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Developmental Biology
Promyelocytic Leukemia Protein Is an Essential Regulator of Stem Cell Pluripotency and Somatic Cell Reprogramming
Apr 10, 2017   Stem Cell Reports
Hadjimichael C, Chanoumidou K, Nikolaou C, Klonizakis A, Theodosi GI, Makatounakis T, Papamatheakis J, Kretsovali A
Promyelocytic Leukemia Protein Is an Essential Regulator of Stem Cell Pluripotency and Somatic Cell Reprogramming
Apr 10, 2017
Stem Cell Reports
Promyelocytic leukemia protein (PML), the main constituent of PML nuclear bodies, regulates various physiological processes in different cell types. However, little is known about its functions in embryonic stem cells (ESC). Here, we report that PML contributes to ESC self-renewal maintenance by controlling cell-cycle progression and sustaining the expression of crucial pluripotency factors. Transcriptomic analysis and gain- or loss-of-function approaches showed that PML-deficient ESC exhibit morphological, metabolic, and growth properties distinct to naive and closer to the primed pluripotent state. During differentiation of embryoid bodies, PML influences cell-fate decisions between mesoderm and endoderm by controlling the expression of Tbx3. PML loss compromises the reprogramming ability of embryonic fibroblasts to induced pluripotent stem cells by inhibiting the transforming growth factor β pathway at the very early stages. Collectively, these results designate PML as a member of the regulatory network for ESC naive pluripotency and somatic cell reprogramming. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Modeling Developmental and Tumorigenic Aspects of Trilateral Retinoblastoma via Human Embryonic Stem Cells
Apr 10, 2017   Stem Cell Reports
Avior Y, Lezmi E, Yanuka D, Benvenisty N
Modeling Developmental and Tumorigenic Aspects of Trilateral Retinoblastoma via Human Embryonic Stem Cells
Apr 10, 2017
Stem Cell Reports
Human embryonic stem cells (hESCs) provide a platform for studying human development and understanding mechanisms underlying diseases. Retinoblastoma-1 (RB1) is a key regulator of cell cycling, of which biallelic inactivation initiates retinoblastoma, the most common congenital intraocular malignancy. We developed a model to study the role of RB1 in early development and tumor formation by generating RB1-null hESCs using CRISPR/Cas9. RB1-/- hESCs initiated extremely large teratomas, with neural expansions similar to those of trilateral retinoblastoma tumors, in which retinoblastoma is accompanied by intracranial neural tumors. Teratoma analysis further revealed a role for the transcription factor ZEB1 in RB1-mediated ectoderm differentiation. Furthermore, RB1-/- cells displayed mitochondrial dysfunction similar to poorly differentiated retinoblastomas. Screening more than 100 chemotherapies revealed an RB1-/--specific cell sensitivity to carboplatin, exploiting their mitochondrial dysfunction. Together, our work provides a human pluripotent cell model for retinoblastoma and sheds light on developmental and tumorigenic roles of RB1. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
RARβ2 is required for vertebrate somitogenesis
Apr 22, 2017   Development (Cambridge, England)
Janesick A, Tang W, Nguyen TTL, Blumberg B
RARβ2 is required for vertebrate somitogenesis
Apr 22, 2017
Development (Cambridge, England)
During vertebrate somitogenesis, retinoic acid is known to establish the position of the determination wavefront, controlling where new somites are permitted to form along the anteroposterior body axis. Less is understood about how RAR regulates somite patterning, rostral-caudal boundary setting, specialization of myotome subdivisions, or the specific RAR subtype that is required for somite patterning. Characterizing the function of RARβ has been challenging due to the absence of embryonic phenotypes in murine loss-of-function studies. Using the Xenopus system, we show that RARβ2 plays a specific role in somite number and size, restriction of the presomitic mesoderm anterior border, somite chevron morphology and hypaxial myoblast migration. Rarβ2 is the RAR subtype whose expression is most up-regulated in response to ligand and its localization in the trunk somites positions it at the right time and place to respond to embryonic retinoid levels during somitogenesis. RARβ2 positively regulates Tbx3 a marker of hypaxial muscle, and negatively regulates Tbx6 via Ripply2 to restrict the anterior boundaries of the presomitic mesoderm and caudal progenitor pool. These results demonstrate for the first time an early and essential role for RARβ2 in vertebrate somitogenesis. © 2017. Published by The Company of Biologists Ltd.
