The Effect Of Pax3a Domain Overexpression On Zebrafish Developmental Phenotypes

Regeneration is currently a hot spot in medical research.Zebrafish,a highly regenerative model of vertebrate organisms,is widely used in regenerative research.Our previous study suggests that the damaged zebrafish mandibular tissue can completely restore the original structure through plastical blastema regeneration.Transcriptome and immunohistochemical analyses show that Pax3 a is consistently expressed in all plasticible blastemal cells.Since human PAX3 gene mutations can cause the disease with multiple cell phenotypes,such as Waardenburg Syndrome and tumors,we hypothesize that zebrafish Pax3 a protein might be act as a key regulator of phenotype plasticity of blastema cells,in which Pax3 a protein containing multiple domains could form a signaling complex according to microenvironment and signal changes,and regulate cell phenotypic through various signal pathways as well.To test how Pax3a domains affect embryos and cell phenotypic of zebrafish,we firstly construct four types of expression vector for Pax3 a domains: p PAX-myc(PD3),p GPS-myc(GPD),p HD-myc(HD)and p Pax7-myc(PD7),to observe the phenotypic changes of surface pigment,eyeball,internal and external canthus of zebrafishes after overexpression in embryos for 25 days.Then we conduct in vitro cellular overexpression to observe cell morphology,migration and proliferation.We find that juveniles which are injected with the Pax3 a domain overexpression vector show characteristic phenotypes similar to Waardenburg Syndrome.Compared with the 17%abnormality in control group,PD3 domain over-expressed embryos occur 55% of abnormal pigmentation and eyeball development,including significantly reducing eyelid ratio as well as internal and external canthus ratio.The GPD domainoverexpression group display 29% of hypochromic and eye mutations.These mutation rate is 21% in the HD domain-overexpression group.The PD7 domain overexpression group is 14% of abnormal pigmentation,and has no significant change of eyelid and internal and external canthus ratio.After in vitro transfection in the zebrafish embryo fibroblasts(PAC2 cells),PD3 domain overexpression strongly inhibit cell proliferation and migration.While HD and PD7 domain overexpression do not influence cell proliferation but inhibit cell migration.In contrast,GPD domain overexpression promotes cell migration.The cellular immunofluorescences show that PD3 domain overexpression is gradually diffused and aggregated around the nuclei,accompanied by changes in nuclear morphology and division.To investigate the mechanisms which Pax3a regulates cell phenotypes,quantitative Real-time PCR PCR(q PCR)and co-immunoprecipitation-mass spectrometry are applied to detect gene expression and interaction proteins in related regulatory pathways.The results show:(1)Transcripts of extracellular matrix and macrophage related cytokines are changed : Most cytokines in the PD3 domain group are down-regulated,while IL10,IFNg,CCL19 b,and CXCL8 a are highly expressed in the HD domain group.Thus,JAK-STAT and NFκB pathways may be activated to promote epithelial-mesenchymal transition(EMT).(2)PD3 domain inhibits the TGFβpathway and down-regulates the synthesis of α-smooth muscle actin,fibronectin,heparin sulfate proteoglycan and Collagen I.It also inhibits Wnt but activates apoptosis and autophagy pathways.GPD domain overexpression increases IL1βexpression and activates proteasome and NFκB pathways.(3)Protein mass spectrometry analyses indicate that Pax3 a may interact with melanin-related small eye-deficient transcription factor(MITF),the cleavage-related proteins such as connectin,centrosomin and β-tubulin,and PI3K-Akt kinases pathways.In conclusion,results of these experiments demonstrate that the each Pax3 a protein domain has a characteristic effect on zebrafish embryonic and cell phenotype,and induce the corresponding phenotype-regulatory pathway through specific signal complex components.In addition,our generated PD3 domain overexpression in zebrafish can be used as the human WS disease model,and provides a theoretical and material basis for treatment of rare human genetic disease caused by PAX3 mutation.