Alistair J. Watt

Cardiac-Specific Deletion of Gata4 Reveals Its Requirement for Hypertrophy, Compensation, and Myocyte Viability

The transcription factor GATA4 is a critical regulator of cardiac gene expression where it controls embryonic development, cardiomyocyte differentiation, and stress responsiveness of the adult heart. Traditional deletion of Gata4 caused embryonic lethality associated with endoderm defects and cardiac malformations, precluding an analysis of the role of GATA4 in the adult myocardium. To address the function of GATA4 in the adult heart, Gata4-loxP–targeted mice (Gata4fl/fl) were crossed with mice containing a β-myosin heavy chain (β-MHC) or α-MHC promoter-driven Cre transgene, which produced viable mice that survived into adulthood despite a 95% and 70% loss of GATA4 protein, respectively. However, cardiac-specific deletion of Gata4 resulted in a progressive and dosage-dependent deterioration in cardiac function and dilation in adulthood. Moreover, pressure overload stimulation induced rapid decompensation and heart failure in cardiac-specific Gata4-deleted mice. More provocatively, Gata4-deleted mice were compromised in their ability to hypertrophy following pressure overload or exercise stimulation. Mechanistically, cardiac-specific deletion of Gata4 increased cardiomyocyte TUNEL at baseline in embryos and adults as they aged, as well as dramatically increased TUNEL following pressure overload stimulation. Examination of gene expression profiles in the heart revealed a number of profound alterations in known GATA4-regulated structural genes as well as genes with apoptotic implications. Thus, GATA4 is a necessary regulator of cardiac gene expression, hypertrophy, stress-compensation, and myocyte viability.

GATA6 Is Essential for Embryonic Development of the Liver but Dispensable for Early Heart Formation

ABSTRACT Several lines of evidence suggest that GATA6 has an integral role in controlling development of the mammalian liver. Unfortunately, this proposal has been impossible to address directly because mouse embryos lacking GATA6 die during gastrulation. Here we show that the early embryonic deficiency associated with GATA6-knockout mice can be overcome by providing GATA6-null embryos with a wild-type extraembryonic endoderm with the use of tetraploid embryo complementation. Analysis of rescued Gata6 − / − embryos revealed that, although hepatic specification occurs normally, the specified cells fail to differentiate and the liver bud does not expand. Although GATA6 is expressed in multiple tissues that impact development of the liver, including the heart, septum transversum mesenchyme, and vasculature, all are relatively unaffected by loss of GATA6, which is consistent with a cell-autonomous requirement for GATA6 during hepatogenesis. We also demonstrate that a closely related GATA factor, GATA4, is expressed transiently in the prehepatic endoderm during hepatic specification and then lost during expansion of the hepatic primordium. Our data support the proposal that GATA4 and GATA6 are functionally redundant during hepatic specification but that GATA6 alone is available for liver bud growth and commitment of the endoderm to a hepatic cell fate.

Loss of both GATA4 and GATA6 blocks cardiac myocyte differentiation and results in acardia in mice.

Despite significant advances in identifying signaling molecules that induce cardiogenesis in mammals, the transcription factors that control the onset of cardiac myocyte gene expression have remained elusive. Candidates include the zinc finger transcription factors GATA binding proteins 4 and 6 (GATA4, GATA6). The individual loss of either protein in mice results in lethality prior to the onset of heart development due to defects in the extra-embryonic endoderm; however, when this extra-embryonic deficiency is circumvented using tetraploid embryo complementation, cardiac myocyte differentiation initiates normally. Here we show that these factors have redundant roles in controlling the onset of cardiac myocyte differentiation. As a consequence, Gata4(-/-)Gata6(-/-) embryos completely lack hearts, although second heart field progenitor cells are still generated. Our data support a model whereby GATA4 or GATA6 are essential for expression of the network of transcription factors that regulate the onset of cardiac myocyte gene expression during mammalian development.

