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Aims Congenital heart disease (CHD) is the most common genetic birth defect, which has considerable morbidity and mortality. We focused on deciphering key regulators that govern cardiac progenitors and cardiogenesis. FOXK1 is a forkhead/winged helix transcription factor known to regulate cell cycle kinetics and is restricted to mesodermal progenitors, somites, and heart. In the present study, we define an essential role for FOXK1 during cardiovascular development. Methods and results We used the mouse embryoid body system to differentiate control and Foxk1 KO embryonic stem cells into mesodermal, cardiac progenitor cells and mature cardiac cells. Using flow cytometry, immunohistochemistry, cardiac beating, transcriptional and chromatin immunoprecipitation quantitative polymerase chain reaction assays, bulk RNA sequencing (RNAseq) and assay for transposase-accessible chromatin using sequencing (ATACseq) analyses, FOXK1 was observed to be an important regulator of cardiogenesis. Flow cytometry analyses revealed perturbed cardiogenesis in Foxk1 KO embryoid bodies (EBs). Bulk RNAseq analysis at two developmental stages showed a significant reduction of the cardiac molecular program in Foxk1 KO EBs compared to the control EBs. ATACseq analysis during EB differentiation demonstrated that the chromatin landscape nearby known important regulators of cardiogenesis was significantly relaxed in control EBs compared to Foxk1 KO EBs. Furthermore, we demonstrated that in the absence of FOXK1, cardiac differentiation was markedly impaired by assaying for cardiac Troponin T expression and cardiac contractility. We demonstrate that FOXK1 is an important regulator of cardiogenesis by repressing the Wnt/β-catenin signalling pathway and thereby promoting differentiation. Conclusion These results identify FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development. Mechanistically, FOXK1 represses Wnt signalling to promote the development of cardiac progenitor cells.

The breakthrough high-throughput measurement of the cis-regulatory activity of millions of randomly generated promoters provides an unprecedented opportunity to systematically decode the cis-regulatory logic that determines the expression values. We developed an end-to-end transformer encoder architecture named Proformer to predict the expression values from DNA sequences. Proformer used a Macaron-like Transformer encoder architecture, where two half-step feed forward (FFN) layers were placed at the beginning and the end of each encoder block, and a separable 1D convolution layer was inserted after the first FFN layer and in front of the multi-head attention layer. The slidingk-mers from one-hot encoded sequences were mapped onto a continuous embedding, combined with the learned positional embedding and strand embedding (forward strand vs. reverse complemented strand) as the sequence input. Moreover, Proformer introduced multiple expression heads with mask filling to prevent the transformer models from collapsing when training on relatively small amount of data. We empirically determined that this design had significantly better performance than the conventional design such as using the global pooling layer as the output layer for the regression task. These analyses support the notion that Proformer provides a novel method of learning and enhances our understanding of how cis-regulatory sequences determine the expression values.

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and thus relevant to the treatment of ischemic heart disease. The ETS-related transcription factor 2 (ETV2) is a transcription factor that is both necessary and sufficient for the development of endothelial and blood lineages. However, the molecular mechanism by which ETV2 promotes endothelial and blood development is not known and warrants further investigation. Here, we report that ETV2 functions as a novel pioneer transcription factor that relaxes closed chromatin and regulates endothelial development. Pioneer factors are a subset of transcription factors that bind closed chromatin and promote chromatin relaxation in order to drive the development and reprogramming of cell lineages. By comparing embryonic stem cell differentiation and fibroblast reprogramming models with single cell RNA-seq, ATAC-seq, ChIP-seq and in-vitro nucleosomal binding techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and function as a pioneer factor independent of the cellular context. We also determined that ETV2 executed a pioneering role by recruiting and directly interacting with the ATP-dependent chromatin remodeling enzyme BRG1 to remodel chromatin around endothelial genes to help maintain an open configuration, resulting in increased H3K27ac deposition. Collectively, these results that define a novel pioneer factor and epigenetic regulatory pathways that govern the specification and differentiation of endothelial progenitor cells, will serve as a platform for targeted therapies to promote cardiovascular regeneration.

