A Research In GATA-6That Inhibites The Apoptosis Of Embryonic Stem Cell-derived Embryoid Bodies By Up-regulating BMP-2Expression In The Early Embryonic Development

Apoptosis has a crucial role in embryonic development, thecombined effect of cell proliferation and apoptosis sculpts the facialintricate structure. The apoptotic changes caused by various reasons, mayinterfere with the fusion of the bilateral palatine process and primitivepalate which contributes to the occurrence of cleft palate. Studies haveshown that bone morphogenetic protein (bone morphogenetic protein(BMP)) can lead to the maxilla and palatine bone defects by regulatingapoptosis, then induce cleft palate. While its upstream gene GATA-6contributes to multiple organ derived alloplasia. Since the completelyknocked heterozygous alleles of GATA-6can not survive and embryonicstem cell–derived embryoid bodies were able to simulate the earlyembryonic development accurately, the embryonic stem cell–derivedembryoid bodies is an ideal model for the correlation between cleft palateand GATA-6.GATA-6is a zinc-finger transcription factor essential for earlyembryogenesis. Ablation of GATA-6in mice impairs endodermdifferentiation and causes apoptosis of epiblast cells. Our preliminarystudies showed that the knock-out of GATA-6in EBs could induceapoptosis. And in this research, we found that grafting of normalendoderm cells onto the mutant EBs completely prevented apoptosis andrescued epiblast polarization and cavitation. Laminin-induced assemblyof an ectopic BM on the mutant EBs or grafting with laminin γ1–nullendoderm cells, which do not secrete trimeric laminins and thus cannot make BMs, partially inhibited apoptosis, suggesting that bothendoderm-derived BM and non-BM factors contribute to GATA-6–dependent cell survival.Our microarray analysis demonstratedthat the expression of BMP-2,insulin-like growth factor2(IGF-2),transforming growth factor β(TGF-β), and vascular endothelial growthfactor A (VEGF-A) wasincreased, whereas BMP-4, FGF-4, PDGF-A,PDGF-C, and VEGF-Cwere reduced, during EB differentiation To determine whether theexpression of these growth factors mightbe altered in GATA-6–null EBs,we performed reverse transcription(RT)-PCR and immunoblot analysison2-d EBs. The mRNAs for BMP-4and IGF-2were reduced, whereasthat for FGF-4was unchanged, inthe absence of GATA-6. Of importance,BMP-2mRNA was readily detectable in2-d normal EBs and was nearlyabsentfrom GATA-6–null EBs. To indentify the exact efforts thatdifferent growth factors really make to the apoptosis of EB, we treated1-d GATA-6–null EBs with or without0.1μg/ml BMP-2, FGF-4,epidermalgrowth factor (EGF), IGF-2, PDGF, and TGF-β1,respectively,for24h. Apoptosis was analyzed by whole-mountimmunostaining for cleaved caspase-3since it occurred mostly in theperipheralcells of the mutant EBs. BMP-2treatment markedly inhibitedtheactivation of caspase-3, whereas FGF-4, EGF and PDGF had no effect.Increasing the BMP-2concentration to0.25μg/ml led to further reductionof apoptosis. Treatment of the mutantEBs with IGF-2also moderately reduced caspase-3activation,whereas TGF-β showed the opposite effect.Immunoblot analysisconfirmed that exogenous BMP-2significantlyreduced apoptosisof GATA-6–null EBs, whereas IGF-2had a mild effect.Chip assay showed GATA-6directly binds to the Bmp2promoter, andGATA-6ablation reduces BMP-2expression at both mRNA and proteinlevels. In addition, overexpression of the BMP antagonist noggin or adominant-negative BMP receptor in normal EBs induces apoptosis.The activation of SMAD1/5by phosphorylation is significantlyinhibited in the absence of GATA-6, and this is reversed by exogenousBMP-2. Treatment of normal EBs with SMAD phosphorylation inhibitorincreases apoptosis. To further examine whether the activation of theSMAD pathway provides a survival signal, we cultured1-d wild-type EBsfor24h with5μM ofAkt1/2inhibitor, the MAPK kinase1/2inhibitorU0126, the p38inhibitor SB203580, or the R-SMAD phosphorylationinhibitor DMH1. Among these inhibitors, only DMH1markedly inducedapoptosis, as evidenced by caspase-3activation. This results shows thatBMP-2contributes to the survival of embryonic epithelial cells throughthe activation of Smad pathway.Collectively these results suggest that GATA-6promotes cellsurvival by regulating endoderm expression of BMP-2and BM duringembryonic epithelial morphogenesis and this acts on the phosphorylationof SMAD1/5. The BM in turn induces epiblast polarization and cavitation. GATA-6also directly activates the expression of BMP-2, which acts inconcert with the BM to protect the epiblast from apoptosis. In addition,BMP-2promotes endoderm differentiation in an autocrine manner. Thisfinding is important for further understanding the relevance amongGATA-6, BMP-2and apoptosis, and explore the mechanism of cleftpalate occurrence and new method to treat cleft palate.