Development of the mammalian liver and ventral pancreas is dependent on GATA4

BackgroundIn the mouse, the parenchyma of both the liver and ventral pancreas is specified from adjacent domains of the ventral foregut endoderm. GATA4, a zinc finger transcription factor, is strongly expressed in these endodermal domains and molecular analyses have implicated GATA4 in potentiating liver gene expression during the onset of hepatogenesis. We therefore hypothesized that GATA4 has an integral role in controlling the early stages of pancreatic and liver development.ResultsTo determine whether GATA4 contributes to development of either the pancreas or liver we characterized the formation of pancreatic and hepatic tissues in embryos derived from Gata4-/- ES cells by tetraploid embryo complementation. In the absence of GATA4, development of the liver and ventral pancreas was disrupted. At embryonic day (E) 9.5, the liver bud failed to expand although, contrary to expectations, the hepatic endoderm was able to form a pseudo-stratified epithelial liver bud that expressed hepatic genes. Moreover, as we had shown previously, the embryos lacked septum transversum mesenchyme suggesting that liver defects may be cell non-autonomous. Analyses of pancreatic development revealed a complete absence of the ventral but not the dorsal pancreas in Gata4-/- embryos. Moreover, Gata6-/- embryos displayed a similar, although less dramatic phenotype, suggesting a critical role for multiple GATA factors at the earliest stages of ventral pancreas development.ConclusionThis study defines integral roles for GATA factors in controlling early development of the mammalian liver and pancreas.

Alpha-fetoprotein, the major fetal serum protein, is not essential for embryonic development but is required for female fertility

The alpha-fetoprotein gene (Afp) is a member of a multigenic family that comprises the related genes encoding albumin, alpha-albumin, and vitamin D binding protein. The biological role of this major embryonic serum protein is unknown although numerous speculations have been made. We have used gene targeting to show that AFP is not required for embryonic development. AFP null embryos develop normally, and individually transplanted homozygous embryos can develop in an AFP-deficient microenvironment. Whereas mutant homozygous adult males are viable and fertile, AFP null females are infertile. Our analyses of these mice indicate that the defect is caused by a dysfunction of the hypothalamic/pituitary system, leading to anovulation.

Glucocorticoid receptor is required for foetal heart maturation

Glucocorticoids are vital for the structural and functional maturation of foetal organs, yet excessive foetal exposure is detrimental to adult cardiovascular health. To elucidate the role of glucocorticoid signalling in late-gestation cardiovascular maturation, we have generated mice with conditional disruption of glucocorticoid receptor (GR) in cardiomyocytes and vascular smooth muscle cells using smooth muscle protein 22-driven Cre recombinase (SMGRKO mice) and compared them with mice with global deficiency in GR (GR(-/-)). Echocardiography shows impaired heart function in both SMGRKO and GR(-/-) mice at embryonic day (E)17.5, associated with generalized oedema. Cardiac ultrastructure is markedly disrupted in both SMGRKO and GR(-/-) mice at E17.5, with short, disorganized myofibrils and cardiomyocytes that fail to align in the compact myocardium. Failure to induce critical genes involved in contractile function, calcium handling and energy metabolism underpins this common phenotype. However, although hearts of GR(-/-) mice are smaller, with 22% reduced ventricular volume at E17.5, SMGRKO hearts are normally sized. Moreover, while levels of mRNA encoding atrial natriuretic peptide are reduced in E17.5 GR(-/-) hearts, they are normal in foetal SMGRKO hearts. These data demonstrate that structural, functional and biochemical maturation of the foetal heart is dependent on glucocorticoid signalling within cardiomyocytes and vascular smooth muscle, though some aspects of heart maturation (size, ANP expression) are independent of GR at these key sites.

A gene trap integration provides an early in situ marker for hepatic specification of the foregut endoderm.

We report the characterization of a gene trap integration that provides an in situ marker for one of the earliest events in liver development. Expression of the reporter gene is observed at the nine-somite stage in the hepatic field of the foregut endoderm. At 10.5 days post-coitus expression is observed exclusively and at high levels in the majority of cells in the developing liver bud. As development proceeds the proportion of expressing cells decreases with expression in adult liver being restricted to a few sporadic cells. This therefore provides the earliest, most specific in situ marker of the hepatic lineage reported to date and will be useful in the further characterization of the inductive events involved in hepatic specification. Molecular characterization of the gene trap insertion suggests that the expression pattern is the result of alternative promoter use in the ankyrin repeat-containing gene, gtar.

