Shan-Mei Xu

Mef2c is a direct transcriptional target of ISL1 and GATA factors in the anterior heart field during mouse embryonic development

The vertebrate heart forms initially as a linear tube derived from a primary heart field in the lateral mesoderm. Recent studies in mouse and chick have demonstrated that the outflow tract and right ventricle originate from a separate source of mesoderm that is anterior to the primary heart field. The discovery of this anterior, or secondary, heart field has led to a greater understanding of the morphogenetic events involved in heart formation; however, many of the underlying molecular events controlling these processes remain to be determined. The MADS domain transcription factor MEF2C is required for proper formation of the cardiac outflow tract and right ventricle, suggesting a key role in anterior heart field development. Therefore, as a first step toward identifying the transcriptional pathways upstream of MEF2C, we introduced a lacZ reporter gene into a bacterial artificial chromosome (BAC) encompassing the murine Mef2c locus and used this recombinant to generate transgenic mice. This BAC transgene was sufficient to recapitulate endogenous Mef2c expression, and comparative sequence analyses revealed multiple regions of significant conservation in the noncoding regions of the BAC. We show that one of these conserved noncoding regions represents a transcriptional enhancer that is sufficient to direct expression of lacZ exclusively to the anterior heart field throughout embryonic development. This conserved enhancer contains two consensus GATA binding sites that are efficiently bound by the zinc finger transcription factor GATA4 and are completely required for enhancer function in vivo. This enhancer also contains two perfect consensus sites for the LIM-homeodomain protein ISL1. We show that these elements are specifically bound by ISL1 and are essential for enhancer function in transgenic embryos. Thus, these findings establish Mef2c as the first direct transcriptional target of ISL1 in the anterior heart field and support a model in which GATA factors and ISL1 serve as the earliest transcriptional regulators controlling outflow tract and right ventricle development.

mef2c is activated directly by myogenic basic helix-loop-helix proteins during skeletal muscle development in vivo

Skeletal muscle development requires the coordinated expression of numerous transcription factors to control the specification of mesodermal progenitor cells to a muscle fate and the differentiation of those committed myoblasts into functional, contractile muscle. Two families of transcription factors play key roles in these processes. The myogenic basic helix-loop-helix (bHLH) proteins, MyoD and Myf5, are required for myoblast specification, while two members of the same family, myogenin and MRF4, play key roles in myoblast differentiation in vivo. All four members of the myogenic bHLH family are sufficient to dominantly induce myogenesis when introduced into a variety of non-muscle cells in culture, however this function requires the activity of a second family of transcriptional regulators, the myocyte enhancer factor 2 (MEF2) family. MEF2 factors are essential for muscle differentiation, and previous studies have shown that MyoD and MEF2 family members function combinatorially to activate transcription and myogenesis. Consistent with these observations, the majority of skeletal muscle genes require both MyoD and MEF2 family members to activate their transcription. A possible exception to this combinatorial model for activation is suggested by the observation that myogenic bHLH factors may be able to independently activate the expression of MEF2. This raises the question as to how mef2 gene transcription is induced by MyoD factors without cooperative activation by MEF2. During skeletal muscle development, mef2c is the first member of the MEF2 family to be expressed. In this study, we have investigated the regulation of a skeletal muscle-specific enhancer from the mouse mef2c gene using a transgenic approach. We show that mef2c is a direct transcriptional target of the MyoD family in vivo via an essential E box in the skeletal muscle enhancer of mef2c, and we show that mef2c is not a direct target for autoregulation by MEF2.

Gata4 expression in lateral mesoderm is downstream of BMP4 and is activated directly by Forkhead and GATA transcription factors through a distal enhancer element.

The GATA family of zinc-finger transcription factors plays key roles in the specification and differentiation of multiple cell types during development. GATA4 is an early regulator of gene expression during the development of endoderm and mesoderm, and genetic studies in mice have demonstrated that GATA4 is required for embryonic development. Despite the importance of GATA4 in tissue specification and differentiation, the mechanisms by which Gata4 expression is activated and the transcription factor pathways upstream of GATA4 remain largely undefined. To identify transcriptional regulators of Gata4 in the mouse, we screened conserved noncoding sequences from the mouse Gata4 gene for enhancer activity in transgenic embryos. Here, we define the regulation of a distal enhancer element from Gata4 that is sufficient to direct expression throughout the lateral mesoderm, beginning at 7.5 days of mouse embryonic development. The activity of this enhancer is initially broad but eventually becomes restricted to the mesenchyme surrounding the liver. We demonstrate that the function of this enhancer in transgenic embryos is dependent upon highly conserved Forkhead and GATA transcription factor binding sites, which are bound by FOXF1 and GATA4, respectively. Furthermore, the activity of the Gata4 lateral mesoderm enhancer is attenuated by the BMP antagonist Noggin, and the enhancer is not activated in Bmp4-null embryos. Thus, these studies establish that Gata4 is a direct transcriptional target of Forkhead and GATA transcription factors in the lateral mesoderm, and demonstrate that Gata4 lateral mesoderm enhancer activation requires BMP4, supporting a model in which GATA4 serves as a downstream effector of BMP signaling in the lateral mesoderm.

