Mudit Gupta

Highly Efficient miRNA-Mediated Reprogramming of Mouse and Human Somatic Cells to Pluripotency

Transcription factor-based cellular reprogramming has opened the way to converting somatic cells to a pluripotent state, but has faced limitations resulting from the requirement for transcription factors and the relative inefficiency of the process. We show here that expression of the miR302/367 cluster rapidly and efficiently reprograms mouse and human somatic cells to an iPSC state without a requirement for exogenous transcription factors. This miRNA-based reprogramming approach is two orders of magnitude more efficient than standard Oct4/Sox2/Klf4/Myc-mediated methods. Mouse and human miR302/367 iPSCs display similar characteristics to Oct4/Sox2/Klf4/Myc-iPSCs, including pluripotency marker expression, teratoma formation, and, for mouse cells, chimera contribution and germline contribution. We found that miR367 expression is required for miR302/367-mediated reprogramming and activates Oct4 gene expression, and that suppression of Hdac2 is also required. Thus, our data show that miRNA and Hdac-mediated pathways can cooperate in a powerful way to reprogram somatic cells to pluripotency.

Plasticity of Hopx+ Type I alveolar cells to regenerate Type II cells in the lung

The plasticity of differentiated cells in adult tissues undergoing repair is an area of intense research. Pulmonary alveolar Type II cells produce surfactant and function as progenitors in the adult, demonstrating both self-renewal and differentiation into gas exchanging Type I cells. In vivo, Type I cells are thought to be terminally differentiated and their ability to give rise to alternate lineages has not been reported. Here, we show that Hopx becomes restricted to Type I cells during development. However, unexpectedly, lineage-labeled Hopx+ cells both proliferate and generate Type II cells during adult alveolar regrowth following partial pneumonectomy. In clonal 3D culture, single Hopx+ Type I cells generate organoids composed of Type I and Type II cells, a process modulated by TGFβ signaling. These findings demonstrate unanticipated plasticity of Type I cells and a bi-directional lineage relationship between distinct differentiated alveolar epithelial cell types in vivo and in single cell culture.

Integration of Bmp and Wnt signaling by Hopx specifies commitment of cardiomyoblasts

Making cardiomyocytes In the heart, multiple cell types work together. Cardiac progenitor cells give rise to cardiomyocyte, endothelial, or smooth muscle lineages. However, the identity of a marker specific to cardiomyocyte formation has been elusive. Jain et al. now identify a specialized progenitor population that is committed exclusively to forming cardiomyocytes. They also identify the niche signals that promote lineage commitment and the mechanisms involved in making cardiomyocytes. The findings may help in the development of future cell-based regenerative therapeutics for heart disease. Science, this issue 10.1126/science.aaa6071 Identification of the committed cardiomyoblast that retains proliferative potential may inform cardiac regenerative therapeutics. INTRODUCTION Cardiac progenitor cells are multipotent, and lineage analyses of murine and chick cardiac development have demonstrated that these cells give rise to the cardiac endothelium, smooth muscle, and cardiomyocytes. However, the mechanisms governing commitment to the myocyte lineage in vivo remain largely unknown. Further understanding of these mechanisms, and of the identity of progenitors committed to the myocyte lineage, may advance cardiac regenerative therapies. RATIONALE Hopx is an atypical homeodomain expressed in cardiac mesoderm shortly after cardiac progenitor cells are first evident. Previous studies have demonstrated that Hopx functions as a nuclear transcription co-repressor and is expressed in adult, +4 intestinal stem cells and hair follicle bulge stem cells. We compare lineage tracing of multipotent cardiac progenitor cells marked by Islet1 and Nkx2-5 expression with lineage tracing of Hopx+ cells. We also perform functional studies of Hopx from endogenous tissue and differentiated embryoid bodies to identify mechanisms promoting commitment and myogenesis. RESULTS We define and characterize a Hopx-expressing cardiomyoblast intermediate that is committed to the cardiomyocyte fate. Hopx+ is initially expressed in a subset of cardiac progenitor cells residing in the precardiac mesoderm prior to the expression of troponin T, a component of the contractile sarcomere apparatus of myocytes. Lineage-tracing experiments demonstrate that Hopx+ cells give rise to cardiac myocytes exclusively. Early Hopx+ cardiomyoblasts expand during cardiogenesis. Overexpression of Hopx in cardiac progenitor cells leads to an increase in myocytes, whereas Hopx deficiency compromises myogenesis. Whole-genome analysis reveals that Hopx occupies regulatory regions of multiple Wnt-related genes, and Hopx–/– cardiac tissues are characterized by an expansion of Wnt signaling. Restoration of Wnt levels during differentiation of Hopx–/– embryoid bodies partially rescues myogenesis. Wnt signaling is a potent regulator of stemness of cardiac progenitor cells, and our data suggest that Hopx promotes myogenesis by repressing Wnt signaling. Cardiac progenitor cells down-regulate Wnt signaling as they enter the cardiac outflow tract, coincident with the expression of Hopx. The outflow tract is also enriched for bone morphogenetic protein (Bmp) signaling, known to influence differentiation of myocytes. Hopx physically interacts with activated Smad complexes in vitro and in vivo. Exogenous Bmp4 represses Wnt signaling in cardiac explants, and Bmp4-mediated Wnt repression requires Hopx. Thus, Hopx functions to couple Bmp signaling to repression of Wnt. CONCLUSION Our work defines an intermediate cardiac progenitor that expresses Hopx and is committed exclusively to the myocyte fate. Therefore, akin to an erythroblast in hematopoietic differentiation, we have termed these committed cardiac progenitor cells “cardiomyoblasts.” The ability to identify committed, but undifferentiated, cardiomyocyte precursors may facilitate development of cardiac regenerative therapies, including those using embryonic stem cells and induced pluripotent stem cells. Hopx functions to promote myogenesis by physically interacting with Smad proteins to repress Wnt signaling. Our findings raise the possibility that Hopx-mediated integration of Bmp signaling to repress Wnt may be active in other progenitor populations and may potentially underlie the tumor suppressor function of Hopx. Lineage tracing of Hopx+ cells. Images depicting lineage tracing of early Hopx+ cardiomyoblasts that give rise to myocytes in the left ventricle and atria. Some images are duplicated and pseudocolored. Cardiac progenitor cells are multipotent and give rise to cardiac endothelium, smooth muscle, and cardiomyocytes. Here, we define and characterize the cardiomyoblast intermediate that is committed to the cardiomyocyte fate, and we characterize the niche signals that regulate commitment. Cardiomyoblasts express Hopx, which functions to coordinate local Bmp signals to inhibit the Wnt pathway, thus promoting cardiomyogenesis. Hopx integrates Bmp and Wnt signaling by physically interacting with activated Smads and repressing Wnt genes. The identification of the committed cardiomyoblast that retains proliferative potential will inform cardiac regenerative therapeutics. In addition, Bmp signals characterize adult stem cell niches in other tissues where Hopx-mediated inhibition of Wnt is likely to contribute to stem cell quiescence and to explain the role of Hopx as a tumor suppressor.