Because post-mortem human skeletal muscle is not viable, autologous muscle grafts are typically required in tissue reconstruction after muscle loss due to disease or injury. However, the use of autologous tissue often leads to donor-site morbidity. Here, we show that intraspecies and interspecies chimaeric pig embryos lacking native skeletal muscle can be produced by deleting the MYF5, MYOD and MYF6 genes in the embryos via CRISPR, followed by somatic-cell nuclear transfer and the delivery of exogenous cells (porcine blastomeres or human induced pluripotent stem cells) via blastocyst complementation. The generated intraspecies chimaeras were viable and displayed normal histology, morphology and function. Human:pig chimaeras generated with TP53-null human induced pluripotent stem cells led to higher chimaerism efficiency, with embryos collected at embryonic days 20 and 27 containing humanized muscle, as confirmed by immunohistochemical and molecular analyses. Human:pig chimaeras may facilitate the production of exogenic organs for research and xenotransplantation. Human:pig embryos can be produced by deleting the MYF5, MYOD and MYF6 genes in pig embryos via CRISPR and somatic-cell nuclear transfer, followed by the delivery of TP53-null human induced pluripotent stem cells via blastocyst complementation.

Objective: Endothelial progenitors migrate early during embryogenesis to form the primary vascular plexus. The regulatory mechanisms that govern their migration are not completely defined. Here, we describe a novel role for ETV2 (Ets variant transcription factor 2) in cell migration and provide evidence for an ETV2 -Rhoj network as a mechanism responsible for this process. Approach and Results: Analysis of RNAseq datasets showed robust enrichment of migratory/motility pathways following overexpression of ETV2 during mesodermal differentiation. We then analyzed ETV2 chromatin immunoprecipitation-seq and assay for transposase accessible chromatin-seq datasets, which showed enrichment of chromatin immunoprecipitation-seq peaks with increased chromatin accessibility in migratory genes following overexpression of ETV2. Migratory assays showed that overexpression of ETV2 enhanced cell migration in mouse embryonic stem cells, embryoid bodies, and mouse embryonic fibroblasts. Knockout of Etv2 led to migratory defects of Etv2-EYFP + angioblasts to their predefined regions of developing embryos relative to wild-type controls at embryonic day (E) 8.5, supporting its role during migration. Mechanistically, we showed that ETV2 binds the promoter region of Rhoj serving as an upstream regulator of cell migration. Single-cell RNAseq analysis of Etv2-EYFP + sorted cells revealed coexpression of Etv2 and Rhoj in endothelial progenitors at E7.75 and E8.25. Overexpression of ETV2 led to a robust increase in Rhoj in both embryoid bodies and mouse embryonic fibroblasts, whereas, its expression was abolished in the Etv2 knockout embryoid bodies. Finally, shRNA-mediated knockdown of Rhoj resulted in migration defects, which were partially rescued by overexpression of ETV2. Conclusions: These results define an ETV2 -Rhoj cascade, which is important for the regulation of endothelial progenitor cell migration.

The scarcity of donor organs may be addressed in the future by using pigs to grow humanized organs with lower potential for immunological rejection after transplantation in humans. Previous studies have demonstrated that interspecies complementation of rodent blastocysts lacking a developmental regulatory gene can generate xenogeneic pancreas and kidney1,2. However, such organs contain host endothelium, a source of immune rejection. We used gene editing and somatic cell nuclear transfer to engineer porcine embryos deficient in ETV2, a master regulator of hematoendothelial lineages3-7. ETV2-null pig embryos lacked hematoendothelial lineages and were embryonic lethal. Blastocyst complementation with wild-type porcine blastomeres generated viable chimeric embryos whose hematoendothelial cells were entirely donor-derived. ETV2-null blastocysts were injected with human induced pluripotent stem cells (hiPSCs) or hiPSCs overexpressing the antiapoptotic factor BCL2, transferred to synchronized gilts and analyzed between embryonic day 17 and embryonic day 18. In these embryos, all endothelial cells were of human origin.

Background: The neonatal porcine heart possesses proliferative cardiomyocyte capacity to recover heart function and structure after an acute myocardial injury during two days of birth. In this study, we have extended the myocardial regenerative window to postnatal day 28 (P28) by a double injury pig model (AR P1 MI P28 ), which performed apical resection at P1 (AR P1 ) followed by LAD ligation at P28 (MI P28 ). However, the molecular mechanism of regenerative window extension is unknown. Methods: The AR P1 MI P28 hearts showed no visible sign of myocardial damage or malfunction 4 weeks after LAD ligation. The nuclear RNA contents were isolated from hearts that received none, singe, and double injury hearts. They performed single nuclei RNA-seq to characterize the gene-expression profile of the AR P1 MI P28 in cardiomyocytes. Results: Gene-expression profiles revealed that regenerating AR P1 MI P28 hearts contained different subpopulations of cardiomyocytes early after 2 nd injury. Louvain clustering identified the six cardiomyocyte clusters (CM1-CM6). The CM6, CM3, and CM2 clusters have mainly comprised fetal (92.4%), CTL-P1 (95.7%), and CTL-P56 (96.2%) cardiomyocytes. In contrast, the CM1 cluster has comprised heterogeneous cardiomyocyte groups from all other animal groups, including less than 1% of fetal and CTL-P1 cardiomyocytes. The CM5 and CM4 clusters have mainly comprised AR P1 MI P28 -P35 (double injury hearts harvested at P35) cardiomyocytes, 97.8% and 68.6%, respectively. Sparse modeling analysis revealed that AR and MI injury, both alone and in combination, appeared to extend the regenerative capacity of cardiomyocytes and perturb cardiomyocyte maturation. Conclusion: We have demonstrated that the unique CM5 cluster appeared only after the 2 nd injury of AR P1 MI P28 -P35 hearts. This cell cluster was characterized by a highly expressed isoenzyme of the glycolytic enzyme pyruvate kinase M2 (Pkm2) and skeletal muscle filamentous gene Nebulin. Transient expression of the CM5 cluster induces by the AR P1 might contribute to the sustained remuscularization and myocardial function recovery following an acute MI in large mammals.

