GATA4 As A Novel Regulator Involved In The Development Of Neural Crest Derivatives Via Barx1

Conditional loss-of-function models for GATA4 genes could be applied to reveal the role of GATA4 in the development of the derivatives of NCCs.To knockout GATA4 specifically in NCCs,we crossed Wnt1-Cre males with Gata4^fl/fll/fl females.We examined the expression patterns of GATA4 in the mouse NCC-derived craniofacial tissue using immunohistochemical analysis at embryonic day 14.5?E14.5?,postnatal day 1?P1?,and P14.GATA4 was expressed in the cells of the mandible,the teeth and the palate.This may indicate that GATA4 plays an essential role in NCCs during the embryo maxillofacial development stage.Compared with control Gata4^fl/fl?wild-type,WT?littermates at P21,Wnt1-cre;Gata4^fl/fl mutants exhibited short stature.We subsequently performed Alcian Blue and Alizarin Red staining at P1 to examine the role of GATA4 in cranial development.Based on the results,the mutants exhibited widened cranial frontal sutures and mandibular retrognathism.By micro-computed tomography?micro-CT?scanning analysis,immunofluorescence staining and histochemical staining,we analyzed the differences of bone mineralization,tooth root development and heart development between the two groups.Micro-CT analysis revealed a drastic decrease in mineralization of the mandible,frontal bone,and palate in mutants,and the mutants'teeth were grossly smaller than those of controls.Von kossa,total collagen,and collagen I staining revealed a substantial decrease in mineralization in the mandible of P1Wnt1-cre;Gata4^fl/fl mice compared with that in control mice.Strikingly,the absence of GATA4 in NCCs induced severe cardiac defects with a prominent ventricular septal defect?VSD?and enlarged heart by H&E staining.We hypothesized that GATA4 may have a direct effect on early NCC development,in addition to its roles in migratory NCC.To validate our hypothesis,we analyzed the pharyngeal arch 1?pa1?by staining for SOX9,a marker of early migrating NCCs,in E9.5 embryos.Our results showed no differences in NCC migration into the pa1 between WT and mutant mice.Additionally,phosphohistone H3?PHH3?and terminal deoxynucleotidyl transferase dUTP nick end labeling?TUNEL?staining at E10.5 also showed no significant differences in cell proliferation and death between Wnt1-cre;Gata4^fl/fl and WT mice.Therefore,these results suggested that the deletion of GATA4 had no effect on NCC migration into pa1.Instead,phenotypic changes due to GATA4 conditional knockout were likely to occur at a later developmental stage.Thus,we performed the PHH3 and TUNEL staining in E14.5 embryos at the mandibular area.The results showed that the specific loss of GATA4 in NCCs decreased cell proliferation in the mandible;however,the apoptosis levels were comparable between mutant and WT mice.Taken together,these observations suggest that GATA4 is essential for the development of NCC-derived craniomaxillofacial derivatives through its role in promoting proliferation at the developmental stage.To provide further evidence of the function of GATA4 in vitro,GATA4expression was reduced in primary embryonic NCCs collected from adult WT mice by using a small hairpin RNA?shRNA?-mediated knockdown approach.The results indicated that GATA4 knockdown decreased the proliferative ability of NCCs,as measured by cell counting kit-8?CCK-8?assay,compared with that of the control group on culture day 4.However,Annexin V-fluorescein isothiocyanate?FITC?/propidium iodide staining showed no significant difference in the percentages of apoptotic cells between the two groups.Moreover,shGATA4-treated NCCs exhibited slower scratch closure,indicating that the migratory ability was attenuated.The osteogenic differentiation ability of NCCs treated with shGATA4 followed by mineralization for 14 days was significantly decreased compared with the control group by Alizarin red staining.Furthermore,we extracted the mRNA from the pa1 of WT and mutant mice at E10.5 and performed real-time quantitative polymerase chain reaction?qRT-PCR?analysis for nine key genes of interest including Sox9,Snail2,Ets1,Msx1,AP-2,Twist,Nestin,Pax3,and HNK-1,which are known to play important roles in the development of NCCs,and the mRNA expression levels of these key genes?except Nestin,Pax3,and HNK-1?were decreased dramatically after GATA4 knockdown.Taken together,the results indicated that GATA4 is critical for the function of NCCs.Based