The Functional Study Of OTUD5 In Early Stage Of Embryonic Development

Background:We screened five patients with mutations in gene OTUD5,one of them presented with immunodeficiency,while the rest of them manifested as global developmental delay,which had a set of syndromes involving multiple systems such as brain malformations,intellectual disability and polydactyly.Combining with the pre-existing functional study of OTUD5,we consider that OTUD5 may be a new pathogenic gene.Objective:This study aims to explore the causal relationship between the mechanism of OTUD5 and patients'phenotypes using OTUD5 functionally defective model organisms,then to further identify the pathogenesis role of OTUD5 in the process of early embryonic development.Method:We knocked down otud5a and otud5b respectively in zebrafish by injecting Morpholino into one-cell embryos,then analyzed the morphants'phenotypes through direct observation under the microscope and whole-mount in situ hybridization.We conducted rescue experiments with m RNA from human OTUD5 or zebrafish otud5a and otud5b,in order to confirm whether the phenotypes were resulted from OTUD5knock-down.We also generated mutants of otud5a and otud5b by CRISPR/CAS9 gene editing technology.The mutants'phenotypes were observed and compared with previous morphants',and the rescue experiments were conducted in mutants as well.Finally,the phenotypes shared by morphants and mutants in zebrafish were compared with that of patients',leading to a conclusion of the relationship between OTUD5 and patients'phenotypes.Results:Knock-down of otud5a or otud5b resulted in a series of phenotypes,including smaller eyes,shrunken midbrain hindbrain boundary,enlarged ventricle,reduced pigment,as well as dysplastic branchial arch,however,rescue experiment using OTUD5 m RNA from both human and zebrafish failed to reverse these phenotypes.Nevertheless,knocking down of otud5a or otud5b in p53 mutants didn't cause shrunken midbrain hindbrain boundary or enlarged ventricle.In addition,mutants of otud5a and otud5b did not exhibit similar phenotypes compared with morphants.Whole-mount in situ hybridization and RT-q PCR revealed that transcriptional levels of neural crest markers sox9b and sox10 were reduced,which was more significant in otud5b mutants with upf3a knock down.Human OTUD5 m RNA could rescue the expression of these neural crest markers.Conclusion:otud5a or otud5b knock-down zebrafish displayed disordered neural development phenotypes including dysplasia in central nervous system and neural crest,similar to that of patients with OTUD5 loss-of-function variants.Shrunken midbrain hindbrain boundary and enlarged ventricle in morphants were due to the up-regulated p53-dependent apoptosis.The genetic compensation response(GCR)may account for phenotypic discrepancy between morphants and knock-out mutants.