Actomyosin-based tissue folding requires a multicellular myosin gradient
Apr 22, 2017   Development (Cambridge, England)
Heer NC, Miller PW, Chanet S, Stoop N, Dunkel J, Martin AC
Actomyosin-based tissue folding requires a multicellular myosin gradient
Apr 22, 2017
Development (Cambridge, England)
Tissue folding promotes three-dimensional (3D) form during development. In many cases, folding is associated with myosin accumulation at the apical surface of epithelial cells, as seen in the vertebrate neural tube and the Drosophila ventral furrow. This type of folding is characterized by constriction of apical cell surfaces, and the resulting cell shape change is thought to cause tissue folding. Here, we use quantitative microscopy to measure the pattern of transcription, signaling, myosin activation, and cell shape in the Drosophila mesoderm. We found that cells within the ventral domain accumulate different amounts of active apical non-muscle myosin 2 depending on distance from the ventral midline. This gradient in active myosin depends on a newly quantified gradient in upstream signaling proteins. A 3D continuum model of the embryo with induced contractility demonstrates that contractility gradients, but not contractility per se, promote changes to surface curvature and folding. As predicted by the model, experimental broadening of the myosin domain in vivo disrupts tissue curvature where myosin is uniform. Our data argue that apical contractility gradients are important for tissue folding. © 2017. Published by The Company of Biologists Ltd.
Uncoupling neurogenic gene networks in the Drosophila embryo
Apr 21, 2017   Genes & Development
Rogers WA, Goyal Y, Yamaya K, Shvartsman SY, Levine MS
Uncoupling neurogenic gene networks in the Drosophila embryo
Apr 21, 2017
Genes & Development
The EGF signaling pathway specifies neuronal identities in the Drosophila embryo by regulating developmental patterning genes such as intermediate neurons defective (ind). EGFR is activated in the ventral midline and neurogenic ectoderm by the Spitz ligand, which is processed by the Rhomboid protease. CRISPR/Cas9 was used to delete defined rhomboid enhancers mediating expression at each site of Spitz processing. Surprisingly, the neurogenic ectoderm, not the ventral midline, was found to be the dominant source of EGF patterning activity. We suggest that Drosophila is undergoing an evolutionary transition in central nervous system (CNS)-organizing activity from the ventral midline to the neurogenic ectoderm. © 2017 Rogers et al.; Published by Cold Spring Harbor Laboratory Press.
