GATA6 belongs to a family of zinc finger transcription factors that play important roles in transducing nuclear events that regulate cellular differentiation and embryonic morphogenesis in vertebrate species. To examine the function of GATA6 during embryonic development, gene targeting was used to generate GATA6-deficient (GATA6^−/−) ES cells and mice harboring a null mutation in GATA6. Differentiated embryoid bodies derived from GATA6^−/− ES cells lack a covering layer of visceral endoderm and severely attenuate, or fail to express, genes encoding early and late endodermal markers, including HNF4, GATA4, α-fetoprotein (AFP), and HNF3β. Homozygous GATA6^−/− mice died between embryonic day (E) 6.5 and E7.5 and exhibited a specific defect in endoderm differentiation including severely down-regulated expression of GATA4 and absence of HNF4 gene expression. Moreover, widespread programmed cell death was observed within the embryonic ectoderm of GATA6-deficient embryos, a finding also observed in HNF4-deficient embryos. Consistent with these data, forced expression of GATA6 activated the HNF4 promoter in nonendodermal cells. Finally, to examine the function of GATA6 during later embryonic development,GATA6^−/−-C57BL/6 chimeric mice were generated. lacZ-taggedGATA6^−/− ES cells contributed to all embryonic tissues with the exception of the endodermally derived bronchial epithelium. Taken together, these data suggest a model in which GATA6 lies upstream of HNF4 in a transcriptional cascade that regulates differentiation of the visceral endoderm. In addition, these data demonstrate that GATA6 is required for establishment of the endodermally derived bronchial epithelium.

Phylogenetic network analysis and understanding of waterfowl migration patterns suggest that the Eurasian H5N8 clade 2.3.4.4 avian influenza virus emerged in late 2013 in China, spread in early 2014 to South Korea and Japan, and reached Siberia and Beringia by summer 2014 via migratory birds. Three genetically distinct subgroups emerged and subsequently spread along different flyways during fall 2014 into Europe, North America, and East Asia, respectively. All three subgroups reappeared in Japan, a wintering site for waterfowl from Eurasia and parts of North America.

The SAP family transcription factor myocardin functionally synergizes with serum response factor (SRF) and plays an important role in cardiac development. To determine the function of myocardin in the smooth muscle cell (SMC) lineage, we mapped the pattern of myocardin gene expression and examined the molecular mechanisms underlying transcriptional activity of myocardin in SMCs and embryonic stem (ES) cells. The human and murine myocardin genes were expressed in vascular and visceral SMCs at levels equivalent to or exceeding those observed in the heart. During embryonic development, the myocardin gene was expressed abundantly in a precise, developmentally regulated pattern in SMCs. Forced expression of myocardin transactivated multiple SMC-specific transcriptional regulatory elements in non-SMCs. By contrast, myocardin-induced transactivation was not observed in SRF^−/− ES cells but could be rescued by forced expression of SRF or the SRF DNA-binding domain. Furthermore, expression of a dominant-negative myocardin mutant protein or small-interfering-RNA-induced myocardin knockdown significantly reduced SM22α promoter activity in SMCs. Most importantly, forced expression of myocardin activated expression of the SM22α, smooth muscle α-actin, and calponin-h1 genes in undifferentiated mouse ES cells. Taken together, these data demonstrate that myocardin plays an important role in the SRF-dependent transcriptional program that regulates SMC development and differentiation.

SM22 alpha is expressed exclusively in smooth muscle-containing tissues of adult animals and is one of the earliest markers of differentiated smooth muscle cells (SMCs). To examine the molecular mechanisms that regulate SMC-specific gene expression, we have isolated and structurally characterized the murine SM22 alpha gene. SM22 alpha is a 6.2-kilobase single copy gene composed of five exons. SM22 alpha mRNA is expressed at high levels in the aorta, uterus, lung, and intestine, and in primary cultures of rat aortic SMCs, and the SMC line, A7r5. In contrast to genes encoding SMC contractile proteins, SM22 alpha gene expression is not decreased in proliferating SMCs. Transient transfection experiments demonstrated that 441 base pairs of SM22 alpha 5'-flanking sequence was necessary and sufficient to program high level transcription of a luciferase reporter gene in both primary rat aortic SMCs and A7r5 cells. DNA sequence analyses revealed that the 441-base pair promoter contains two CArG/SRF boxes, a CACC box, and one potential MEF-2 binding site, cis-acting elements which are each important regulators of striated muscle transcription. Taken together, these studies have identified the murine SM22 alpha promoter as an excellent model system for studies of developmentally regulated, lineage-specific gene expression in SMCs.