IGF signaling directs ventricular cardiomyocyte proliferation during embryonic heart development.

Secreted factors from the epicardium are believed to be important in directing heart ventricular cardiomyocyte proliferation and morphogenesis, although the specific factors involved have not been identified or characterized adequately. We found that IGF2 is the most prominent mitogen made by primary mouse embryonic epicardial cells and by a newly derived immortalized mouse embryonic epicardial cell line called MEC1. In vivo, Igf2 is expressed in the embryonic mouse epicardium during midgestation heart development. Using a whole embryo culture assay in the presence of inhibitors, we confirmed that IGF signaling is required to activate the ERK proliferation pathway in the developing heart, and that the epicardium is required for this response. Global disruption of the Igf2 gene, or conditional disruption of the two IGF receptor genes Igf1r and Insr together in the myocardium, each resulted in a significant decrease in ventricular wall proliferation and in ventricular wall hypoplasia. Ventricular cardiomyocyte proliferation in mutant embryos was restored to normal at E14.5, concurrent with the establishment of coronary circulation. Our results define IGF2 as a previously unexplored epicardial mitogen that is required for normal ventricular chamber development.

Knockdown of Hspa9, a del(5q31.2) gene, results in a decrease in hematopoietic progenitors in mice.

Heterozygous deletions spanning chromosome 5q31.2 occur frequently in the myelodysplastic syndromes (MDS) and are highly associated with progression to acute myeloid leukemia (AML) when p53 is mutated. Mutagenesis screens in zebrafish and mice identified Hspa9 as a del(5q31.2) candidate gene that may contribute to MDS and AML pathogenesis, respectively. To test whether HSPA9 haploinsufficiency recapitulates the features of ineffective hematopoiesis observed in MDS, we knocked down the expression of HSPA9 in primary human hematopoietic cells and in a murine bone marrow-transplantation model using lentivirally mediated gene silencing. Knockdown of HSPA9 in human cells significantly delayed the maturation of erythroid precursors, but not myeloid or megakaryocytic precursors, and suppressed cell growth by 6-fold secondary to an increase in apoptosis and a decrease in the cycling of cells compared with control cells. Erythroid precursors, B lymphocytes, and the bone marrow progenitors c-kit(+)/lineage(-)/Sca-1(+) (KLS) and megakaryocyte/erythrocyte progenitor (MEP) were significantly reduced in a murine Hspa9-knockdown model. These abnormalities suggest that cooperating gene mutations are necessary for del(5q31.2) MDS cells to gain clonal dominance in the bone marrow. Our results demonstrate that Hspa9 haploinsufficiency alters the hematopoietic progenitor pool in mice and contributes to abnormal hematopoiesis.

Phylogenetic analysis of three complete gap junction gene families reveals lineage-specific duplications and highly supported gene classes.

Gap junctions, composed of connexin proteins in chordates, are the most ubiquitous form of intercellular communication. Complete connexin gene families have been identified from human (20) and mouse (19), revealing significant diversity in gap junction channels. We searched current databases and identified 37 putative zebrafish connexin genes, almost twice the number found in mammals. Phylogenetic comparison of entire connexin gene families from human, mouse, and zebrafish revealed 23 zebrafish relatives of 16 mammalian connexins, and 14 connexins apparently unique to zebrafish. We found evidence for duplication events in all genomes, as well as evidence for recent tandem duplication events in the zebrafish, indicating that the complexity of the connexin family is growing. The identification of a third complete connexin gene family provides novel insight into the evolution of connexins, and sheds light into the phenotypic evolution of intercellular communication via gap junctions.

The role of erythropoietin in regulating angiogenesis.

Erythropoietin (EPO) is an essential growth factor that regulates erythrocyte production in mammals. In this study, we demonstrate a novel role of EPO in regulating angiogenesis in vivo. Epo and Epo receptor (EpoR) are expressed in the vasculature during embryogenesis. Deletion of Epo or EpoR leads to angiogenic defects starting at E10.5, 2 days before ventricular hypoplasia and 3 days before the onset of the embryonic lethal phenotype. Overall, angiogenesis was severely affected in the mutant embryos: vascular anomalies included decreased complexity of the vessel networks. However, de novo vasculogenesis remained intact, consistent with the differential expression of Epo and EpoR during the early stages of embryonic development. The aforementioned angiogenesis defect can be partially rescued by expressing human EPO during embryogenesis. Moreover, Ang-1 expression is regulated by EPO/EPOR under normoxic conditions. Taken together, our results suggest important roles of EPO and EPOR in angiogenesis.

Epicardial induction of fetal cardiomyocyte proliferation via a retinoic acid-inducible trophic factor.

Mouse embryos lacking the retinoic acid receptor RXRalpha properly undergo the early steps of heart development, but then fail to initiate a proliferative expansion of cardiomyocytes that normally results in the formation of the compact zone of the ventricular chamber wall. RXRalpha(-/-) embryos have a hypoplastic ventricular chamber and die in midgestation from cardiac insufficiency. In this study, we have investigated the underlying mechanistic basis of this phenotype. We find that interference with retinoic acid receptor function in the epicardium of transgenic embryos recapitulates the hypoplastic phenotype of RXRalpha deficient embryos. We further show that wild type primary epicardial cells, and an established epicardial cell line (EMC cells), secrete trophic protein factors into conditioned media that stimulate thymidine incorporation in primary fetal cardiomyocytes, and thymidine incorporation, cell cycle progression, and induction of cyclin D1 and E activity in NIH3T3 cells. In contrast, primary epicardial cells derived from RXRalpha(-/-) embryos and an EMC subline constitutively expressing a dominant negative receptor construct both fail to secrete activity into conditioned media. The production of trophic factors is induced by retinoic acid treatment and is inhibited by a retinoid receptor antagonist. Fetal atrial and ventricular myocytes both respond to epicardial-derived trophic signaling, although postnatal cardiomyocytes are nonresponsive. We therefore propose that the fetal epicardium, in response to retinoic acid and in a manner requiring the activity of RXRalpha, secretes trophic factors which drive fetal cardiomyocyte proliferation and promote ventricular chamber morphogenesis.