Decreased cell adhesion promotes angiogenesis in a Pyk2-dependent manner

Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.

Semaphorin 3d and semaphorin 3e direct endothelial motility through distinct molecular signaling pathways.

Background: Class 3 semaphorins are guidance molecules for endothelial cells. Results: In multiple endothelial cell assays, semaphorin 3d requires neuropilin 1 or PI3K/Akt but not plexin D1, whereas semaphorin 3e requires plexin D1 but not neuropilin 1 or PI3K/Akt. Conclusion: Semaphorin 3d and 3e utilize different pathways to mediate similar effects in endothelial cells. Significance: Related guidance molecules utilize distinct mechanisms to repel endothelial cells. Class 3 semaphorins were initially described as axonal growth cone guidance molecules that signal through plexin and neuropilin coreceptors and since then have been established to be regulators of vascular development. Semaphorin 3e (Sema3e) has been shown previously to repel endothelial cells and is the only class 3 semaphorin known to be capable of signaling via a plexin receptor without a neuropilin coreceptor. Sema3e signals through plexin D1 (Plxnd1) to regulate vascular patterning by modulating the cytoskeleton and focal adhesion structures. We showed recently that semaphorin 3d (Sema3d) mediates endothelial cell repulsion and pulmonary vein patterning during embryogenesis. Here we show that Sema3d and Sema3e affect human umbilical vein endothelial cells similarly but through distinct molecular signaling pathways. Time-lapse imaging studies show that both Sema3d and Sema3e can inhibit cell motility and migration, and tube formation assays indicate that both can impede tubulogenesis. Endothelial cells incubated with either Sema3d or Sema3e demonstrate a loss of actin stress fibers and focal adhesions. However, the addition of neuropilin 1 (Nrp1)-blocking antibody or siRNA knockdown of Nrp1 inhibits Sema3d-mediated, but not Sema3e-mediated, cytoskeletal reorganization, and siRNA knockdown of Nrp1 abrogates Sema3d-mediated, but not Sema3e-mediated, inhibition of tubulogenesis. On the other hand, endothelial cells deficient in Plxnd1 are resistant to endothelial repulsion mediated by Sema3e but not Sema3d. Unlike Sema3e, Sema3d incubation results in phosphorylation of Akt in human umbilical vein endothelial cells, and inhibition of the PI3K/Akt pathway blocks the endothelial guidance and cytoskeletal reorganization functions of Sema3d but not Sema3e.

Murine craniofacial development requires Hdac3-mediated repression of Msx gene expression.

