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During cardiac development and morphogenesis, cardiac progenitor cells differentiate into cardiomyocytes that expand in number and size to generate the fully formed heart. Much is known about the factors that regulate initial differentiation of cardiomyocytes, and there is ongoing research to identify how these fetal and immature cardiomyocytes develop into fully functioning, mature cells. Accumulating evidence indicates that maturation limits proliferation and conversely proliferation occurs rarely in cardiomyocytes of the adult myocardium. We term this oppositional interplay the proliferation-maturation dichotomy. Here we review the factors that are involved in this interplay and discuss how a better understanding of the proliferation-maturation dichotomy could advance the utility of human induced pluripotent stem cell–derived cardiomyocytes for modeling in 3-dimensional engineered cardiac tissues to obtain truly adult-level function.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Urodele amphibians have a tremendous capacity for the regeneration of appendages, including limb and tail, following injury. While studies have focused on the cellular and morphological changes during appendicular regeneration, the signaling mechanisms that govern these cytoarchitectural changes during the regenerative response are unclear. In this study, we describe the essential role of hedgehog (Hh) and Wnt signaling pathways following tail amputation in the newt. Quantitative PCR studies revealed that members of both the Hh and Wnt signaling pathways, including the following: shh, ihh, ptc-1, wnt-3a, β-catenin, axin2, frizzled (frzd)-1, and frzd-2 transcripts, were induced following injury. Continuous pharmacological-mediated inhibition of Hh signaling resulted in spike-like regenerates with no evidence of tissue patterning, whereas activation of Hh signaling enhanced the regenerative process. Pharmacological-mediated temporal inhibition experiments demonstrated that the Hh-mediated patterning of the regenerating tail occurs early during regeneration and Hh signals are continuously required for proliferation of the blastemal progenitors. BrdU incorporation and PCNA immunohistochemical studies demonstrated that Hh signaling regulates the cellular proliferation of the blastemal cells following amputation. Similarly, Wnt inhibition resulted in perturbed regeneration, whereas its activation promoted tail regeneration. Using an inhibitor-activator strategy, we demonstrated that the Wnt pathway is likely to be upstream of the Hh pathway and together these signaling pathways function in a coordinated manner to facilitate tail regeneration. Mechanistically, the Wnt signaling pathway activated the Hh signaling pathway that included ihh and ptc-1 during the tail regenerative process. Collectively, our results demonstrate the absolute requirement of signaling pathways that are essential in the regulation of tail regeneration.

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

Regulatory networks that govern embryonic development have been well defined. While a common hypothesis supports the notion that the embryonic regulatory cascades are reexpressed following injury and tissue regeneration, the mechanistic regulatory pathways that mediate the regenerative response in higher organisms remain undefined. Relative to mammals, lower vertebrates, including zebrafish and newts, have a tremendous regenerative capacity to repair and regenerate a number of organs including: appendages, retina, heart, jaw and nervous system. Elucidation of the pathways that govern regeneration in these lower organisms may provide cues that will enhance the capacity for the regeneration of mammalian organs. Signaling pathways, such as the hedgehog pathway, have been shown to play critical functions during development and during regeneration in lower organisms. These signaling pathways have been shown to modulate multiple processes including cellular origin, positional identity and cellular maturation. The present review will focus on the cellular and molecular regulation of the hedgehog (HH) signaling pathway and its interaction with other signaling factors during appendage development and regeneration.

Remodeling of the pre-existing primitive vasculature is necessary for the formation of a complex branched vascular architecture. However, the factors that modulate these processes are incompletely defined. Previously, we defined the role of microRNAs (miRNAs) in endothelial specification. In the present study, we further examined the Etv2-Cre mediated ablation of Dicer L/L and characterized the perturbed vascular patterning in the embryo proper and yolk-sac. We mechanistically defined an important role for miR-130a , an Etv2 downstream target, in the mediation of vascular patterning and angiogenesis in vitro and in vivo . Inducible overexpression of miR-130a resulted in robust induction of vascular sprouts and angiogenesis with increased uptake of acetylated-LDL. Mechanistically, miR-130a directly regulates Jarid2 expression by binding to its 3’-UTR region. CRISPR/Cas9 mediated knockout of miR-130a showed increased levels of Jarid2 in the ES/EB system. Further, the levels of Jarid2 transcripts were increased in the Etv2-null embryos at E8.5. In the in vivo settings, injection of miR-130a specific morpholinos in zebrafish embryos resulted in perturbed vascular patterning with reduced levels of endothelial transcripts in the miR-130a morphants. qPCR and in situ hybridization techniques demonstrated increased expression of jarid2a in the miR-130a morphants in vivo . These findings demonstrate a critical role for Etv2-miR-130a-Jarid2 in vascular patterning both in vitro and in vivo .

