Identification of Dmrt2a downstream genes during zebrafish early development using a timely controlled approach

Abstract

BackgroundDmrt2a is a zinc finger like transcription factor with several roles during zebrafish early development: left-right asymmetry, synchronisation of the somite clock genes and fast muscle differentiation. Despite the described functions, Dmrt2a mechanism of action is unknown. Therefore, with this work, we propose to identify Dmrt2a downstream genes during zebrafish early development.ResultsWe generated and validated a heat-shock inducible transgenic line, to timely control dmrt2a overexpression, and dmrt2a mutant lines.We characterised dmrt2a overexpression phenotype and verified that it was very similar to the one described after knockdown of this gene, with left-right asymmetry defects and desynchronisation of somite clock genes. Additionally, we identified a new phenotype of somite border malformation.We generated several dmrt2a mutant lines, but we only detected a weak to negligible phenotype. As dmrt2a has a paralog gene, dmrt2b, with similar functions and expression pattern, we evaluated the possibility of redundancy. We found that dmrt2b does not seem to compensate the lack of dmrt2a. Furthermore, we took advantage of one of our mutant lines to confirm dmrt2a morpholino specificity, which was previously shown to be a robust knockdown tool in two independent studies.Using the described genetic tools to perform and validate a microarray, we were able to identify six genes downstream of Dmrt2a: foxj1b, pxdc1b, cxcl12b, etv2, foxc1b and cyp1a.ConclusionsIn this work, we generated and validated several genetic tools for dmrt2a and identified six genes downstream of this transcription factor. The identified genes will be crucial to the future understanding of Dmrt2a mechanism of action in zebrafish.Electronic supplementary materialThe online version of this article (10.1186/s12861-018-0173-5) contains supplementary material, which is available to authorized users.

The zebrafish dmrt family genes have cooperative and antagonistic roles in sex determination and oogenesis

“…Zebrafish dmrt2a and dmrt2b mutants and morphants have been previously reported, some of which suggest roles in embryonic development. In contrast to the dmrt2a-/- lethality we report here, prior work suggested that zebrafish dmrt2a mutants were normal and viable, with only a small number of embryos exhibiting left-right patterning defects (Pinto et al . 2018).…”

dmrt2 and myf5 Link Early Somitogenesis to Left-Right Axis Determination in Xenopus laevis

“…It is most likely that Dmrt2-dependent LRO morphogenesis is conserved in fish. This notion is supported by three recent publications: 1) zebrafish with diminished dmrt2 levels develop LR and somitogenesis defects ( Saúde et al, 2005 ; Liu et al, 2009 ); 2) the zebrafish LRO expresses dmrt2 ( Lourenço et al, 2010 ), and 3) in zebrafish, the master control gene for the biogenesis of motile cilia foxj1 , is a transcriptional target of Dmrt2 ( Pinto et al, 2018 ). Surprisingly, dmrt2 knockout mice do not exhibit LR defects, suggesting that mouse LRO morphogenesis is independent of Dmrt2 ( Lourenço et al, 2010 ).…”

“…Although the pattern of ciliation was unaltered in dmrt2 morphants ( Figure 1I ), cilia were substantially shortened to about 2–2.5 µm ( Figures 1I, J ), providing an explanation for flow deficiency. Recently, the master regulator of motile cilia, foxj1, was shown to be a transcriptional target of Dmrt2 in fish ( Pinto et al, 2018 ). The loss of flow and shortened cilia in dmrt2 morphants could therefore reflect impaired foxj1 expression in the cLRO precursor cells at gastrula stages.…”

“…We detected dmrt2 transcripts in both cell layers ( Supplemental Figures S1A, B ) which did not allow for a differentiation between both modes. However, since foxj1 is exclusively expressed in SM cells ( Stubbs et al, 2008 ; Beyer et al, 2012 ) and foxj1 is a transcriptional target of Dmrt2 in fish ( Pinto et al, 2018 ), a cell-autonomous Dmrt2 activity to induce foxj1 expression seems plausible. Importantly, this likely applies to the axial part of the SM, the cLRO precursor cells, but not to lateral SM cells which are fated to develop into sLRO tissue.…”

High Polymorphism in the Dmrt2a Gene Is Incompletely Sex-Linked in Spotted Scat, Scatophagus argus

“…On the other hand, Dmrt2a is farther away from Dmrt1 , which means more time may be needed for the differentiation to occur. Although in zebrafish Dmrt2a mutation is not lethal in experimental conditions [ 52 ], we cannot rule out the possibility of Dmrt2a exerting an essential function in developmental processes or adaptable characteristics in the natural environment. The critical function of Dmrt2a might restrict the accumulation of the dysfunctional mutation on its sequences.…”