Comparative analysis of gene expression profiles for several migrating cell types identifies cell migration regulators
Apr 21, 2017   Mechanisms Of Development
Bae YK, Macabenta F, Curtis HL, Stathopoulos A
Comparative analysis of gene expression profiles for several migrating cell types identifies cell migration regulators
Apr 21, 2017
Mechanisms Of Development
Cell migration is an instrumental process that ensures cells are properly positioned to support the specification of distinct tissue types during development. To provide insight, we used fluorescence activated cell sorting (FACS) to isolate two migrating cell types from the Drosophila embryo: caudal visceral mesoderm (CVM) cells, precursors of longitudinal muscles of the gut, and hemocytes (HCs), the Drosophila equivalent of blood cells. ~350 genes were identified from each of the sorted samples using RNA-seq, and in situ hybridization was used to confirm expression within each cell type or, alternatively, within other interacting, co-sorted cell types. To start, the two gene expression profiling datasets were compared to identify cell migration regulators that are potentially generally-acting. 73 genes were present in both CVM cell and HC gene expression profiles, including the transcription factor zinc finger homeodomain-1 (zfh1). Comparisons with gene expression profiles of Drosophila border cells that migrate during oogenesis had a more limited overlap, with only the genes neyo (neo) and singed (sn) found to be expressed in border cells as well as CVM cells and HCs, respectively. Neo encodes a protein with Zona pellucida domain linked to cell polarity, while sn encodes an actin binding protein. Tissue specific RNAi expression coupled with live in vivo imaging was used to confirm cell-autonomous roles for zfh1 and neo in supporting CVM cell migration, whereas previous studies had demonstrated a role for Sn in supporting HC migration. In addition, comparisons were made to migrating cells from vertebrates. Seven genes were found expressed by chick neural crest cells, CVM cells, and HCs including extracellular matrix (ECM) proteins and proteases. In summary, we show that genes shared in common between CVM cells, HCs, and other migrating cell types can help identify regulators of cell migration. Our analyses show that neo in addition to zfh1 and sn studied previously impact cell migration. This study also suggests that modification of the extracellular milieu may be a fundamental requirement for cells that undergo cell streaming migratory behaviors. Copyright © 2017. Published by Elsevier B.V.
Models of global gene expression define major domains of cell type and tissue identity
Apr 20, 2017   Nucleic Acids Research
Hutchins AP, Yang Z, Li Y, He F, Fu X, Wang X, Li D, Liu K, He J, Wang Y, Chen J, Esteban MA, Pei D
Models of global gene expression define major domains of cell type and tissue identity
Apr 20, 2017
Nucleic Acids Research
The current classification of cells in an organism is largely based on their anatomic and developmental origin. Cells types and tissues are traditionally classified into those that arise from the three embryonic germ layers, the ectoderm, mesoderm and endoderm, but this model does not take into account the organization of cell type-specific patterns of gene expression. Here, we present computational models for cell type and tissue specification derived from a collection of 921 RNA-sequencing samples from 272 distinct mouse cell types or tissues. In an unbiased fashion, this analysis accurately predicts the three known germ layers. Unexpectedly, this analysis also suggests that in total there are eight major domains of cell type-specification, corresponding to the neurectoderm, neural crest, surface ectoderm, endoderm, mesoderm, blood mesoderm, germ cells and the embryonic domain. Further, we identify putative genes responsible for specifying the domain and the cell type. This model has implications for understanding trans-lineage differentiation for stem cells, developmental cell biology and regenerative medicine. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
Critical involvement of ZEB2 in collagen fibrillogenesis: the molecular similarity between Mowat-Wilson syndrome and Ehlers-Danlos syndrome
Apr 19, 2017   Scientific Reports
Teraishi M, Takaishi M, Nakajima K, Ikeda M, Higashi Y,   . . . . . .   , Mizuno S, Fukada T, Furukawa T, Wakamatsu N, Sano S
Critical involvement of ZEB2 in collagen fibrillogenesis: the molecular similarity between Mowat-Wilson syndrome and Ehlers-Danlos syndrome
Apr 19, 2017
Scientific Reports
Mowat-Wilson syndrome (MOWS) is a congenital disease caused by de novo heterozygous loss of function mutations or deletions of the ZEB2 gene. MOWS patients show multiple anomalies including intellectual disability, a distinctive facial appearance, microcephaly, congenital heart defects and Hirschsprung disease. However, the skin manifestation(s) of patients with MOWS has not been documented in detail. Here, we recognized that MOWS patients exhibit many Ehlers-Danlos syndrome (EDS)-like symptoms, such as skin hyperextensibility, atrophic scars and joint hypermobility. MOWS patients showed a thinner dermal thickness and electron microscopy revealed miniaturized collagen fibrils. Notably, mice with a mesoderm-specific deletion of the Zeb2 gene (Zeb2-cKO) demonstrated redundant skin, dermal hypoplasia and miniaturized collagen fibrils similar to those of MOWS patients. Dermal fibroblasts derived from Zeb2-cKO mice showed a decreased expression of extracellular matrix (ECM) molecules, such as collagens, whereas molecules involved in degradation of the ECM, such as matrix metalloproteinases (MMPs), were up-regulated. Furthermore, bleomycin-induced skin fibrosis was attenuated in Zeb2-cKO mice. We conclude that MOWS patients exhibit an EDS-like skin phenotype through alterations of collagen fibrillogenesis due to ZEB2 mutations or deletions.