Background: Congenital heart disease (CHD) is the most common genetic birth defect and has considerable morbidity and mortality. Therefore, it is essential to define the mechanisms governing the specification and differentiation of mesodermal and cardiac progenitors to develop targeted therapies for CHD. FOXK1 is a forkhead/winged helix transcription factor known to regulate cell cycle kinetics, myogenic stem cell proliferation and tumorigenesis. During development, FOXK1 expression is restricted to mesodermal progenitors, somites and the heart. The role for FOXK1 in the developing heart is unknown and warrants investigation. In the present study, we describe a novel role for FOXK1 as an essential regulator of cardiovascular development. Approach and Results: We used a mouse embryoid body (EB) system to differentiate WT and Foxk1 null ESCs into mesodermal and cardiac progenitor cells. Flow cytometry analysis for FLK1+PDGFRa+ showed that in the absence of FOXK1, the cardiac lineage was significantly affected. Bulk RNAseq analysis of D3 and D5 EBs showed a significant induction of the cardiac molecular program in WT EBs compared to the Foxk1 null EBs. Since forkhead transcription factors have been shown to be important epigenetic regulators (i.e. pioneer factors), we performed ATACseq of D3 and D5 EBs, which showed that the chromatin landscape nearby known important regulators of cardiogenesis (Isl1, Gata4, Hand1, Hand2, etc.) was significantly relaxed in WT EBs compared to Foxk1 null EBs. Furthermore, we demonstrated that in the absence of Foxk1 , cardiac differentiation was markedly impaired by assaying for cTnT expression and cardiac contractility which was essentially absent in Foxk1 null EBs compared to WT EBs at D10 of differentiation. Additionally, using transcriptional, Co-IP and pulldown assays we demonstrated that FOXK1 is an important regulator of Notch and Wnt/β-catenin signaling pathways during cardiogenesis. These results were further supported as the Foxk1 null embryo was lethal at E9.5 and had perturbed heart development. Conclusions: These results identify FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development using EB assays and gene disruption technologies.

Background: Ets transcription factors function as important developmental regulators and are known to be modulators of cell fate. Previously, in conjunction with co-factors, we have described Ets variant 2 (Etv2) as an essential regulator of the hematopoietic and endothelial lineages. But the mechanism and the Etv2 interacting partners involved in achieving this critical function remains poorly understood. Results: Using a yeast two-hybrid assay, we identified Vascular Endothelial Zinc Finger 1 (Vezf1) as an interacting factor with Etv2. Vezf1 is a conserved C2H2 zinc finger transcription factor known to regulate the formation, proliferation, and migration of endothelial cells. We verified Vezf1 as a binding partner of Etv2 through co-immunoprecipitation and GST-pull down studies. Bioinformatics analysis of ChIP-seq and Etv2-expressing single cell RNA sequencing was conducted to identify candidate genes containing both Etv2 and Vezf1 binding motifs in their regulatory regions. Histone deacetylase 7 (Hdac7) and angiomotin like protein 2 (Amotl2) were identified as candidate genes and have previously been shown to function as regulators of endothelial development. RT-qPCR analysis showed upregulation of Hdac7 and Amotl2 in response to doxycycline inducible Etv2 and Vezf1; whereas significant reduction of expression of these two genes was observed in the Etv2 and Vezf1 knockout cells. Chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assays (EMSA) confirmed Etv2-Vezf1 adjacent binding sites in the promoters of Hdac7 and Amotl2. Histone Acetyl transferase (HAT) assays was performed to investigate Etv2-Vezf1 on global histone acetylation conditions in doxycycline inducible embryoid bodies. Vezf1 overexpression results in a significant reduction of acetylated histones. Additionally, we have undertaken ATAC-Seq (Assay for Transposase-Accessible Chromatin using sequencing) and Vezf1 ChIP-Seq studies to profile the epigenetic modifications and genome-wide occupancy of Vezf1 in the presence or absence of Etv2 in endothelial progenitor cells. Conclusion: In summary, this study identifies Vezf1 as a novel binding partner of Etv2 and we further demonstrate their combined role in the regulation of downstream target genes, Amotl2 and Hdac7, during endothelial development.

