THE IMPACT OF ECOLOGICALLY RELEVANT THERMAL STRESS ON GENE EXPRESSION AND MORPHOGENESIS IN HALIOTIS ASININA

There is an increasing weight of evidence that environmental cues influence developmental pathways to produce phenotypes that are not dictated solely by the genotype. The heat shock proteins (Hsps) are an environmentally responsive family that chaperone the members of a range of developmental pathways, thus they provide ideal candidates to study the role of the environment in regulating developmental processes. Heat shock proteins buffer organisms from environmental fluctuations, through preventing protein aggregation, actively refolding unfolded proteins and ultimately preventing apoptotic degradation. Additionally, Hsps are expressed extensively in tissues that are undergoing morphogenesis, rapid cellular division, and migration, throughout the development of a broad range of taxa. Moreover, exposure to elevated temperatures during embryogenesis can result in developmental abnormalities, particularly in the tissues that express the Hsps at physiological temperatures. I have studied the role of heat shock proteins in the development of the gastropod Haliotis asinina. The major Hsp genes - Hsp90, Hsp70 and the heat shock transcription factor gene, HSF were cloned and their expression was determined through in situ hybridisation. HasHsp90A, HasHsp70 and HasHSF are coexpressed in a dynamic and extensive pattern during early development in tissues that include the prototroch, foot and mantle. H. asinina were continually exposed to elevated temperatures throughout early development, which resulted in varying degrees of disorganisation of the tissues that normally express Hsps. Surprisingly, heat shock does not alter the spatial expression of HasHsp90A or HasHsp70, and the Hsps do not appear to confer significant protection to the tissues in which they were expressed. This finding supports the theory that heat shock may titrate the Hsps from their developmental roles, subsequently contributing to the heat-induced developmental abnormalities. Sustained exposure to heat shock resulted in complex and variable effects on tissue organisation. To pinpoint the most heat sensitive developmental processes, I performed brief heat shocks that focussed around specific stages of the development of a single tissue, the prototroch. Normal prototroch development was initially characterised for H. asinina through in situ hybridisation using Has-Cal, which encodes Calmodulin, a marker of prototroch development. Embryos were heat shocked during three different windows of prototroch development: two during putative trochoblast specification (1-16 cells and 16-64 cells), and one during trochoblast migration (4-9 hours post fertilisation (hpf)). The application of heat shock during the specification stages consistently resulted in disorganisation of the prototroch, however, heat shock during trochoblast migration did not. It was concluded that heat shock impacts the establishment of polarity in this tissue, which has downstream effects on migration, rather than migration itself. Embryonic exposure to heat shock results in the development of exogastrulae, which allowed the examination of normal inductive processes in H. asinina. Heat shock leads to the development of exogastrulae through an unknown mechanism, so the heat shocked exogastrulae were compared to exogastrulae that had developed as a result of pharmacological inhibition of the induction of 3D, the molluscan organizer. The two forms of exogastrulae share similar morphological characteristics, including an anterior constriction of the posttrochal region, disorganisation of the posttrochal ectoderm and quadri-radialisation of the pretrochal region. 3D inhibited larvae are also characterised by the absence of mesoderm, indicated by a lack of Mox expression, and subsequently muscle (indicated by an absence of F-actin) and the adult nervous system, (indicated by a loss of complexity in anti-serotonin staining in veliger larvae). Ironically, many heat shocked larvae that show stereotypical characteristics of 3D inhibition possess mesoderm, muscles, and an adult nervous system. This indicates that gastropod morphogenesis involves yet uncharacterised, organisational influences that are independent to 3D. A microarray study compared the global gene expression profiles of the 3D inhibited larvae to the morphologically similar heat shocked larvae. Of the 5547 clones on the abalone microarray chip, there was only one clone in common between 3D inhibited larvae and heat shock exogastrulae. Thus these treatments that produce similar morphological outcomes, have fundamentally different underlying gene expression profiles. The microarray experiment resulted in the identification of a broad range of heat responsive mediators of morphogenesis including cytoskeletal components, organisers of cytoskeletal components, and the upstream organizers of polarity and morphogenesis. It is likely that the observed alterations in the expression of these genes, contributed to the heat induced developmental defects of the prototroch, and the development of exogastrulae. Thus heat shock can impact morphogenesis in molluscs at many levels. This study provides evidence that the major heat shock proteins have an ancient and pleiotropic role in morphogenesis, and that heat shock impacts heavily on morphogenesis, possibly through the titration of Hsp function. This study also indicates that Hsps are an environmentally responsive family, which interact with a range of developmental pathways that may be useful in characterising a range of environmentally mediated phenotypes in the future