on the aforementioned in vivo and in vitro results,we sought to examine the potential mechanism underlying the function of GATA4 in NCCs.Thus,iTRAQ?isobaric tags for relative and absolute quantification?analysis was used to identify proteomic changes after GATA4 knockdown in NCCs.Among the identified 1888proteins,a group of 43 proteins was significantly upregulated,whereas 19 proteins were significantly downregulated in NCCs after GATA4 knockdown.Our results revealed that BARX1 was among the downregulated proteins after GATA4knockdown.Because BARX1 is widely expressed in the branchial arches and is important for molar,cartilage,and jaw development,we chose BARX1 for further analysis.In order to verify the proteomic difference observed by iTRAQ analysis,we further measured the expression levels of Barx1 after GATA4 knockdown using qRT-PCR and western blot assays,respectively.Our results showed that the expression of Barx1 decreased significantly both at mRNA and protein levels after GATA4 knockdown.Several putative GATA4-binding sites upstream of the transcription start site of the mouse Barx1 promoter were acquired from the JASPAR database,and two predicted binding sites with high scores were selected for further investigation.Dual-luciferase assay,electrophoretic mobility shift assay?EMSA?and chromatin immunoprecipitation?ChIP?assay were performed to analyze the respective promoter regions.In the dual-luciferase assay,overexpression of GATA4enhanced the transcription of Barx1 compared with that in the controls.The mutant GATA4 core binding sequence of the Barx1 promoter reduced the induction via both binding sites.As revealed by EMSA,in the first predicted binding site,a retarded band was observed in the lane containing the GATA4-Barx1.Chromatin immunoprecipitation?ChIP?assay was carried out using NCCs.ChIP analysis was performed using anti-GATA4 antibody,and then the binding sequence of the Barx1promoter was obtained by PCR amplification.The ChIP assay demonstrated a significant increase in the binding efficiency,confirming the binding of GATA4 to the Barx1 promoter.To confirm that gata4 expression promotes the development of neural crest derivatives via barx1,we adopted a loss-of-function analysis of gata4 in zebrafish.To knockdown the expression of gata4 in zebrafish embryos,we used gata4 MO?morpholino?.Zebrafish embryos injected with a standard control MO were used as controls.First,we examined the knockdown efficiency of gata4 MO by qRT-PCR and confirmed that the expression of gata4 was significantly reduced,indicating that gata4 MO inhibited the function of gata4.Subsequently,we observed the morphological defects induced by gata4 MO at 72 h post fertilization?hpf?.Compared with the control embryos,gata4 MO embryos were smaller and displayed a prominent indentation in the lower jaw,fewer iridophores and edema around the heart.Alcian blue staining indicated that the first pair of the cartilaginous pharyngeal arch?mandibular arch?was smaller in gata4 MO-injected zebrafish at 96 hpf than in controls,with a decrease in length leading to mandibular retrognathism.These results indicate that gata4 is required for the normal development of NC derivatives.The results indicated that gata4 may promote the development of NC derivatives via barx1.Thus,we examined the expression of barx1 in zebrafish.Whole-mount in situ hybridization showed that barx1 expression in the control group at 24 hpf was normal in each of the pharyngeal arches.However,the gata4 MO group displayed a significantly lower expression of barx1 in these areas,and the results were further confirmed by qRT-PCR analysis.To determine whether barx1 expression was regulated by gata4,rescue experiments were carried out by co-injecting gata4 MO and barx1 mRNA.Notably,administration of barx1 mRNA could partially rescue the defects caused by gata4 deficiency.The length of the mandible and the number of the iridophores in the eyes and the body were also partly rescued compared with those in the gata4 MO group.In summary,our findings using two independent animal models indicated that Barx1 is a downstream effector of GATA4,which thereby trans-activates Barx1expression and regulates NC development.Uncovering the relationship between GATA4 and Barx1 in NCCs during development is critical to understand the complex morphogenic event.It may also provide direct insight into the causes and potential treatments of similar disorders in humans.