Duchenne muscular dystrophy (DMD) is an X chromosome-linked disease, and it is the most common form of muscular dystrophy caused by genetic mutations in the Dmd gene (1). The Dmd gene contains 79 exons, spans 2.4 Mb, and is the single largest gene in the human genome (2). This gene encodes for dystrophin, which is a component of the dystrophin–glycoprotein complex that provides structural stability to the cell membrane (by connecting the cytoskeleton and the extracellular matrix), and a dysfunctional or absent dystrophin protein leads to progressive muscle wasting with cycles of muscle degeneration and regeneration that ultimately fail (3, 4). Young boys with DMD lose their ability to ambulate, become wheelchair bound, and die prematurely (1, 5). This disease affects skeletal muscle, which is the single largest organ in the body, and it normally has a remarkable capacity for regeneration (6). In response to a severe injury caused by genetic disorders, trauma, or exposure to toxins that destroys over 90% of the muscle, the cytoarchitecture of the injured tissue is completely restored and is indistinguishable compared to uninjured muscle within a 2- to 4-wk period. This regenerative capacity is due to the myogenic stem cell population (i.e., satellite cells) that is resident in adult skeletal muscle, and with repeated DMD-mediated degeneration and regeneration this regenerative process ultimately is exhausted (6). While a number of studies have enhanced our understanding of the myogenic stem cell population, new insights are needed regarding the factors that govern muscle regeneration and potentially serve as therapies for diseases like DMD. In PNAS, Chemello et al. (7) use emerging technologies to provide a discovery science platform. First, they engineered a mouse model by deleting exon 51 of the Dmd gene. This gene-edited mouse (ΔEx51) was viable, lacked dystrophin in skeletal muscle and heart, … [↵][1]1To whom correspondence may be addressed. Email: garry{at}umn.edu. [1]: #xref-corresp-1-1

Etv2, an Ets-transcription factor, governs the specification of the earliest hemato-endothelial progenitors during embryogenesis. While the transcriptional networks during hemato-endothelial development have been well described, the mechanistic details are incompletely defined. In the present study, we described a new role for Etv2 as a regulator of cellular proliferation via Yes1 in mesodermal lineages. Analysis of an Etv2-ChIPseq dataset revealed significant enrichment of Etv2 peaks in the upstream regions of cell cycle regulatory genes relative to non-cell cycle genes. Our bulk-RNAseq analysis using the doxycycline-inducible Etv2 ES/EB system showed increased levels of cell cycle genes including E2f4 and Ccne1 as early as 6 h following Etv2 induction. Further, EdU-incorporation studies demonstrated that the induction of Etv2 resulted in a ~2.5-fold increase in cellular proliferation, supporting a proliferative role for Etv2 during differentiation. Next, we identified Yes1 as the top-ranked candidate that was expressed in Etv2-EYFP+ cells at E7.75 and E8.25 using single cell RNA-seq analysis. Doxycycline-mediated induction of Etv2 led to an increase in Yes1 transcripts in a dose-dependent fashion. In contrast, the level of Yes1 was reduced in Etv2 null embryoid bodies. Using bioinformatics algorithms, biochemical, and molecular biology techniques, we show that Etv2 binds to the promoter region of Yes1 and functions as a direct upstream transcriptional regulator of Yes1 during embryogenesis. These studies enhance our understanding of the mechanisms whereby Etv2 governs mesodermal fate decisions early during embryogenesis.

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and is thus relevant to the treatment of ischaemic diseases, injury-induced regeneration and solid tumour growth. Previously, we demonstrated that ETV2 is an essential transcription factor for the development of cardiac, endothelial and haematopoietic lineages. Here we report that ETV2 functions as a pioneer factor that relaxes closed chromatin and regulates endothelial development. By comparing engineered embryonic stem cell differentiation and reprogramming models with multi-omics techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and recruit BRG1. BRG1 recruitment remodelled chromatin around endothelial genes and helped to maintain an open configuration, resulting in increased H3K27ac deposition. Collectively, these results will serve as a platform for the development of therapeutic initiatives directed towards cardiovascular diseases and solid tumours.