The adult mammalian heart has a limited regenerative capacity due primarily to reduced cardiomyocyte (CM) proliferation. Here, we demonstrated hedgehog (HH) signaling pathway as an essential regulator of heart regeneration and CM proliferation. We undertook genome-wide screening using a novel algorithm, bootstrap, which showed an induction of HH signals in the regenerating newt heart. Blockade of HH signaling in the resected newt heart resulted in complete ablation of cardiac regeneration and scar formation. EdU-labeling revealed that inhibition of the HH pathway significantly reduced CM proliferation by 3-fold (n=4 at each time period post-injury). In mammals, cardiac specific loss- and gain-of-function of HH signals demonstrated its role in CM proliferation and regeneration in the postnatal heart. Genetic deletion of floxed-Smoothened ( Smo L/L ) allele at postnatal day 2 (P2) inhibited neonatal heart regeneration with impaired cardiac function and scarring following injury. Conversely, induction of constitutively active Smoothened (SmoM2) at P7 stimulated CM proliferation by 2.5-fold (n=3) and regeneration after myocardial infarction during the non-regenerative window. Lineage-tracing experiments showed that activation of Smo contributed to heart regeneration by promoting proliferation of the pre-existing cardiomyocytes. Activation of HH signals in the cultured CM at P1 and P7 showed an increased proliferative response by 2- and 3-fold (n=4; 1900 cells evaluated for each condition), respectively. Mechanistically, ChIP-seq analysis revealed that HH signals promoted the proliferative program by directly regulating the expression of cyclin-dependent kinases including cyclinD2, cyclinE1 and Cdc7. Finally, activation of HH signaling in the terminally differentiated hiPSC-derived CM resulted in an increase in the number of α-Actinin + /EdU + and α-Actinin + /Ki67 + cells by 2.5-fold (n=3; 645 cells assessed for each condition) and 3-fold (n=3; 685 cells assessed for each condition), respectively. These studies defined an evolutionarily conserved function of HH signaling from newt to mouse to human, as a key regulator of cardiomyocyte proliferation and regeneration that may serve as a platform for regenerative therapies.

The field of regenerative medicine holds tremendous promise for the treatment of chronic diseases. While the adult mammalian heart has limited regenerative capacity, previous studies have focused on cellular therapeutic strategies in an attempt to modulate cardiac regeneration. An alternative strategy relies on the modulation of endogenous stem/progenitor cells or signaling pathways to promote cardiac regeneration. Several organisms, including the newt, have an incomparable capacity for the regeneration of differentiated tissues. An enhanced understanding of the signals, pathways, and factors that mediate the regenerative response in these organisms may be useful in modulating the regenerative response of mammalian organs including the injured adult heart.

SummaryMicroRNAs (miRNAs) are known to regulate critical developmental stages during embryogenesis. Here, we defined an Etv2-miR-130a cascade that regulates mesodermal specification and determination. Ablation of Dicer in the Etv2-expressing precursors resulted in altered mesodermal lineages and embryonic lethality. We identified miR-130a as a direct target of Etv2 and demonstrated its role in the segregation of bipotent hemato-endothelial progenitors toward the endothelial lineage. Gain-of-function experiments demonstrated that miR-130a promoted the endothelial program at the expense of the cardiac program without impacting the hematopoietic lineages. In contrast, CRISPR/Cas9-mediated knockout of miR-130a demonstrated a reduction of the endothelial program without affecting hematopoiesis. Mechanistically, miR-130a directly suppressed Pdgfra expression and promoted the endothelial program by blocking Pdgfra signaling. Inhibition or activation of Pdgfra signaling phenocopied the miR-130a overexpression and knockout phenotypes, respectively. In summary, we report the function of a miRNA that specifically promotes the divergence of the hemato-endothelial progenitor to the endothelial lineage.

