Molecular tools to dissect the role of Dmrt2a and Dmrt2b in the left-right axis formation in zebrafish

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

We tend to view the vertebrate body as bilaterally symmetric, but in fact, this only happens from the outside. Internally, most of the organs from heart to liver are asymmetrically positioned. Skeleton and its associated muscles, symmetric structures of the vertebrate body, have its origins in the transient symmetric blocks of mesoderm called somites whereas the asymmetric morphogenesis of the internal organs is due to asymmetric gene expression in the lateral plate mesoderm (LPM). Previous studies using Morpholino (MO) technology have shown that dmrt2a is involved in these two processes in zebrafish. When Dmrt2a levels are reduced, asymmetric gene expression in the LPM becomes randomized and symmetric gene expression in the presomitic mesoderm (PSM) is disrupted. The paralogous of dmrt2a, the fish specific dmrt2b has been shown to be involved in regulating asymmetric gene expression in the LPM as well. Here we used the recent Transcription activator-like effector nucleases (TALENs) technology to generate dmrt2a and dmrt2b mutant alleles that will allow us in the future to uncover the downstream effectors of these transcription factors using high-throughput experiments. In addition, we overexpressed dmrt2a at the one-cell stage to characterize asymmetry versus symmetry phenotypes. The results show clearly the ability of TALEN technology to generate mutant alleles in zebrafish. Nevertheless, dmrt2a and dmrt2b homozygous mutants developed so far fail to recapitulate their previously described MO phenotypes which raise the question on what molecular mechanism(s) allow(s) zebrafish to cope with frameshift mutations. The overexpression of dmrt2a shows that a time window of opportunity during which symmetric embryonic territories are able to respond to asymmetric signals does exist during embryonic development.