Background: Endothelial progenitors migrate early during embryogenesis to form the primary vascular plexus. ETV2, an Ets-transcription factor, governs the specification of the earliest hemato-endothelial progenitors during embryogenesis. The regulatory mechanisms that govern their migration are undefined. In the present study, we describe a novel role for ETV2 in cell migration and provide evidence for an ETV2 -Rhoj network as a mechanism responsible for this process. Approach and Results: We analyzed our RNAseq datasets, which revealed robust enrichment of migratory/motility pathways following overexpression of ETV2 during mesodermal differentiation. We then analyzed ETV2 ChIPseq and ATACseq datasets, which showed enrichment of ChIPseq peaks with increased chromatin accessibility in migratory genes following overexpression of ETV2. Additionally, scratch and sprouting assays showed that overexpression of ETV2 enhanced cell migration in mouse embryonic stem cells (ESCs), embryoid bodies (EBs) and mouse embryonic fibroblasts (MEFs). Knockout of Etv2 led to migratory defects of Etv2-EYFP + angioblasts to their pre-defined regions of developing embryos relative to wildtype controls at embryonic day (E) 8.5, supporting its role during migration. Mechanistically, we showed that ETV2 binds to the promoter region of Rhoj serving as an upstream regulator of cell migration. Single cell RNAseq analysis of Etv2-EYFP + sorted cells revealed co-expression of Etv2 and Rhoj in endothelial progenitors at E7.75 and E8.25. Overexpression of ETV2 led to a robust increase in Rhoj in both EBs and MEFs, whereas, its expression was abolished in the Etv2 knockout EBs. Finally, shRNA-mediated knockdown of Rhoj resulted in migratory defects which were rescued by overexpression of ETV2. Conclusions: These results define an ETV2 -Rhoj cascade, which is important for the regulation of endothelial progenitor cell migration during embryogenesis.

Background: Genetic mutations perturb the multipotent progenitors, which results in congenital cardiovascular disease. Therefore, it is essential to decipher the pioneer factors and the regulatory pathways that govern the specification and differentiation of mesodermal progenitors and use this information to develop targeted therapies to promote cardiovascular regeneration. Etv2 as an essential transcription factor for the development of cardiac, endothelial and hematopoietic lineages. In the present study, we used ES/EB differentiation and MEF reprogramming systems, to define Etv2 as a novel pioneer factor that relaxes the closed chromatin and drives endothelial development. Results: Using the iHA-Etv2 ES cell line, we engineered a mouse that inducibly overexpresses ETV2. The bulk RNA-seq, single cell RNA-seq data and ATAC-seq experiments showed that inducing Etv2 in MEFs and ES/EBs activated the downstream endothelial marker genes and promoted the development of endothelial lineages, supporting the notion that Etv2 functioned as a master regulator to drive the endothelial lineage development in different cellular contexts. We found that similar to other known pioneer factors, Etv2 was intrinsically able to target and bind the nucleosomes, and this capability appeared to be independent of the cellular context. To further define the mechanism, we performed Etv2, Brg1 and H3K27ac ChIP-seq analyses during MEF reprogramming and ES/EB differentiation. We found that Brg1 maintains and stabilizes the binding of Etv2 on the nucleosome, and Etv2 requires Brg1 to activate downstream genes during reprograming. Conclusion: In these studies, we defined Etv2 as a novel pioneer factor that relaxed the closed chromatin and promoted the endothelial lineage in both ES/EB differentiation and MEF reprogramming. The definition of these mechanisms will enhance our understanding of cardiovascular development and regeneration and serve as a platform for therapeutic applications for patients with congenital or aging related cardiovascular diseases.

Because post-mortem human skeletal muscle is not viable, autologous muscle grafts are typically required in tissue reconstruction after muscle loss due to disease or injury. However, the use of autologous tissue often leads to donor-site morbidity. Here, we show that intraspecies and interspecies chimaeric pig embryos lacking native skeletal muscle can be produced by deleting the MYF5, MYOD and MYF6 genes in the embryos via CRISPR, followed by somatic-cell nuclear transfer and the delivery of exogenous cells (porcine blastomeres or human induced pluripotent stem cells) via blastocyst complementation. The generated intraspecies chimaeras were viable and displayed normal histology, morphology and function. Human:pig chimaeras generated with TP53-null human induced pluripotent stem cells led to higher chimaerism efficiency, with embryos collected at embryonic days 20 and 27 containing humanized muscle, as confirmed by immunohistochemical and molecular analyses. Human:pig chimaeras may facilitate the production of exogenic organs for research and xenotransplantation.

