imprint:
[Erscheinungsort nicht ermittelbar]: [Verlag nicht ermittelbar], 2019
Language:
English
Identifier:
Origination:
University thesis:
Dissertation, 2019
Footnote:
Description:
Dmrt2a is a zinc finger like transcription factor associated with left-right asymmetry establishment, bilateral synchronisation of the somite clock genes and fast muscle differentiation in zebrafish. Despite the described functions, Dmrt2a mechanism of action during zebrafish embryonic development is unknown. In an effort to identify Dmrt2a most immediate downstream genes, we developed several genetic tools: two heat-shock inducible transgenic lines – Tg(hsp70:HA-dmrt2a) and Tg(hsp70:Venus-dmrt2a) – that allowed temporal regulation of dmrt2a overexpression, and several stable dmrt2a mutant lines. We validated and characterised both transgenic lines and focusing in Tg(hsp70:HA-dmrt2a) we characterised with finer detail dmrt2a gain-of-function phenotype. We observed that the gain-of-function phenotype we obtained was very similar to the previously described dmrt2a loss-of-function phenotype – desynchronisation of somite clock genes and left-right asymmetry defects –, suggesting a need to fine tune dmrt2a levels to assure a normal development. Plus, we identified a new phenotype of somite border malformation that suggests that Dmrt2a might be involved in the somite epithelialization process. Using the TALEN technology, we generated several dmrt2a mutant lines and characterized their phenotype, however, the mutants we generated had only a very mild to negligible phenotype when comparing to the previously described dmrt2a knockdown phenotype using the antisense morpholino technology. This mild phenotype could be due to the action of a compensatory gene that could overcome the lack of dmrt2a. Its fish-specific paralog gene – dmrt2b – is expressed in similar territories and has similar functions therefore, we generated and characterised a single dmrt2b mutant line and two double mutant lines between dmrt2b and two of the different dmrt2a mutants we had available. However, as previously described using the antisense morpholino technology, indeed dmrt2a and dmrt2b are not redundant during embryonic development. Therefore, other gene(s) could be compensating for the lack of dmrt2a or, despite the mutations, a partially functional protein can still be produced. Therefore, in order to esxiv tablish a solid loss-of-function approach to identify Dmrt2a downstream genes, we proceeded to evaluate the specificity of the previously used dmrt2a morpholino. Taking advantage of dmrt2a mutants, we validated dmrt2a morpholino proving it specific and non-toxic in the evaluated conditions. With our genetic tools selected and validated, we used Tg(hsp70:HA-dmrt2a) in a microarray approach. From the microarray data, we identified that Dmrt2a acts mainly as a repressor during development, and obtained two different lists of genes that were further validated using quantitative PCR and dmrt2a antisense morpholino. Only genes that were validated by quantitative PCR, and that were down-regulated in the gain-offunction and up-regulated in the loss-of-function approach, or vice-versa, were considered to be truly downstream of Dmrt2a. Using this system we were able to validate six genes: etv2, foxj1b, pxdc1b, cxcl12b, foxc1b and cyp1a. Together, etv2, foxc1b and cxcl12b genes suggest a new role for Dmrt2a in vascular development. Furthermore, foxj1b can establish a link between Dmrt2a and the left-right asymmetry defects observed after dmrt2a gain and loss-of-function.