Alveolar Type II Cells Possess the Capability of Initiating Lung Tumor Development

Identifying cells of tumor origin is a fundamental question in tumor biology. Answers to this central question will not only advance our understanding of tumor initiation and progression but also have important therapeutic implications. In this study, we aimed to uncover the cells of origin of lung adenocarcinoma, a major subtype of non-small cell lung cancer. To this end, we developed new mouse models of lung adenocarcinoma that enabled selective manipulation of gene activity in surfactant associated protein C (SPC)-expressing cells, including alveolar type II cells and bronchioalveolar stem cells (BASCs) that reside at the bronchioalveolar duct junction (BADJ). Our findings showed that activation of oncogenic Kras alone or in combination with the removal of the tumor suppressor p53 in SPC+ cells resulted in development of alveolar tumors. Similarly, sustained EGF signaling in SPC+ cells led to alveolar tumors. By contrast, BASCs failed to proliferate or produce tumors under these conditions. Importantly, in a mouse strain in which Kras/p53 activity was selectively altered in type II cells but not BASCs, alveolar tumors developed while BADJs retained normal architecture. These results confirm and extend previous findings and support a model in which lung adenocarcinoma can initiate in alveolar type II cells. Our results establish the foundation for elucidating the molecular mechanisms by which lung cancer initiates and progresses in a specific lung cell type.

ETS-Dependent Regulation of a Distal Gata4 Cardiac Enhancer

The developing heart contains an inner tube of specialized endothelium known as endocardium, which performs multiple essential functions. In spite of the essential role of the endocardium in heart development and function, the transcriptional pathways that regulate its development remain largely undefined. GATA4 is a zinc finger transcription factor that is expressed in multiple cardiovascular lineages and is required for endocardial cushion development and embryonic viability, but the transcriptional pathways upstream of Gata4 in the endocardium and its derivatives in the endocardial cushions are unknown. Here, we describe a distal enhancer from the mouse Gata4 gene that is briefly active in multiple cardiac lineages early in cardiac development but restricts to the endocardium where it remains active through cardiogenesis. The activity of this Gata4 cardiac enhancer in transgenic embryos and in cultured aortic endothelial cells is dependent on four ETS sites. To identify which ETS transcription factors might be involved in Gata4 regulation via the ETS sites in the enhancer, we determined the expression profile of 24 distinct ETS factors in embryonic mouse hearts. Among multiple ETS transcripts present, ETS1, FLI1, ETV1, ETV5, ERG, and ETV6 were the most abundant in the early embryonic heart. We found that ETS1, FLI1, and ERG were strongly expressed in the heart at embryonic day 8.5 and that ETS1 and ERG bound to the endogenous Gata4 enhancer in cultured endothelial cells. Thus, these studies define the ETS expression profile in the early embryonic heart and identify an ETS-dependent enhancer from the Gata4 locus.

Isl1 is a direct transcriptional target of Forkhead transcription factors in second-heart-field-derived mesoderm.

The cells of the second heart field (SHF) contribute to the outflow tract and right ventricle, as well as to parts of the left ventricle and atria. Isl1, a member of the LIM-homeodomain transcription factor family, is expressed early in this cardiac progenitor population and functions near the top of a transcriptional pathway essential for heart development. Isl1 is required for the survival and migration of SHF-derived cells into the early developing heart at the inflow and outflow poles. Despite this important role for Isl1 in early heart formation, the transcriptional regulation of Isl1 has remained largely undefined. Therefore, to identify transcription factors that regulate Isl1 expression in vivo, we screened the conserved noncoding sequences from the mouse Isl1 locus for enhancer activity in transgenic mouse embryos. Here, we report the identification of an enhancer from the mouse Isl1 gene that is sufficient to direct expression to the SHF and its derivatives. The Isl1 SHF enhancer contains three consensus Forkhead transcription factor binding sites that are efficiently and specifically bound by Forkhead transcription factors. Importantly, the activity of the enhancer is dependent on these three Forkhead binding sites in transgenic mouse embryos. Thus, these studies demonstrate that Isl1 is a direct transcriptional target of Forkhead transcription factors in the SHF and establish a transcriptional pathway upstream of Isl1 in the SHF.

The MADS box transcription factor MEF2C regulates melanocyte development and is a direct transcriptional target and partner of SOX10.