Summary The recent advent of CRISPR and other molecular tools enabled the reconstruction of cell lineages based on induced DNA mutations and promises to solve the ones of more complex organisms. To date, no lineage reconstruction algorithms have been rigorously examined for their performance and robustness across dataset types and number of cells. To benchmark such methods, we decided to organize a DREAM challenge using in vitro experimental intMEMOIR recordings and in silico data for a C. elegans lineage tree of about 1,000 cells and a Mus musculus tree of 10,000 cells. Some of the 22 approaches submitted had excellent performance, but structural features of the trees prevented optimal reconstructions. Using smaller sub-trees as training sets proved to be a good approach for tuning algorithms to reconstruct larger trees. The simulation and reconstruction methods here generated delineate a potential way forward for solving larger cell lineage trees such as in mouse.

Blind enucleation is used in porcine somatic cell nuclear transfer (SCNT) to remove the metaphase II (MII) spindle from the oocyte. Deviation of the MII spindle location, however, leads to incomplete enucleation (IE). Here, we report that the rate of complete enucleation (CE) using the blind method was 80.2 ± 1.7%, although this significantly increased when the polar body-MII deviation was minimized (≦45°). While it is established that IE embryos will not survive to full term, the effect of IE on early stage development is unknown. We have previously demonstrated in mice and pigs that ETV2 deletion results in embryonic lethality due to the lack of hematoendothelial lineages. We observed that ETV2-null cloned embryos derived from blindly and incompletely enucleated oocytes had both WT and mutant sequences at E18 and, using FISH analysis, we observed triploidy. We also compared SCNT embryos generated from either CE or intentionally IE oocytes using the spindle viewer system. We observed a higher in vitro blastocyst rate in the IE versus the CE-SCNT embryos (31.9 ± 3.2% vs 21.0 ± 2.1%). Based on known processes in normal fertilization, we infer that the IE-SCNT embryos extruded the haploid second PB after fusion with donor fibroblasts and formed a near-triploid aneuploid nucleus in each blastomere. These studies demonstrate the peri-implantation survival of residual haploid nuclei following IE and emphasize the need for complete enucleation especially for the analysis of SCNT embryos in the peri-implantation stage and will, further, impact the field of reverse xenotransplantation.

The mammalian heart has a limited regenerative capacity and typically progresses to heart failure following injury. Here, we defined a hedgehog (HH)-Gli1-Mycn network for cardiomyocyte proliferation and heart regeneration from amphibians to mammals. Using a genome-wide screen, we verified that HH signaling was essential for heart regeneration in the injured newt. Next, pharmacological and genetic loss- and gain-of-function of HH signaling demonstrated the essential requirement for HH signaling in the neonatal, adolescent, and adult mouse heart regeneration, and in the proliferation of hiPSC-derived cardiomyocytes. Fate-mapping and molecular biological studies revealed that HH signaling, via a HH-Gli1-Mycn network, contributed to heart regeneration by inducing proliferation of pre-existing cardiomyocytes and not by de novo cardiomyogenesis. Further, Mycn mRNA transfection experiments recapitulated the effects of HH signaling and promoted adult cardiomyocyte proliferation. These studies defined an evolutionarily conserved function of HH signaling that may serve as a platform for human regenerative therapies., Due to the limited proliferation capacity of adult mammalian cardiomyocytes, the human heart has negligible regenerative capacity after injury. Here the authors show that a Hedgehog-Gli1-Mycn signaling cascade regulates cardiomyocyte proliferation and cardiac regeneration from amphibians to mammals.

Background: Single cell RNA-seq (scRNA-seq) over specified time periods has been widely used to dissect the cell populations during mammalian embryogenesis. Integrating such scRNA-seq data from different developmental stages and from different laboratories is critical to comprehensively define and understand the molecular dynamics and systematically reconstruct the lineage trajectories. Methods: Here, we describe a novel algorithm to integrate heterogenous temporal scRNA-seq datasets and to preserve the global developmental trajectories. scNCA not only successfully corrected the batch effects, but also preserved the global structure of gene expression. For both balanced and imbalanced synthetic scRNA-seq data, we found that scNCA had significantly better performance of batch correction than mnnCorrect and Seurat alignment, on mixing cells from different batches, revealing the global trajectories, and bringing together the cells from the same lineage. Results: We applied this algorithm and approach to integrate 3,387 single cells from seven heterogenous temporal scRNA-seq datasets, and reconstructed the cell atlas of early mouse cardiovascular development from E6.5 to E9.5. Using this integrated atlas, we identified an Etv2 downstream target, Ebf1 , as an important transcription factor for mouse endothelial development. Conclusions: In summary, we presented scNCA as a novel tool to correct the batch effect of temporal scRNA-seq. We used scNCA to integrate 3,387 single cells from seven heterogenous temporal scRNA-seq datasets of mouse early cardiovascular development , and identified an Etv2 downstream target, Ebf1 , as an important transcription factor for mouse endothelial development. We provide the R/TensorFlow implementation of scNCA at https://github.com/gongx030/scNCA. The integrated mouse early cardiovascular development data can be explored at https://heartmap.umn.edu/scNCA.