IP3R-mediated Ca2+ signals govern hematopoietic and cardiac divergence of Flk1+ cells via the calcineurin-NFATc3-Etv2 pathway
Apr 18, 2017   Journal Of Molecular Cell Biology
Wang YJ, Huang J, Liu W, Kou X, Tang H, Wang H, Yu X, Gao S, Ouyang K, Yang HT
IP3R-mediated Ca2+ signals govern hematopoietic and cardiac divergence of Flk1+ cells via the calcineurin-NFATc3-Etv2 pathway
Apr 18, 2017
Journal Of Molecular Cell Biology
Ca2+ signals participate in various cellular processes with spatial and temporal dynamics, among which, inositol 1,4,5-trisphosphate receptors (IP3Rs)-mediated Ca2+ signals are essential for early development. However, the underlying mechanisms of IP3R-regulated cell fate decision remain largely unknown. Here we report that IP3Rs are required for the hematopoietic and cardiac fate divergence of mouse embryonic stem cells (mESCs). Deletion of IP3Rs (IP3R-tKO) reduced Flk1+/PDGFRα- hematopoietic mesoderm, c-Kit+/CD41+ hematopoietic progenitor cell population, and the colony-forming unit activity, but increased cardiac progenitor markers as well as cardiomyocytes. Concomitantly, the expression of a key regulator of hematopoiesis, Etv2, was reduced in IP3R-tKO cells, which could be rescued by the activation of Ca2+ signals and calcineurin or overexpression of constitutively active form of NFATc3. Furthermore, IP3R-tKO impaired specific targeting of Etv2 by NFATc3 via its evolutionarily conserved cis-element in differentiating ESCs. Importantly, the activation of Ca2+-calcineurin-NFAT pathway reversed the phenotype of IP3R-tKO cells. These findings reveal an unrecognized governing role of IP3Rs in hematopoietic and cardiac fate commitment via IP3Rs-Ca2+-calcineurin-NFATc3-Etv2 pathway. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
ERK signalling modulates epigenome to drive epithelial to mesenchymal transition
Apr 18, 2017   Oncotarget
Navandar M, Garding A, Sahu SK, Pataskar A, Schick S, Tiwari VK
ERK signalling modulates epigenome to drive epithelial to mesenchymal transition
Apr 18, 2017
Oncotarget
The series of events that allow the conversion from adherent epithelial cells into migratory cells is collectively known as epithelial-mesenchymal transition (EMT). EMT is employed during embryonic development such as for gastrulation and neural crest migration and is misused in diseases, such as cancer metastasis. ERK signalling is known to be essential for EMT, however its influence on the epigenetic and transcriptional programme underlying EMT is poorly understood. Here, using a comprehensive genome-wide analysis of H3K27ac mark and gene expression in mammary epithelial cells undergoing EMT, we found that ERK signalling is essential for the epigenetic reprogramming underlying hallmark gene expression and phenotypic changes of EMT. We show that the chemical inhibition of Erk signalling during EMT prevents the loss and gain of the H3K27ac mark at regulatory regions of epithelial and mesenchymal genes, respectively, and results in a transcriptome and epigenome closer to those of epithelial cells. Further computational analyses identified a distinct set of transcription factor motifs enriched at distal regulatory regions that are epigenetically remodelled by ERK signalling. Altogether, our findings reveal an ERK-dependent epigenetic remodelling of regulatory elements that results in a gene expression programme essential for driving EMT.