Waardenburg syndromes are characterized by pigmentation and autosensory hearing defects, and mutations in genes encoding transcription factors that control neural crest specification and differentiation are often associated with Waardenburg and related disorders. For example, mutations in SOX10 result in a severe form of Waardenburg syndrome, Type IV, also known as Waardenburg-Hirschsprung disease, characterized by pigmentation and other neural crest defects, including defective innervation of the gut. SOX10 controls neural crest development through interactions with other transcription factors. The MADS box transcription factor MEF2C is an important regulator of brain, skeleton, lymphocyte and cardiovascular development and is required in the neural crest for craniofacial development. Here, we establish a novel role for MEF2C in melanocyte development. Inactivation of Mef2c in the neural crest of mice results in reduced expression of melanocyte genes during development and a significant loss of pigmentation at birth due to defective differentiation and reduced abundance of melanocytes. We identify a transcriptional enhancer of Mef2c that directs expression to the neural crest and its derivatives, including melanocytes, in transgenic mouse embryos. This novel Mef2c neural crest enhancer contains three functional SOX binding sites and a single essential MEF2 site. We demonstrate that Mef2c is a direct transcriptional target of SOX10 and MEF2 via this evolutionarily conserved enhancer. Furthermore, we show that SOX10 and MEF2C physically interact and function cooperatively to activate the Mef2c gene in a feed-forward transcriptional circuit, suggesting that MEF2C might serve as a potentiator of the transcriptional pathways affected in Waardenburg syndromes.

Direct transcriptional regulation of Gata4 during early endoderm specification is controlled by FoxA2 binding to an intronic enhancer

The embryonic endoderm is a multipotent progenitor cell population that gives rise to the epithelia of the digestive and respiratory tracts, the liver and the pancreas. Among the transcription factors that have been shown to be important for endoderm development and gut morphogenesis is GATA4. Despite the important role of GATA4 in endoderm development, its transcriptional regulation is not well understood. In this study, we identified an intronic enhancer from the mouse Gata4 gene that directs expression to the definitive endoderm in the early embryo. The activity of this enhancer is initially broad in all endodermal progenitors, as demonstrated by fate mapping analysis using the Cre/loxP system, but becomes restricted to the dorsal foregut and midgut, and associated organs such as dorsal pancreas and stomach. The function of the intronic Gata4 enhancer is dependent upon a conserved Forkhead transcription factor-binding site, which is bound by recombinant FoxA2 in vitro. These studies identify Gata4 as a direct transcriptional target of FoxA2 in the hierarchy of the transcriptional regulatory network that controls the development of the definitive endoderm.

An endoderm-specific transcriptional enhancer from the mouse Gata4 gene requires GATA and homeodomain protein-binding sites for function in vivo.

Several transcription factors function in the specification and differentiation of the endoderm, including the zinc finger transcription factor GATA4. Despite its essential role in endoderm development, the transcriptional control of the Gata4 gene in the developing endoderm and its derivatives remains incompletely understood. Here, we identify a distal enhancer from the Gata4 gene, which directs expression exclusively to the visceral and definitive endoderm of transgenic mouse embryos. The activity of this enhancer is initially broad within the definitive endoderm but later restricts to developing endoderm‐derived tissues, including pancreas, glandular stomach, and duodenum. The activity of this enhancer in vivo is dependent on evolutionarily‐conserved HOX‐ and GATA‐binding sites, which are bound by PDX‐1 and GATA4, respectively. These studies establish Gata4 as a direct transcriptional target of homeodomain and GATA transcription factors in the endoderm and support a model in which GATA4 functions in the transcriptional network for pancreas formation. Developmental Dynamics 238:2588–2598, 2009.

An arterial-specific enhancer of the human endothelin converting enzyme 1 (ECE1) gene is synergistically activated by Sox17, FoxC2, and Etv2.

Endothelin-converting enzyme-1 (Ece-1), a crucial component of the Endothelin signaling pathway, is required for embryonic development and is an important regulator of vascular tone, yet the transcriptional regulation of the ECE1 gene has remained largely unknown. Here, we define the activity and regulation of an enhancer from the human ECE1 locus in vivo. The enhancer identified here becomes active in endothelial progenitor cells shortly after their initial specification and is dependent on a conserved FOX:ETS motif, a composite binding site for Forkhead transcription factors and the Ets transcription factor Etv2, for activity in vivo. The ECE1 FOX:ETS motif is bound and cooperatively activated by FoxC2 and Etv2, but unlike other described FOX:ETS-dependent enhancers, ECE1 enhancer activity becomes restricted to arterial endothelium and endocardium by embryonic day 9.5 in transgenic mouse embryos. The ECE1 endothelial enhancer also contains an evolutionarily-conserved, consensus SOX binding site, which is required for activity in transgenic mouse embryos. Importantly, the ECE1 SOX site is bound and activated by Sox17, a transcription factor involved in endothelial cell differentiation and an important regulator of arterial identity. Moreover, the ECE1 enhancer is cooperatively activated by the combinatorial action of FoxC2, Etv2, and Sox17. Although Sox17 is required for arterial identity, few direct transcriptional targets have been identified in endothelial cells. Thus, this work has important implications for our understanding of endothelial specification and arterial subspecification.