Craniofacial development is characterized by reciprocal interactions between neural crest cells and neighboring cell populations of ectodermal, endodermal and mesodermal origin. Various genetic pathways play critical roles in coordinating the development of cranial structures by modulating the growth, survival and differentiation of neural crest cells. However, the regulation of these pathways, particularly at the epigenomic level, remains poorly understood. Using murine genetics, we show that neural crest cells exhibit a requirement for the class I histone deacetylase Hdac3 during craniofacial development. Mice in which Hdac3 has been conditionally deleted in neural crest demonstrate fully penetrant craniofacial abnormalities, including microcephaly, cleft secondary palate and dental hypoplasia. Consistent with these abnormalities, we observe dysregulation of cell cycle genes and increased apoptosis in neural crest structures in mutant embryos. Known regulators of cell cycle progression and apoptosis in neural crest, including Msx1, Msx2 and Bmp4, are upregulated in Hdac3-deficient cranial mesenchyme. These results suggest that Hdac3 serves as a critical regulator of craniofacial morphogenesis, in part by repressing core apoptotic pathways in cranial neural crest cells.

Coronary vasculature patterning requires a novel endothelial ErbB2 holoreceptor

Organogenesis and regeneration require coordination of cellular proliferation, regulated in part by secreted growth factors and cognate receptors, with tissue nutrient supply provided by expansion and patterning of blood vessels. Here we reveal unexpected combinatorial integration of a growth factor co-receptor with a heterodimeric partner and ligand known to regulate angiogenesis and vascular patterning. We show that ErbB2, which can mediate epidermal growth factor (EGF) and neuregulin signalling in multiple tissues, is unexpectedly expressed by endothelial cells where it partners with neuropilin 1 (Nrp1) to form a functional receptor for the vascular guidance molecule semaphorin 3d (Sema3d). Loss of Sema3d leads to improper patterning of the coronary veins, a phenotype recapitulated by endothelial loss of ErbB2. These findings have implications for possible cardiovascular side-effects of anti-ErbB2 therapies commonly used for cancer, and provide an example of integration at the molecular level of pathways involved in tissue growth and vascular patterning.

Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction

Progenitor cells differentiate into specialized cell types through coordinated expression of lineage-specific genes and modification of complex chromatin configurations. We demonstrate that a histone deacetylase (Hdac3) organizes heterochromatin at the nuclear lamina during cardiac progenitor lineage restriction. Specification of cardiomyocytes is associated with reorganization of peripheral heterochromatin, and independent of deacetylase activity, Hdac3 tethers peripheral heterochromatin containing lineage-relevant genes to the nuclear lamina. Deletion of Hdac3 in cardiac progenitor cells releases genomic regions from the nuclear periphery, leading to precocious cardiac gene expression and differentiation into cardiomyocytes; in contrast, restricting Hdac3 to the nuclear periphery rescues myogenesis in progenitors otherwise lacking Hdac3. Our results suggest that availability of genomic regions for activation by lineage-specific factors is regulated in part through dynamic chromatin-nuclear lamina interactions and that competence of a progenitor cell to respond to differentiation signals may depend upon coordinated movement of responding gene loci away from the nuclear periphery.

Erratum to Pax3 and hippo signaling coordinate melanocyte gene expression in neural crest [Cell Reports 9, 26, (2014) 1885-1895]

Loss of Pax3, a developmentally regulated transcription factor expressed in pre-migratory neural crest, results in severe developmental defects and embryonic lethality. Although Pax3 mutations produce profound phenotypes, the intrinsic transcriptional activation exhibited by Pax3 is surprisingly modest. We postulated the existence of transcriptional co-activators that function with Pax3 to mediate developmental functions. A high-throughput screen identified the Hippo effector proteins Taz and Yap65 as Pax3 co-activators. Synergistic co-activation of target genes by Pax3Taz/Yap65 requires DNA binding by Pax3, is Tead-independent, and is regulated by Hippo kinases Mst1 and Lats2. In vivo, Pax3 and Yap65 co-localize in the nucleus of neural crest progenitors in the dorsal neural tube. Neural crest deletion of Taz and Yap65 results in embryonic lethal neural crest defects and decreased expression of the Pax3 target gene, Mitf. These results suggest that Pax3 activity is regulated by the Hippo pathway and that Pax factors are Hippo effectors.

Combinatorial Identification of Broad Association Regions with ChIP-seq Data

Motivation: Differentiation of cells into different cell types involves many types of chromatin modifications, and mapping these modifications is a key computational task as researchers uncover different aspects of that process. Modifications associated with heterochromatin formation pose new challenges in this context because we must define very broad regions that have only a moderately stronger signal than the rest of the chromatin. Lamin-associated domains (LADs) are a prime example of such regions. Results: We present Combinatorial Identification of Broad Association Regions (CIBAR), a new method to identify these types of broad regions. CIBAR is based on an efficient solution to a natural combinatorial problem, which adapts to widely variable yields of reads from ChIP-seq data and the associated controls and performs competitively with previous methods, including DamID, which has been used in many publications on LADs but cannot be applied in most in vivo situations.