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Search for endotherm genes involved in early patterning of vertebrates.

de Sousa Nunes, R.M.D.; (2004) Search for endotherm genes involved in early patterning of vertebrates. Doctoral thesis , University of London. Green open access

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In addition to giving rise to the gut, the endoderm plays a crucial role in embryonic axis determination. The murine extra-embryonic endoderm is thought to provide an early positional cue defining the antero-posterior axis of the embryo. The axial mesendoderm, which emanates from the gastrula organizer, populates the midline of the embryo and patterns it in all three axes. Later, maintenance and refinement of the antero-posterior axis of the brain requires the embryonic endoderm (reviewed in Martinez-Barbera and Beddington, 2001). Genes expressed in the endoderm are responsible for imparting it with its patterning properties. It is therefore useful to identify the expression profile of the endoderm. To this end, a cDNA library was made from 7.5 days post-coitum mouse endoderm (Harrison et al., 1995). Many clones from this library were sequenced and constitute a set of expression sequence tags (ESTs). I screened these ESTs in silico for non- essential molecules whose role in embryonic patterning had not been determined. I then screened clones obeying these criteria by whole-mount in situ hybridisation on 6.5 - 9.5 dpc mouse embryos. Restricted expression was displayed by 18% of the clones (from the total of my work and that of two other students). The restricted expression patterns encountered are presented. One of the restricted genes I encountered in the mouse in situ hybridisation screen was that encoding the serum and glucocorticoid-regulated kinase (Sgk). I was very interested in the expression pattern of Sgk since it was asymmetric in the visceral endoderm at the onset of gastrulation. Sgk expression presented other interesting features, such as being exclusively expressed in angioblasts at 9.5 dpc. I constructed a targeting vector in order to analyse Sgk function in mouse by a loss-of-function approach. I targeted embryonic stem (ES) cells with this construct and recovered neomycin-resistant clones. One of these is possibly a clone where homologous recombination took place at the Sgk locus. I cloned zebrafish orthologues of some of the restrictedly expressed endoderm genes. I functionally screened these genes in zebrafish by a loss-of-function approach, using antisense morpholino oligonucleotides (MOs). I uncovered several molecules required for proper early embryonic development, one of which I studied in more detail. This was Nop seven-associated protein 2 (Nsa2), a eukaryotic protein involved in ribosome biogenesis (Harnpicharnchai et al., 2001). Zebrafish nsa2 morphants have slowed epiboly and early patterning defects. Furthermore, nsa2 morphant cells gradually die by apoptosis. A smaller embryo develops from surviving nsa2 morphant cells during the first day of development, after which presumably all cells die. The phenotype of nsa2 zebrafish morphants is analogous to that of morphants for the ribosomal proteins RpL19 and RpS5, which when mutated in fly cause the Minute phenotype. I describe the zebrafish Minute phenotype and hypothesize that nsa2 is likely to be a Minute.

Type: Thesis (Doctoral)
Title: Search for endotherm genes involved in early patterning of vertebrates.
Identifier: PQ ETD:602567
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by Proquest
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Cell and Developmental Biology
URI: https://discovery.ucl.ac.uk/id/eprint/1446642
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