Ets transcription factors function as important developmental regulators and are known to be modulators of cell fate. Previously, in conjunction with co-factors, we have described Ets variant 2 (Etv2) as an essential regulator of the hematopoietic and endothelial lineages. But the mechanism and the Etv2 interacting partners involved in achieving this critical function remains poorly understood. Through Yeast two-hybrid analysis we identified Vascular Endothelial Zinc Finger 1 (Vezf1) as an interacting factor with Etv2. Vezf1 is a conserved C2H2 zinc finger transcription factor known to regulate the formation, proliferation, and migration of endothelial cells through several gene targets. We verified Vezf1 as a binding partner of Etv2 through co-immunoprecipitation and GST-pull down studies. Bioinformatic analysis of ChIP-seq and Etv2-expressing single cell RNA sequencing was conducted to identify candidate genes containing both Etv2 and Vezf1 binding motifs in their regulatory regions. Histone deacetylase 7 (Hdac7) and angiomotin like protein 2 (Amotl2) were identified as genes of interest involved in endothelial development. Hdac7 is a conserved class II deacetylase that functions in endothelial cell adhesion and vascular integrity. Amotl2 is a Motin family protein that functions in angiogenesis, endothelial cell polarity and migration. RT-qPCR analysis showed upregulation of Hdac7 and Amotl2 in response to doxycycline inducible Etv2 and Vezf1; whereas significant reduction of expression of these two genes was observed in the Etv2 and Vezf1 knockout cells. Chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assays (EMSA) confirmed Etv2-Vezf1 adjacent binding sites in the promoters of Hdac7 and Amotl2. Histone Acetyl transferase (HAT) assays was performed to investigate Etv2-Vezf1 on global histone acetylation conditions in doxycycline inducible embryoid bodies. Vezf1 overexpression results in a significant reduction of acetylated histone. In summary, this study identifies Vezf1 as a novel binding partner of Etv2 and their combined role in regulating downstream target genes Amotl2 and Hdac7 in hematoendothelial development.

Etv2, an Ets-transcription factor, governs the specification of the earliest hemato-endothelial progenitors during embryogenesis. While the transcriptional networks during hemato-endothelial development have been well described, the mechanistic details are incompletely defined. In the present study, we described a new role for Etv2 as a regulator of cellular proliferation via Yes1 in mesodermal lineages. Analysis of an Etv2-ChIPseq dataset revealed significant enrichment of Etv2 peaks in the upstream regions of cell cycle regulatory genes relative to non-cell cycle genes. Our bulk-RNAseq analysis using the doxycycline-inducible Etv2 ES/EB system showed increased levels of cell cycle genes including E2f4, Gadd45g and Ccne1 as early as 6h following Etv2 induction. Further, EdU-incorporation studies demonstrated that the induction of Etv2 resulted in a ~2.5-fold increase in cellular proliferation, supporting a proliferative role for Etv2 during differentiation. Next, we identified Yes1 as the top-ranked candidate that was expressed in Etv2-EYFP + cells at E7.75 and E8.25 using single cell RNA-seq analysis. Doxycycline-mediated induction of Etv2 led to an increase in Yes1 transcripts in a dose-dependent fashion. In contrast, the level of Yes1 was reduced in Etv2 null embryoid bodies. Using bioinformatics algorithms, biochemical, and molecular biology techniques, we show that Etv2 binds to the promoter region of Yes1 and functions as a direct upstream regulator of Yes1 during embryogenesis. These studies enhance our understanding of the mechanisms whereby Etv2 governs mesodermal fate decisions early during embryogenesis.