Molecular control of gut formation in the spider parasteatoda tepidariorum
Apr 22, 2017   Genesis (New York, N.Y. : 2000)
Feitosa NM, Pechmann M, Schwager EE, Tobias-Santos V, McGregor AP, Damen WGM, Nunes da Fonseca R
Molecular control of gut formation in the spider parasteatoda tepidariorum
Apr 22, 2017
Genesis (New York, N.Y. : 2000)
The development of a digestive system is an essential feature of bilaterians. Studies of the molecular control of gut formation in arthropods have been studied in detail in the fruit fly Drosophila melanogaster. However, little is known in other arthropods, especially in noninsect arthropods. To better understand the evolution of arthropod alimentary system, we investigate the molecular control of gut development in the spider Parasteatoda tepidariorum (Pt), the primary chelicerate model species for developmental studies. Orthologs of the ectodermal genes Pt-wingless (Pt-wg) and Pt-hedgehog (Pt-hh), of the endodermal genes, Pt-serpent (Pt-srp) and Pt-hepatocyte-nuclear factor-4 (Pt-hnf4) and of the mesodermal gene Pt-twist (Pt-twi) are expressed in the same germ layers during spider gut development as in D. melanogaster. Thus, our expression data suggest that the downstream molecular components involved in gut development in arthropods are conserved. However, Pt-forkhead (Pt-fkh) expression and function in spiders is considerably different from its D. melanogaster ortholog. Pt-fkh is expressed before gastrulation in a cell population that gives rise to endodermal and mesodermal precursors, suggesting a possible role for this factor in specification of both germ layers. To test this hypothesis, we knocked down Pt-fkh via RNA interference. Pt-fkh RNAi embryos not only fail to develop a proper gut, but also lack the mesodermal Pt-twi expressing cells. Thus, in spiders Pt-fkh specifies endodermal and mesodermal germ layers. We discuss the implications of these findings for the evolution and development of gut formation in Ecdysozoans. © 2017 Wiley Periodicals, Inc.
Asymmetric and Unequal Cell Divisions in Ascidian Embryos
Apr 14, 2017   Results And Problems In Cell Differentiation
Negishi T, Nishida H
Asymmetric and Unequal Cell Divisions in Ascidian Embryos
Apr 14, 2017
Results And Problems In Cell Differentiation
Asymmetric cell division during embryogenesis contributes to cell diversity by generating daughter cells that adopt distinct developmental fates. In this chapter, we summarize current knowledge of three examples of asymmetric cell division occurring in ascidian early embryos: (1) Three successive cell divisions that are asymmetric in terms of cell fate and unequal in cell size in the germline lineage at the embryo posterior pole. A subcellular structure, the centrosome-attracting body (CAB), and maternal PEM mRNAs localized within it control both the positioning of the cell division planes and segregation of the germ cell fates. (2) Asymmetric cell divisions involving endoderm and mesoderm germ layer separation. Asymmetric partitioning of zygotically expressed mRNA for Not, a homeodomain transcription factor, promotes the mesoderm fate and suppresses the endoderm fate. This asymmetric partitioning is mediated by transient nuclear migration toward the mesodermal pole of the mother cell, where the mRNA is delivered. In this case, there is no special regulation of cleavage plane orientation. (3) Asymmetric cell divisions in the marginal region of the vegetal hemisphere. The directed extracellular FGF and ephrin signals polarize the mother cells, inducing distinct fates in a pair of daughter cells (nerve versus notochord and mesenchyme versus muscle). The directions of cell division are regulated and oriented but independently of FGF and ephrin signaling. In these examples, polarization of the mother cells is facilitated by localized maternal factors, by delivery of transcripts from the nucleus to one pole of each cell, and by directed extracellular signals. Two cellular processes-asymmetric fate allocation and orientation of the cell division plane-are coupled by a single factor in the first example, but these processes are regulated independently in the third example. Thus, various modes of asymmetric cell division operate even at the early developmental stages in this single type of organism.
