Inhibition Of GCR Reveals New Functions Of Yap1 In Zebrafish Development

The genetic compensation response(GCR)is a new mechanism of genetic robustness,which was firstly described in zebrafish to expound phenotypic discrepancies between gene-knockout and gene-knockdown,whereby the nonsense mutation,but not gene-knockdown,can activate the transcriptional upregulation of related genes.The phenomenon was also found in other model systems including Arabidopsis and mice.Even important for the survival of organism,GCR has impeded the study of gene function.In 2019,the potential molecular mechanisms of GCR were published by Stainier's lab and our lab at the same time.Both of us revealed that mutations generated with premature termination codon(PTC)and the nucleotide sequence homology are two prerequisites for GCR activation;Nonsense-mediated m RNA decay pathway(NMD)is involved in GCR and the upregulation of compensatory gene is associated with the modification of H3K4me3at their transcription start site(TSS).However,there is a key discrepancy in the two findings.Stainier's lab proved that Upf1-mediated NMD degradation branch triggered GCR.Our lab found that Upf3a-mediated non-degradation branch,but not NMD degradation branch,was involved in GCR activation.So,this is a controversial scientific questions which need to furtherly clarify in urgent.Here,we found that injection with capped or uncapped m RNA or nonsense mutation m RNA can not activate homologous gene expression,proving that m RNA degradation did not trigger GCR.Then,the expression of taz was found to be significantly upregulated in the yap1^-/-,indicating the activation of GCR.Knockout of upf3a but not upf1 was able to block taz upregulation in yap1^-/-by constructing upf3a^-/-;yap1^-/-and upf1^-/-;yap1^-/-mutants.Notably,a severe edema and small liver phenotype were observed in upf3a^-/-;yap1^-/-.Knockdown of taz in the yap1^-/-resulted in a similar edema and small liver phenotype.Those experiments further confirmed that the Upf3a-mediated non-degradation pathway is involved in GCR activation.In addition,we used the upf3a^-/-;yap1^-/-to study the molecular mechanism of edema caused by yap1 loss of function.The upf3a^-/-;yap1^-/-showed defects in glomerulus development,ion transport and protein reabsorption.Overexpression of yap1 or taz was able to rescue the edema phenotype and the glomerulus development defects in upf3a^-/-;yap1^-/-.Further studies found that the expressional pattern of bmpx overlaps with yap1 at early stage and the transcription of bmpx was significantly downregulated in upf3a^-/-;yap1^-/-.Knockdown of bmpx resulted in edema and glomerulus developmental defects which is similar to upf3a^-/-;yap1^-/-'s phenotypes.Overexpression of yap1 or taz was able to upregulate bmpx transcription.Ch IP-q PCR analysis showed that Yap1 was enriched at the TEAD-binding site of the bmpx promoter region.Taken together,the thesis confirms that m RNA degradation does not trigger GCR;Upf3a-mediated non-degradation branch,but not Upf1-mediated degradation branch,is involved in GCR activation.Inhibiting GCR by knockout of upf3a reveals that yap1promotes zebrafish glomerulus development through activating Bmp signals to avoid nephrotic syndrome.This study demonstrates that knockout of upf3a can be used to reveal the phenotype of mutants which trigger GCR and using this approach reveals new functions of yap1 in kidney development.