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No description available.

No description available.

No description available.

No description available.

The heart is the first functional organ of embryogenesis in many vertebrates, however little is known about the early specification events of cardiogenesis. Evidence in the chick and amphibian suggests a requirement for the anterior endoderm in cardiac induction to direct mesoderm toward a cardiac fate. Furthermore, the signals responsible for specification and their mode of action are unknown. Several signalling pathways, including FGF, Nodal, BMP and Wnt have been implicated. However, as these pathways have other roles in early embryogenesis a specific role in cardiac induction has been difficult to define. We have devised a model testing the cardiac-inducing activity of the anterior endoderm addressing its ability to re-specify pluripotent embryonic ectoderm upon conjugation. We have shown that the anterior endoderm is sufficient to induce robust expression of cardiac markers and formation of contractile tissue in the responder. Characterisation of the model showed the anterior endoderm produces a specific signal skeletal muscle is not induced, distinguishing it from general mesoderm induction. The cardiac-inducing capacity of the anterior endoderm was not uniform as it was restricted to the most anterior regions of the anterior endoderm, correlating with expression of Hex. The cardiac-inducing signal requires two hours of interaction with the responding tissue during gastrulation to produce an effect. Further involvement of the anterior endoderm beyond specification of cardiac precursors was not required. The model provided the basis to investigate the early signalling events of specification. Whereas BMP signalling was not necessary for cardiac induction by the endoderm, an essential requirement for FGF and Nodal pathways was shown. Timed inhibition revealed both were required during the first hour of conjugation, while sustained ERK activation was needed for at least four hours. In addition it was shown that elevated Wnt/p-catenin signalling during specification had no effect, while sustained activation antagonised cardiogenesis. Further analysis revealed Wnt/p-catenin has no direct role in specification, but suppression or low activity was required prior to the onset of cardiac differentiation. Therefore, this work established a simple and experimentally amenable assay for elucidating the mechanisms of cardiac specification.

Using <italic>Xenopus laevis</italic> embryos, the aim of this project is to establish a new experimental model to help understand the mechanism that regulates cardiac cell diversification and heart morphogenesis. In order to achieve these goals we use two assays. The cardiogenesis assay involves the use of animal cap explants excised from the animal pole of blastua embryos. Here it is shown that GATA-4 reliably induces the expression of ventricular and proepicardial markers, providing an assay to study the mechanisms of cardiac cell fate diversification. However, cardiomyocytes generated in animal pole explants do not undergo significant morphogenesis and physiological maturation. In order to study these later aspects of heart development we required a different assay to generate a structure similar to the heart. Using GATA-4 injected AC explants transplanted into host embryos we obtained secondary beating hearts in which regionally restricted cardiac gene expression was observed in addition to growth and a limited degree of morphogenesis. We demonstrated that the host plays an essential role as it provides a wide range of permissive regions which allow the development of the SH. Moreover, we also showed that the competence to generate a secondary heart is lost in reaggregates transplanted at stage 28. The host cells however do not contribute to the SH indicating that the role of the host is providing signals which allow the development of the SH.

Cartilagineous long bone rudiments, of the chick embryo, were used as a model for the mammalian epiphyseal growth plate. Both contain 3 zones of chondrocytes which, therefore allows the cartilege rudiment to be considered as an expanded growth plate. The involvement of each zone in the growth of the rudiment was determined at the cellular level, by counting cell numbers in histological sections of the ulna. Studies in the cell kinetics of the rudiments, in vitro, demonstrated that cell division was confined, mainly to the zone of rounded cells, with little or no division observed in the zones of cell flattening and hypertrophy. It is proposed that the morphogenesis of the early cartilage long bone rudiment is influenced by the structure of it's surrounding perichondrium, rather than by propert intrinsic to the constituent chondrocytes. The perichondrium is thought to exert its influence though a process termed 'directed dilation', whereby circumferential expansion is resisted and longtitudinal growth is favoured. Ultrastructural examinations show that the perichondrium of a long bone rudiment has a variable structure: distinct at the diaphysis and loose at the epiphysis. By contrast, the perichondrium surrounding heckel's cartilage, which has only one type of chondrocyte, appears to have a uniform structure. Evidence for the lack of intrinsic property determining morphogenesis comes from the observation that chondrocytes, from various cartilage elements, behave identically in vitro, underappropriate conditions. In addition, contrary to reports that that the expression of a cartilage phenotype is dependant on high cell densities and histogenic interactions, it is proposed that the maintenance of a rounded cell configuration is sufficient to elicit phenotypic expression. Experimental evidence suggests that the 3 zones of cells are set up by combination of interactions with the perichondrium and by a signal specific to the cartilage matrix.

Nitric oxide (NO) is a highly diffusible signalling molecule that serves a wide range of physiological functions. During development, biosynthetic enzymes for NO are often expressed in nascent neurons, although little is known of how this molecule regulates in vivo nervous system development. This thesis aims to address this problem by examining the functional roles of NO signalling in the zebrafish embryo, focusing specifically on nitrergic regulation of spinal locomotor network assembly. The first aim of this study is to characterise the spatiotemporal distribution of NO synthase 1 (NOS1), the enzyme responsible for NO biosynthesis in the zebrafish nervous system. NOS1 transcript and protein was observed in discrete regions of the brain as well as a distinct class of spinal interneuron from early stages of embryonic development. The second aim was to examine functional roles of NO signalling during in vivo spinal cord development. Using molecular antisense and pharmacological approaches, NO levels were disrupted during early life and the consequences to spinal circuit maturation assessed. NO was found to specifically regulate the growth of spinal motoneurons that innervate axial trunk muscles. Abrogation of NO signalling dramatically increased the number of motor axon branches formed within the muscle across the first three days of life whilst exogenous elevation of NO levels had the opposite effect. The third aim was to determine downstream signalling pathways underpinning NOs effects. Pharmacological studies revealed that NO regulates motoneuron branching through the cyclic guanosine monophosphate pathway and subsequent analysis revealed that this pathway can modify neuromuscular synapse density, with high NO levels suppressing synaptogenesis and retarding locomotor maturation and low NO levels having the converse effect. In summary, the work presented in this thesis identifies a novel and important role for NO signalling, demonstrating that it functions to sculpt neuromuscular synapse assembly and modify locomotor maturation.

No description available.