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Identification and Functional Analyses of Novel Genes Involved in Haemovascular Development

Eve, AMJ; (2016) Identification and Functional Analyses of Novel Genes Involved in Haemovascular Development. Doctoral thesis , UCL (University College London).

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Characterising the transcriptome of the haemangioblast is of therapeutic interest. By profiling the precursor to both blood and endothelial cells, important candidate genes can be identified for further study, and contribute to our understanding of vascular development and disease. Using previously generated RNA-sequencing data, novel genes with roles in haemovascular development were functionally characterised. Previously, the Wnt signalling antagonist Tmem88a was shown to be necessary for primitive erythropoiesis. Using qPCR and in situ hybridisation the expression profile of tmem88a was characterised. Morpholino knockdown of tmem88a in the zebrafish reduces the number of erythrocytes and myelocytes, according to qPCR and histological stains. Erythro-myelopoiesis was rescued by small molecule inhibition of the Wnt signalling pathway. In addition, a tmem88a mutant line was generated using TALENs, and the tmem88a mutant did not phenocopy tmem88a knockdown embryos. Although the tmem88a mutant is yet to be validated as a genetic knockout at the protein level, this may be an example of off-target effects from morpholino injection. Future work will validate the mutant and investigate discrepancies between morphant and mutant phenotypes. A morpholino knockdown screen showed that laminin γ3 is necessary for zebrafish para- chordal chain development. Use of the CRISPR-Cas9 system in F0 embryos also caused the same lymphatic phenotype, although a homozygous knockout line was not generated. The expression profile of lamc3 was characterised using in situ hybridisation and qPCR. Single-blastomere injections determined that γ3 was required in a non-cell autonomous fashion. Analyses using morpholino knockdown showed that γ3-deficient embryos had improper migration of the rostral primary motor neurons, preventing formation of the parachordal chain and thoracic duct. Furthermore, Netrin-1 protein was delocalised in lamc3 knockdown embryos, explaining the latter phenotypes. Here, I suggest a model whereby γ3 binds Netrin-1 to facilitate migration of motor neurons, and lymphatic endothelial cells.

Type: Thesis (Doctoral)
Title: Identification and Functional Analyses of Novel Genes Involved in Haemovascular Development
Event: University College London
Language: English
Keywords: zebrafish, vasculogenesis, lymphangiogenesis, haematopoiesis, tmem88a, laminin gamma-3, wnt signalling, endothelial cell, cell guidance, netrin-1
URI: http://discovery.ucl.ac.uk/id/eprint/1473223
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