Pattern formation is influenced by transcriptional regulation as well as by morphogenetic mechanisms that shape organ primordia, although factors that link these processes remain under-appreciated. Here we show that, apart from their established transcriptional roles in pattern formation, IRX3/5 help to shape the limb bud primordium by promoting the separation and intercalation of dividing mesodermal cells. Surprisingly, IRX3/5 are required for appropriate cell cycle progression and chromatid segregation during mitosis, possibly in a nontranscriptional manner. IRX3/5 associate with, promote the abundance of, and share overlapping functions with coregulators of cell division such as the cohesin subunits SMC1, SMC3, NIPBL and CUX1. The findings imply that IRX3/5 coordinate early limb bud morphogenesis with skeletal pattern formation.

Single cell RNA-seq (scRNA-seq) over specified time periods has been widely used to dissect the cell populations during mammalian embryogenesis. Integrating such scRNA-seq data from different developmental stages and from different laboratories is critical to comprehensively define and understand the molecular dynamics and systematically reconstruct the lineage trajectories. Here, we describe a novel algorithm to integrate heterogenous temporal scRNA-seq datasets and to preserve the global developmental trajectories. We applied this algorithm and approach to integrate 3,387 single cells from seven heterogenous temporal scRNA-seq datasets, and reconstructed the cell atlas of early mouse cardiovascular development from E6.5 to E9.5. Using this integrated atlas, we identified an Etv2 downstream target, Ebf1, as an important transcription factor for mouse endothelial development.

Bi-potential neuromesodermal progenitors (NMPs) produce both neural and paraxial mesodermal progenitors in the trunk and tail during vertebrate body elongation. We show that Sall4, a pluripotency-related transcription factor gene, has multiple roles in regulating NMPs and their descendants in post-gastrulation mouse embryos. Sall4 deletion using TCre caused body/tail truncation, reminiscent of early depletion of NMPs, suggesting its role in NMP maintenance. This phenotype became significant at the time of the trunk to tail transition, suggesting that Sall4 maintenance of NMPs enables tail formation. Sall4 mutants exhibit expanded neural and reduced mesodermal tissues, indicating its role in NMP differentiation balance. Mechanistically, we show that Sall4 promotion of WNT/ß-catenin signaling contributes to NMP maintenance and differentiation balance. RNA-Seq and SALL4 ChIP-Seq analyses support the notion that Sall4 regulates both mesodermal and neural development. Furthermore, in the mesodermal compartment, genes regulating presomitic mesoderm differentiation are downregulated in Sall4 mutants. In the neural compartment, we show that differentiation of NMPs towards post-mitotic neuron is accelerated in Sall4 mutants. Our results collectively provide evidence supporting the role of Sall4 in regulating NMPs and their descendants.

Background Although cardiomyopathy has emerged as a leading cause of death in Duchenne muscular dystrophy (DMD), limited studies and therapies have emerged for dystrophic heart failure. Objectives The purpose of this study was to model DMD cardiomyopathy using DMD patient-specific human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and to identify physiological changes and future drug therapies. Methods To explore and define therapies for DMD cardiomyopathy, the authors used DMD patient-specific hiPSC-derived cardiomyocytes to examine the physiological response to adrenergic agonists and β-blocker treatment. The authors further examined these agents in vivo using wild-type and mdx mouse models. Results At baseline and following adrenergic stimulation, DMD hiPSC-derived cardiomyocytes had a significant increase in arrhythmic calcium traces compared to isogenic controls. Furthermore, these arrhythmias were significantly decreased with propranolol treatment. Using telemetry monitoring, the authors observed that mdx mice, which lack dystrophin, had an arrhythmic death when stimulated with isoproterenol; the lethal arrhythmias were rescued, in part, by propranolol pre-treatment. Using single-cell and bulk RNA sequencing (RNA-seq), the authors compared DMD and control hiPSC-derived cardiomyocytes, mdx mice, and control mice (in the presence or absence of propranolol and isoproterenol) and defined pathways that were perturbed under baseline conditions and pathways that were normalized after propranolol treatment in the mdx model. The authors also undertook transcriptome analysis of human DMD left ventricle samples and found that DMD hiPSC-derived cardiomyocytes have dysregulated pathways similar to the human DMD heart. The authors further determined that relatively few patients with DMD see a cardiovascular specialist or receive β-blocker therapy. Conclusions The results highlight mechanisms and therapeutic interventions from human to animal and back to human in the dystrophic heart. These results may serve as a prelude for an adequately powered clinical study that examines the impact of β-blocker therapy in patients with dystrophinopathies.

The scarcity of donor organs may be addressed in the future by using pigs to grow humanized organs with lower potential for immunological rejection after transplantation in humans. Previous studies have demonstrated that interspecies complementation of rodent blastocysts lacking a developmental regulatory gene can generate xenogeneic pancreas and kidney