Background: In contrast to adult mammalian heart, lower vertebrates such as newt exhibit a dramatic capacity for regeneration in response to injury. Understanding the underlying mechanisms and signaling pathways in newt could form the basis for regenerative therapies in mammals. In the present study, we explored the role of hedgehog (HH) signaling during cardiac regeneration. Methods and Results: To investigate cardiac regeneration (CR) in vivo, we performed ventricular resection studies in adult newt heart. Whole mount and histochemical studies revealed CR within 30 d of resection injury. Functional assessment by echocardiographic analysis showed improved ejection fraction from 22 ± 5% at 4 d to 42 ± 3% at 30 d of regeneration relative to control (60 ± 3%). Gene clustering and qRT-PCR analysis at 7d post injury indicated enrichment of hedgehog (HH) signaling factors including Shh and ptch1 by 3- and 2-fold respectively, suggesting a critical requirement of HH signaling at early stages of CR. We determined HH signaling is essential as pharmacological inhibition of the HH pathway resulted in complete ablation of CR. Using EdU-labeling and immunohistochemical analyses, we showed that HH signaling regulates proliferation of both epicardial and myocardial cells, as inhibition of the HH pathway reduced EdU-positive nuclei from 15 ± 2% to 5 ± 3%. In contrast, qRT-PCR analysis from murine hearts postnatal day (P) 2 to P14, showed reduced levels of HH signaling factors and cell-cycle associated mRNAs by 3.5-fold with concomitant increase of p21 by 2-fold. This implies HH signals are required for the proliferative process. Consistent with the newt studies, activation of HH in murine neonatal ventricular cardiomyocytes (NVCM) promoted cardiomyocyte proliferation, increasing positive nuclei from 10 ± 2% to 25 ± 3%, while inhibition of HH signaling reduced positivity to 6 ± 3%. qRT-PCR analysis established that activation of HH signaling in NVCM resulted in up regulation of transcripts associated with cardiomyocyte proliferation. Conclusion: These data indicated that HH signaling pathways modulate cardiomyocyte proliferation providing potential therapeutic targets for achieving mammalian cardiac regeneration.

αB-crystallin, a member of the small heat shock protein family, has been implicated in various biological functions including response to heat shock, differentiation and apoptosis, the mechanisms of which have not been well understood. Myoblasts, the precursor cells in muscle regeneration, when subjected to growth factor deprivation differentiate to form myotubes or undergo apoptosis. During differentiation, myoblasts express elevated levels of αB-crystallin as well as TNF-α but the connecting link between these proteins in cell signaling is not clearly understood. We have therefore investigated the role of αB-crystallin in TNF-α induced regulation of NF-κB.We demonstrate that in response to TNF-α treatment, αB-crystallin associates with IKKβ and activate its kinase activity, facilitating the degradation of phosphorylated I-kBα, a prime step in NF-κB activation. Reducing the level of αB-crystallin using the RNAi approach reduces the translocation of p65, further confirming the role of αB-crystallin in NF-κB activation. Our study shows that the ability of αB-crystallin to activate NF-κB depends on its phosphorylation status. The present study shows that αB-crystallin-dependent NF-κB activation protects myoblasts from TNF-α induced cytoxicity by enhancing the expression of the anti-apoptotic protein, Bcl 2. Thus, our study identifies yet another mechanism by which αB-crystallin exerts its anti-apoptotic activity.

Disruption of cytoskeletal assembly is one of the early effects of any stress that can ultimately lead to cell death. Stabilization of cytoskeletal assembly, therefore, is a critical event that regulates cell survival under stress. alphaB-crystallin, a small heat shock protein, has been shown to associate with cytoskeletal proteins under normal and stress conditions. Earlier reports suggest that alphaB-crystallin could prevent stress-induced aggregation of actin in vitro. However, the molecular mechanisms by which alphaB-crystallin stabilizes actin filaments in vivo are not known. Using the H9C2 rat cardiomyoblast cell line as a model system, we show that upon heat stress, alphaB-crystallin preferentially partitions from the soluble cytosolic fraction to the insoluble cytoskeletal protein-rich fraction. Confocal microscopic analysis shows that alphaB-crystallin associates with actin filaments during heat stress and the extent of association increases with time. Further, immunoprecipitation experiments show that alphaB-crystallin interacts directly with actin. Treatment of heat-stressed H9C2 cells with the actin depolymerzing agent, cytochalasin B, failed to disorganize actin. We show that this association of alphaB-crystallin with actin is dependent on its phosphorylation status, as treatment of cells with MAPK inhibitors SB202190 or PD98059 results in abrogation of this association. Our results indicate that alphaB-crystallin regulates actin filament dynamics in vivo and protects cells from stress-induced death. Further, our studies suggest that the association of alphaB-crystallin with actin helps maintenance of pinocytosis, a physiological function essential for survival of cells.

L-Asparaginase (isozyme II) from Escherichia coli is an important therapeutic enzyme used in the treatment of leukemia. Extracellular expression of recombinant asparaginase was obtained by fusing the gene coding for asparaginase to an efficient pelB leader sequence and an N-terminal 6x histidine tag cloned under the T7lac promoter. Media composition and the induction strategy had a major influence on the specificity and efficiency of secretion of recombinant asparaginase. Induction of the cells with 0.1 mM IPTG at late log phase of growth in TB media resulted in fourfold higher extracellular activity in comparison to growing the cells in LB media followed by induction during the mid log phase. Using an optimized expression strategy a yield of 20,950 UI/L of recombinant asparaginase was obtained from the extracellular medium. The recombinant protein was purified from the culture supernatant in a single step using Ni-NTA affinity chromatography which gave an overall yield of 95 mg/L of purified protein, with a recovery of 86%. This is approximately 8-fold higher to the previously reported data in literature. The fluorescence spectra, analytical size exclusion chromatography, and the specific activity of the purified protein were observed to be similar to the native protein which demonstrated that the protein had folded properly and was present in its active tetramer form in the culture supernatant.

