSB) in the primitive streak region, and the Chordoneural Hinge (CNH) in its descendant, the tail bud. At primitive streak stages, the Caudal Lateral Ectoderm (CLE) on either side of the primitive streak itself is also fated for neurectoderm and mesoderm. However, fate mapping studies in mouse and chick have suggested that these progenitors are more transitory than those in the NSB and CNH, leading to the idea that two different types of progenitor cell exist in the primitive streak region; long-­‐term (stem cell-­‐like) and transient progenitors. In this thesis, I have examined the potency of the CLE cells by heterotopically grafting them into the NSB. Anterior CLE cells are exquisitely sensitive to their position and differentiate predominantly as neurectoderm, mesoderm, or both, depending on their exact location in the NSB. Most significantly, their descendants are retained in the CNH, indicating that CLE cells show equal potential to NSB progenitors on transplantation to the border environment. The relationship between fate and potency within the streak stage embryo suggest a mechanism by which stem cells are maintained not only by their intrinsic stem cell program, but are also influenced by their location. Furthermore, I have investigated the expression of candidate markers of NM-­‐progenitors, and have found that the combined expression of Sox2 and T genes in the progenitor area coincides with observed NM-­‐potency, and could serve as a marker for this stem cell population. Over time, axial elongation comes to a halt and NM-­‐progenitors are thereafter not longer active. It is still unclear how exactly this process occurs and specifically whether axial elongation ceases because NM progenitors are eliminated. I have investigated the events occurring immediately before the end of axial elongation. Morphological and gene expression analysis shows that apoptosis reaches a peak only after the complete axis has been laid down, and is not dramatically elevated in the progenitors themselves before that. In order to test signalling pathways that lead to progenitor maintenance, I have developed an in vitro tail growth assay that recapitulates in vivo development, as measured by several morphometric criteria. I show that, even though FGF signalling is critical for most cells in the tail bud including NM-­‐progenitors, it is not sufficient for NM-­‐ progenitor maintenance. In contrast, exposing tail buds to elevated Wnt/β-­‐catenin signalling does prolong the lifetime of NM-­‐progenitors in the ageing tail bud, as judged by the presence of Sox2-­‐T double-­‐positive cells. In this regard we have found that the time of cessation coincides with the disappearance of Sox2-­‐T double-­‐positive cells, the disappearance of Wnt3a and concomitant neuralisation of the progenitor region. This data suggest an important governing role for Wnt signalling in both maintenance and fate decision of NM progenitors. Thus the disappearance of Wnt signalling in the tail bud over time could well be the main reason for triggering the halt of caudal elongation.

No description available.

The morphogenetic events that give rise to the early post-implantation mouse embryo (egg cylinder) have not been thoroughly studied and our knowledge is restricted to "snap-shot" descriptions of embryos recovered at different stages of implantation from the mother. A central feature of the egg cylinder is the pro-amniotic cavity, which spans the embryo and participates in formation of the extraembryonic membranes. The major aims of my PhD studies have been to reveal how this cavity is formed (Aim 1) and then how the egg cylinder grows (Aim 2). In order to address how the pro-amniotic cavity forms (Aim 1), I first characterised in detail development of the architecture of the extra-embryonic ectoderm (ExE), which has to be remodelled to permit cavity formation. My findings indicate that the ExE comprises cells in direct contact with a basement membrane and cells that lie deeper in the tissue. The ExE originates in the polar trophectoderm, a monolayer covering the epiblast of the blastocyst, which expands and undergoes invagination to form a slit-like cavity. By carrying out analyses of fixed specimens and live imaging of cultured embryos, I have found that the epiblast and ExE cavity extend towards each other through the formation and resolution of multiple rosette structures. This leads to the fusion of the ExE and epiblast cavities to form the unified pro-amniotic cavity. I show that this process is dependent on signalling cues stemming from the underlying basement membrane that activate the b1-integrin signalling pathway to regulate cell polarity, ExE tissue architecture and rosette formation. In addition to the basement membrane's role in b1-integrin signalling, it also has physical functions that I characterise in the second part of my study (Aim 2). High resolution imaging revealed that the basement membrane underlying the epiblast is highly perforated during the implantation stages. These perforations are initially evenly distributed and then accumulate asymmetrically at the future posterior part of the embryo, just prior to gastrulation. Finally, I demonstrate that remodelling of the basement membrane requires the expression of matrix metalloproteinases (MMPs) in the epiblast under the control of Nodal. The anterior visceral endoderm inhibits Nodal signalling and hence MMP inhibition in the anterior. I demonstrate that activity of the MMPs and perforations in the basement membrane are essential for embryo growth. The domain of posterior basement membrane perforations persists beyond gastrulation suggesting a potential role for these perforations in primitive streak formation and extension. Together, my studies bring new important insights into the understanding of early mouse embryo morphogenesis.