GATA4/FOG2 transcriptional complex regulates Lhx9 gene expression in murine heart development

<p>Abstract</p> <p>Background</p> <p>GATA4 and FOG2 proteins are required for normal cardiac development in mice. It has been proposed that GATA4/FOG2 transcription complex exercises its function through gene activation as well as repression; however, targets of GATA4/FOG2 action in the heart remain elusive.</p> <p>Results</p> <p>Here we report identification of the <it>Lhx9 </it>gene as a direct target of the GATA4/FOG2 complex. We demonstrate that the developing mouse heart normally expresses truncated isoforms of <it>Lhx9 </it>– <it>Lhx9α </it>and <it>Lhx9β</it>, and not the <it>Lhx9-HD </it>isoform that encodes a protein with an intact homeodomain. At E9.5 <it>Lhx9α/β </it>expression is prominent in the epicardial primordium, septum transversum while <it>Lhx9-HD </it>is absent from this tissue; in the E11.5 heart LHX9α/β-positive cells are restricted to the epicardial mesothelium. Thereafter in the control hearts <it>Lhx9α/β </it>epicardial expression is promptly down-regulated; in contrast, mouse mutants with <it>Fog2 </it>gene loss fail to repress <it>Lhx9α/β </it>expression. Chromatin immunoprecipitation from the E11.5 hearts demonstrated that <it>Lhx9 </it>is a direct target for GATA4 and FOG2. In transient transfection studies the expression driven by the cis-regulatory regions of <it>Lhx9 </it>was repressed by FOG2 in the presence of intact GATA4, but not the GATA4^kimutant that is impaired in its ability to bind FOG2.</p> <p>Conclusion</p> <p>In summary, the <it>Lhx9 </it>gene represents the first direct target of the GATA4/FOG2 repressor complex in cardiac development.</p

Ovarian Development in Mice Requires the GATA4-FOG2 Transcription Complex

We have demonstrated previously that mammalian sexual differentiation requires both the GATA4 and FOG2 transcriptional regulators to assemble the functioning testis. Here we have determined that the sexual development of female mice is profoundly affected by the loss of GATA4-FOG2 interaction. We have also identified the Dkk1 gene, which encodes a secreted inhibitor of canonical beta-catenin signaling, as a target of GATA4-FOG2 repression in the developing ovary. The tissue-specific ablation of the beta-catenin gene in the gonads disrupts female development. In Gata4(ki/ki); Dkk1(-/-) or Fog2(-/-); Dkk1(-/-) embryos, the normal ovarian gene expression pattern is partially restored. Control of ovarian development by the GATA4-FOG2 complex presents a novel insight into the cross-talk between transcriptional regulation and extracellular signaling that occurs in ovarian development

Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods

PurposeThe application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic, with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells, but previous studies have shown variability in iPSC propensity to differentiate into RPE.MethodsThis study provides a comparison between 2 different methods for RPE differentiation: (1) a commonly used spontaneous continuously adherent culture (SCAC) protocol and (2) a more rapid, directed differentiation using growth factors. Integration-free iPSC lines were differentiated to RPE, which were characterized with respect to global gene expression, expression of RPE markers, and cellular function.ResultsWe found that all 5 iPSC lines (iPSC-1, iPSC-2, iPSC-3, iPSC-4, and iPSC-12) generated RPE using the directed differentiation protocol; however, 2 of the 5 iPSC lines (iPSC-4 and iPSC-12) did not yield RPE using the SCAC method. Both methods can yield bona fide RPE that expresses signature RPE genes and carry out RPE functions, and are similar, but not identical to fetal RPE. No differences between methods were detected in transcript levels, protein localization, or functional analyses between iPSC-1-RPE, iPSC-2-RPE, and iPSC-3-RPE. Directed iPSC-3-RPE showed enhanced transcript levels of RPE65 compared to directed iPSC-2-RPE and increased BEST1 expression and pigment epithelium-derived factor (PEDF) secretion compared to directed iPSC-1-RPE. In addition, SCAC iPSC-3-RPE secreted more PEDF than SCAC iPSC-1-RPE.ConclusionsThe directed protocol is a more reliable method for differentiating RPE from various pluripotent sources and some iPSC lines are more amenable to RPE differentiation

Abstracts from the NIHR INVOLVE Conference 2017

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Concise Review: Making Stem Cells Retinal: Methods for Deriving Retinal Pigment Epithelium and Implications for Patients With Ocular Disease.

Stem cells provide a potentially unlimited source of cells for treating a plethora of human diseases. Regenerative therapies for retinal degenerative diseases are at the forefront of translation to the clinic, with stem cell-derived retinal pigment epithelium (RPE)-based treatments for age-related macular degeneration (AMD) already showing promise in human patients. Despite our expanding knowledge of stem cell biology, methods for deriving cells, including RPE have remained inefficient. Thus, there has been a push in recent years to develop more directed approaches to deriving cells for therapy. In this concise review, we summarize recent efforts that have been successful in improving RPE derivation efficiency by directing differentiation from human pluripotent stem cells using developmental cues important for normal RPE specification and maturation in vivo. In addition, potential obstacles for clinical translation are discussed. Finally, we review how derivation of RPE from human induced pluripotent stem cells (hiPSCs) provides in vitro models for studying mechanisms of retinal disease and discovering new avenues for treatment