N-Acetylglucosamine kinase (ATP:2-acetamido-2-deoxy-d-glucose 6-phosphotransferase, EC 2.7.1.59) catalyzes the first reaction in the inducible N-acetylglucosamine catabolic pathway of Candida albicans, an obligatory aerobic yeast. As a part of continuing biochemical studies concerning the regulation of gene expression in a simple eukaryote, N-acetylglucosamine kinase has been purified and characterized biochemically. The enzyme has been purified about 300-fold from the crude extract and its molecular weight of 75 000 has been determined by Sephadex G-100 gel filtration. Isolation and analysis procedures are described. The kinase reaction is optimal within a pH range of 7–8. The enzyme is strictly specific for GlcNAc as phosphate acceptor; ATP is the phosphoryl group donor for the kinase reaction and to a lesser extent dATP and CTP. Km values for GlcNAc and ATP are 1.33 mM and 1.82 mM, respectively. The enzyme requires Mg2+, which may be replaced by other bivalent metal ions such as Mn2+, Ca2+, Ba2+ and Co2+ for a lesser degree of effectiveness. The purified enzyme is extremely sensitive to thermal denaturation and becomes completely inactive by heating at 65°C for 2 min. The enzyme is also inactivated by sulphydryl reagents such as p-chloromercuribenzene sulfonic acid and N-ethylmaleimide.

Amphibians have a remarkable capacity for limb regeneration. Following a severe injury, there is complete regeneration with restoration of the patterning and cellular architecture of the amputated limb. While studies have focused on the structural anatomical changes during amphibian limb regeneration, the signaling mechanisms that govern cellular dedifferentiation and blastemal progenitors are unknown. Here, we demonstrate the temporal and spatial requirement for hedgehog (Hh) signaling and its hierarchical correlation with respect to Wnt signaling during newt limb regeneration. While the dedifferentiation process of mature lineages does not depend on Hh signaling, the proliferation and the migration of the dedifferentiated cells are dependent on Hh signaling. Temporally controlled chemical inactivation of the Hh pathway indicates that Hh-mediated antero-posterior (AP) specification occurs early during limb regeneration and that Hh is subsequently required for expansion of the blastemal progenitors. Inhibition of Hh signaling results in G0/G1 arrest with a concomitant reduction in S-phase and G2/M population in myogenic progenitors. Furthermore, Hh inhibition leads to reduced Pax7-positive cells and fewer regenerating fibers relative to control tissue. We demonstrate that activation of Wnt signaling rescues the inhibition of Hh pathway mainly by enhancing proliferative signals, possibly mediated through TCF4 activity. Collectively, our results demonstrate coordinated signaling of Hh and Wnt activities in regulating blastemal progenitors and their hierarchical positioning during limb regeneration.

alphaB-crystallin, a small heat shock protein, plays an important role in muscle homeostasis. It gets up-regulated during muscle differentiation and mice lacking alphaB-crystallin die prematurely with extensive muscle wastage. We have examined the role of alphaB-crystallin in muscle development using C2C12 myoblasts as a model system. Over-expression of alphaB-crystallin delays the muscle differentiation program significantly. C2C12 myoblasts over-expressing alphaB-crystallin (CRYAB-C2C12) display defect in cell-cycle exit upon induction of differentiation. During differentiation, CRYAB-C2C12 cells exhibit sustained level of cyclin D1 and delay in p21 and myogenin expression as compared to C2C12 cells. We find less accumulation of MyoD in CRYAB-C2C12 cells than in C2C12 cells. In vivo protein stability studies reveal faster ubiquitin-proteasome-mediated MyoD degradation in CRYAB-C2C12 cells (t(1/2)=1.42 h) than in C2C12 cells (t(1/2)=2.37 h). Immuno-precipitation experiments showed that MyoD gets ubiquitinated at earlier time points in CRYAB-C2C12 cells than in C2C12 cells. Our data reveal alterations in the synthesis and degradation of MyoD in CRYAB-C2C12 cells. The level of alphaB-crystallin as well as its Ser-59 phosphorylated form increases with increasing time of differentiation. Our studies show, inter alia, that alphaB-crystallin modulates myogenesis by altering MyoD level and provide an interesting insight in its role in myogenesis.