The zebrafish Dtk, Axl and Mer receptor tyrosine kinases

Abstract

Receptor tyrosine kinases (RTKs) constitute a large family of transmembrane receptors which mediate the responses of cells to external stimuli. Activation of RTKs and associated downstream signaling pathways commonly results in cellular proliferation and/or differentiation in both embryonic and adult forms, The Axl subfamily of RTKs is characterised by the presence of two immunoglobulin-like domains and two fibronectin-type III domains in the extracellular region of the receptor. This extracellular domain arrangement has structural similarity with cell adhesion molecules. Three members of the Axl subfamily of RTKs have been identified and have been designated Axl, Dtk and Mer. Transcripts for all three receptors have been detected in a wide range of mammalian or avian embryonic tissues suggestive of developmental roles. All three receptors have also been implicated in neoplastic events. The anticoagulation factor protein S and a related protein called Gas6 have been identified as ligands for Dtk and Axl. The aim of this thesis was to obtain detailed information about the expression patterns of Axl, Dtk and Mer during vertebrate development. It was anticipated that this information would provide insights into the possible developmental roles of these receptors. The zebrafish was chosen as a model animal since many aspects of its embryonic development make it highly suitable for developmental studies. Also, the presence of a Dtk-like gene was detected in the genomic DNA of this species by Southern analysis indicating that it may be possible to isolate zebrafish homologues of Axl subfamily members. Through polymerase chain reactions, zebrafish RTKs with homology to members of the FGFR, PDGFR and Axl subfamilies were isolated. Subsequent library screening resulted in the isolation of partial overlapping cDNAs encoding the full-length zebrafish Dtk cDNA. The amino acid sequences of the coding regions of zebrafish and human Dtk are 48% identical and the kinase domains are 72% identical. Partial cDNAs encoding part of the zebrafish Axl and Mer receptors were also isolated through library screening. The zebrafish Axl cDNA, which encodes part of the kinase domain and part of the C-terminal cytoplasmic domain of the receptor, is 76% identical to human Axl. The zebrafish Mer cDNA, which encodes part of the N-terminal end of the cytoplasmic domain and part of the kinase domain of the receptor, is 77% identical to chicken Mer. The zebrafish Dtk, Axl and Mer cDNAs were used as templates for the synthesis of DIG-labelled RNA probes for use in wholemount in situ hybridisation studies to detect expression patterns during zebrafish development. The results of these analyses show that Dtk is predominantly expressed in the presumptive notochord and tailbud during gastrulation, in a region of the tail in which undifferentiated haematopoietic cells are thought to be located and in parts of the brain during later embryogenesis. Dtk expression within the brain was most obvious in dorsally located ventricular cells of the telencephalon, anterior midbrain cells, the trigeminal ganglion and segmentally arranged clusters of cells in the hindbrain. These results are comparable to previous observations of Dtk expression within mammalian embryonic haematopoietic tissue and in the developing mammalian neocortex. The pattern of expression observed during zebrafish embryogenesis indicates an involvement of Dtk in the formation of the notochord and in parts of the brain, and also in the biology of specific neurons in the hindbrain, including trigeminal ganglion neurons. In addition, experiments designed to detect the distribution patterns of membrane bound ligands for Dtk in whole zebrafish embryos were undertaken. Dtk binding proteins could not be detected by the strategy employed during this study. Axl transcripts were detected throughout the blastoderm of gastrulating zebrafish embryos and throughout the whole embryo during segmentation. At later stages Axl transcripts were restricted to the brain. Previous studies have demonstrated widespread Axl expression in tissues of mesodermal origin in the midgestational mouse embryo. In contrast, Axl transcripts were detected in tissues of ectodermal as well as mesodermal origin in zebrafish embryos. The presence of Axl transcripts throughout the zebrafish gastrula and in the developing brain indicates an involvement for this receptor in developmental events additional to those that have been predicted from mammalian embryonic expression patterns. Mer transcripts were present in numerous, regularly spaced, distinct regions of the yolk syncytial layer of gastrula and early segmentation stage embryos. It is possible that these transcripts encode a cytoplasmic form of the Mer receptor since the nuclei of the yolk syncytial layer are not surrounded by cell membranes. The yolk syncytial layer is important for the cell movements involved in epiboly and shield formation and it is possible that Mer could be acting in pathways which facilitate these processes. Since the yolk syncytial layer is unique to teleost embryos it is suggested that the Mer receptor has been requisitioned for different developmental processes during vertebrate evolution. The results presented in this thesis demonstrate the existence of zebrafish Dtk, Axl and Mer homologues and contribute to our knowledge of the expression patterns of these genes during embryogenesis. This work provides a basis from which to undertake further studies designed to directly investigate the functions of these receptors during vertebrate development.