Functional Study Of Gata6 In The Development Of Zebrafish Pancreas And Heart

Pancreas is one of most important organ in vertebrate which regulates nutrient metabolism and blood glucose homestasis.Cardiovascular system transports oxygen,carbon dioxide,nutrients and hormones.Zebrafish,a small tropical freshwater fish,is an ideal vertebrate model of developmental biology.Their transparent embryos develop outside of mother body,which are convenient for observation.Zebrafish are productive and easy to feed,which is amiable for genetic analysis.The pancreas develops from dosal bud and ventral bud in zebrafish,dorsal bud emerges at 24 hpf and contributes to endocrine cell,ventral bud first appears at 34 hpf and gives rise to exocrine cell and partial endocrine cell.The bilateral cardiac progenitor cells migrate to the dorsal midline to fuse and form a cardiac disc structure with the endocardium within the hole at the central.Cardiac morphogenesis trasforms the cardiac disc into a myocardial tube,and the endocardium forms the inner lining of the myocardial tube.The development of the pancreas and heart is strictly regulated by dynamic signals and transcription factors.gata6 was found expressed in both organs,suggesting that gata6 might invole in the regulation of the development of the pancreas and heart.However,it is poorly understood.gata6 is one member of GATA zinc finger transcription factor family,which is responsible for mesoderm and endoderm specification and differentiation.To investigate the function of gata6 in the development of pancreas and heart,the gene was knock-out through CRISPR/Cas9 technology,and two mutants,with 5bp and 20 bp delete in the exon 2 respectively,were obtained.Each mutant mRNA had a premature stop codon,and coded a truncted protein without zinc finger domain.The gata6?20/?20 mutant embryos displayed defective anterior gut,small liver and pancreas agenesis.Indicated by pancreas differentiation marker trypsin,exocrine pancreas in 3dpf mutant was found to be two separated groups of tissue.To check exocrine progenitor cell,gata6?20/+ was crossed with Tg(ptf1a:GFP).Progenitor cells of ventral bud in wild type embryos emerged as one groups of cells,however,they form two separated ventral pancreas bud in mutant embryos.Previou studies had showed that defective migration of lateral plate mesoderm(LPM)would impair exocrine pancreas development.Thus,the migration of LPM had been checked,and found that it was normal in mutant as wild type.It had been reported that ectopic expression of shh resulted in exocrine pancreas hypoplasia in mouse.Misexpression of shha was found in the gut endoderm in the mutant,however,the phenotyoe had not been rescued by administration of cyclopamine,an inhibitor of Hedgehog signaling pathway.The regulatory mechnism of gata6 on pancreas develoment need further investigation.We also studied the function of gata6 in heart development.At 24 hpf,when wild type embryos started blood circulation,mutant embryos lost it,although the heart beating was normal.Observed on Tg(flk1:GFP)background,endothelial cells of outflow tract in the mutant didn't form a lumen that could allow blood cells flow out from the heart.Subsequent examination found that about 70% mutant embryos recovered circulation between 26 hpf and 48 hpf.Previous studies showed that genetic compensation might happen in mutants,and the expression level of gata5 had been found to be upregulated significantly in heart in the gata6 mutant.Partial knock-down of gata5 was performed in mutant embryos which reduced the ratio of circulation recovered mutant,and this result could be reversed by co-injetion with an optimal dose of gata5 mRNA.It had been reported that vascule could formed a lumen in such a model: angioblasts aggregated in a cord-like structure and gained apical-basal polarity,then the lumen is formed by separation of apical endothelial cell surfaces.The molecular pathway of this modle was VE-cadherin-PKC-F-actin-myosinII.To confirm whether outflow tract formed a lumen in zebrafish agreed with this modle,Blebbistatin,Latrunculin B and Nocodazole,inhibitors of myosin II,F-actin and microtubule respectively,were used to treat wild type embryos from 22 hpf,and all these inhibitors caused a obstructed outflow tract.PKC expression level had been detected in 36 hpf embryos,it was higher in sibling embryos than mutant embryos,and mutant embryo which recovered circulation had more PKC than embryo without circulation.These data suggested that PKC may be directly regulated by gata6 and/or gata5 to control the lumen formation of outflow tract.Taken together,our works provide a useful investigation of gata6 for the study of development of the pancreas and heart.