A murine specific expansion of the Rhox cluster involved in embryonic stem cell biology is under natural selection

BackgroundThe rodent specific reproductive homeobox (Rhox) gene cluster on the X chromosome has been reported to contain twelve homeobox-containing genes, Rhox1-12.ResultsWe have identified a 40 kb genomic region within the Rhox cluster that is duplicated eight times in tandem resulting in the presence of eight paralogues of Rhox2 and Rhox3 and seven paralogues of Rhox4. Transcripts have been identified for the majority of these paralogues and all but three are predicted to produce full-length proteins with functional potential. We predict that there are a total of thirty-two Rhox genes at this genomic location, making it the most gene-rich homoeobox cluster identified in any species. From the 95% sequence similarity between the eight duplicated genomic regions and the synonymous substitution rate of the Rhox2, 3 and 4 paralogues we predict that the duplications occurred after divergence of mouse and rat and represent the youngest homoeobox cluster identified to date. Molecular evolutionary analysis reveals that this cluster is an actively evolving region with Rhox2 and 4 paralogues under diversifying selection and Rhox3 evolving neutrally. The biological importance of this duplication is emphasised by the identification of an important role for Rhox2 and Rhox4 in regulating the initial stages of embryonic stem (ES) cell differentiation.ConclusionThe gene rich Rhox cluster provides the mouse with significant biological novelty that we predict could provide a substrate for speciation. Moreover, this unique cluster may explain species differences in ES cell derivation and maintenance between mouse, rat and human.

Deriving and identifying hepatocytes from embryonic stem cells.

The generation of hepatocytes from embryonic stem cells (ESCs) holds considerable promise for basic and applied research. However, the unequivocal identification of hepatocytes in ESC differentiation strategies has been hampered by a lack of hepatocyte-specific markers. Recent studies are beginning to address this issue with the identification of hepatocyte-specific genes and the production of hepatocytes from intermediate cell types like definitive endoderm. Assuming the successful identification of ESC-derived hepatocytes, the next challenge will be in balancing the proliferation and differentiation of these cells in order to generate usable numbers of functional hepatocytes in vitro.

A Role for MOSPD1 in Mesenchymal Stem Cell Proliferation and Differentiation

Mesenchymal stem cells (MSCs) isolated from many tissues including bone marrow and fat can be expanded in vitro and can differentiate into a range of different cell types such as bone, cartilage, and adipocytes. MSCs can also exhibit immunoregulatory properties when transplanted but, although a number of clinical trials using MSCs are in progress, the molecular mechanisms that control their production, proliferation, and differentiation are poorly understood. We identify MOSPD1 as a new player in this process. We generated MOSPD1‐null embryonic stem cells (ESCs) and demonstrate that they are deficient in their ability to differentiate into a number of cell lineages including osteoblasts, adipocytes, and hematopoietic progenitors. The self‐renewal capacity of MOSPD1‐null ESCs was normal and they exhibited no obvious defects in early germ layer specification nor in epithelial to mesenchymal transition (EMT), indicating that MOSPD1 functions after these key steps in the differentiation process. Mesenchymal stem cell (MSC)‐like cells expressing CD73, CD90, and CD105 were generated from MOSPD1‐null ESCs but their growth rate was significantly impaired implying that MOSPD1 plays a role in MSC proliferation. Phenotypic deficiencies exhibited by MOSPD1‐null ESCs were rescued by exogenous expression of MOSPD1, but not MOSPD3 indicating distinct functional properties of these closely related genes. Our in vitro studies were supported by RNA‐sequencing data that confirmed expression of Mospd1 mRNA in cultured, proliferating perivascular pre‐MSCs isolated from human tissue. This study adds to the growing body of knowledge about the function of this largely uncharacterized protein family and introduces a new player in the control of MSC proliferation and differentiation. Stem Cells 2015;33:3077–3086