The single cell RNA sequencing (scRNA-seq) technique begin a new era by allowing the observation of gene expression at the single cell level. However, there is also a large amount of technical and biological noise. Because of the low number of RNA transcriptomes and the stochastic nature of the gene expression pattern, there is a high chance of missing nonzero entries as zero, which are called dropout events. We develop DrImpute to impute dropout events in scRNA-seq data. We show that DrImpute has significantly better performance on the separation of the dropout zeros from true zeros than existing imputation algorithms. We also demonstrate that DrImpute can significantly improve the performance of existing tools for clustering, visualization and lineage reconstruction of nine published scRNA-seq datasets. DrImpute can serve as a very useful addition to the currently existing statistical tools for single cell RNA-seq analysis. DrImpute is implemented in R and is available at https://github.com/gongx030/DrImpute .

Recent studies support the existence of a common progenitor for the cardiac and endothelial cell lineages, but the underlying transcriptional networks responsible for specification of these cell fates remain unclear. Here we demonstrated that Ets-related protein 71 (Etsrp71), a newly discovered ETS family transcription factor, was a novel downstream target of the homeodomain protein, Nkx2–5. Using genetic mouse models and molecular biological techniques, we demonstrated that Nkx2–5 binds to an evolutionarily conserved Nkx2–5 response element in the Etsrp71 promoter and induces the Etsrp71 gene expression in vitro and in vivo. Etsrp71 was transiently expressed in the endocardium/endothelium of the developing embryo (E7.75-E9.5) and was extinguished during the latter stages of development. Using a gene disruption strategy, we found that Etsrp71 mutant embryos lacked endocardial/endothelial lineages and were nonviable. Moreover, using transgenic technologies and transcriptional and chromatin immunoprecipitation (ChIP) assays, we further established that Tie2 is a direct downstream target of Etsrp71. Collectively, our results uncover a novel functional role for Nkx2–5 and define a transcriptional network that specifies an endocardial/endothelial fate in the developing heart and embryo.

Er71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse model by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the EYFP⁺ population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the number of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed to alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic stem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis.

Stem and progenitor cell populations occupy a specialized niche and are consequently exposed to hypoxic as well as oxidative stresses. We have previously established that the multidrug resistance protein Abcg2 is the molecular determinant of the side population (SP) progenitor cell population. We observed that the cardiac SP cells increase in number more than 3-fold within 3 days of injury. Transcriptome analysis of the SP cells isolated from the injured adult murine heart reveals increased expression of cytoprotective transcripts. Overexpression of Abcg2 results in an increased ability to consume hydrogen peroxide and is associated with increased levels of α-glutathione reductase protein expression. Importantly, overexpression of Abcg2 also conferred a cell survival benefit following exposure to hydrogen peroxide. To further examine the molecular regulation of the Abcg2 gene, we demonstrated that hypoxia-inducible factor (HIF)-2α binds an evolutionary conserved HIF-2α response element in the murine Abcg2 promoter. Transcriptional assays reveal a dose-dependent activation of Abcg2 expression by HIF-2α. These results support the hypothesis that Abcg2 is a direct downstream target of HIF-2α which functions with other factors to initiate a cytoprotective program for this progenitor SP cell population that resides in the adult heart.