Depletion of polycomb repressive complex 2 core component EED impairs fetal hematopoiesis
Apr 13, 2017   Cell Death & Disease
Yu W, Zhang F, Wang S, Fu Y, Chen J, Liang X, Le H, Pu WT, Zhang B
Depletion of polycomb repressive complex 2 core component EED impairs fetal hematopoiesis
Apr 13, 2017
Cell Death & Disease
Polycomb repressive complex 2 (PRC2), a H3K27me3 methyltransferase complex, promotes the development of many organs by silencing ectopic transcription program. However, currently little is known about the role of PRC2 in blood and vascular development. In this study, we interrogated the function of embryonic ectoderm development (EED), a core PRC2 component, in both endothelial and hematopoietic tissues by inactivating a floxed murine EED allele with Tie2Cre, which catalyzes recombination in endothelial and hematopoietic lineages. Murine EEDfl/fl;Tie2Cre (EEDCKO) embryos died at embryonic day (E) 13.5. We did not observe structural abnormalities of blood vessels or cardiac valves, suggesting that EED is dispensable in endothelial cells for initial steps of vascular development. EEDCKO embryos were pale and had abnormal livers. Flow cytometry of fetal liver cells showed that EED depletion significantly impeded erythroid maturation. There was a corresponding increase in myeloid progenitors and granulocytes and macrophages, suggesting an attenuated differentiation path in myeloid lineages. Moreover, EED depletion impaired the generation of hematopoietic stem cells. Collectively, our study demonstrates that within Tie2Cre-recombined embryonic cells, EED is required for proper erythropoiesis and for formation of hematopoietic progenitor and stem cells, but is dispensable for endothelial lineage commitment and early vascular patterning.
Sequential Response to Multiple Developmental Network Circuits Encoded in an Intronic cis-Regulatory Module of Sea Urchin hox11/13b
Apr 13, 2017   Cell Reports
Cui M, Vielmas E, Davidson EH, Peter IS
Sequential Response to Multiple Developmental Network Circuits Encoded in an Intronic cis-Regulatory Module of Sea Urchin hox11/13b
Apr 13, 2017
Cell Reports
Gene expression in different spatial domains is often controlled by separate cis-regulatory modules (CRMs), but regulatory states determining CRM activity are not only distinct in space, they also change continuously during developmental time. Here, we systematically analyzed the regulatory sequences controlling hox11/13b expression and identified a single CRM required throughout embryonic gut development. We show that within this CRM, distinct sets of binding sites recognizing Ets, Tcf, and homeodomain transcription factors control the dynamic spatial expression of hox11/13b in each developmental phase. Several binding sites execute multiple, sometimes contradictory, regulatory functions, depending on the temporal and spatial regulatory context. In addition, we identified a nearby second CRM operating in inter-modular AND logic with the first CRM to control hox11/13b expression in hindgut endoderm. Our results suggest a mechanism for continuous gene expression in response to changing developmental network functions that depends on sequential combinatorial regulation of individual CRMs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
An Activating STAT3 Mutation Causes Neonatal Diabetes through Premature Induction of Pancreatic Differentiation
Apr 13, 2017   Cell Reports
Saarimäki-Vire J, Balboa D, Russell MA, Saarikettu J, Kinnunen M,   . . . . . .   , Hawkins RD, Silvennoinen O, Varjosalo M, Morgan NG, Otonkoski T
An Activating STAT3 Mutation Causes Neonatal Diabetes through Premature Induction of Pancreatic Differentiation
Apr 13, 2017
Cell Reports
Activating germline mutations in STAT3 were recently identified as a cause of neonatal diabetes mellitus associated with beta-cell autoimmunity. We have investigated the effect of an activating mutation, STAT3K392R, on pancreatic development using induced pluripotent stem cells (iPSCs) derived from a patient with neonatal diabetes and pancreatic hypoplasia. Early pancreatic endoderm differentiated similarly from STAT3K392R and healthy-control cells, but in later stages, NEUROG3 expression was upregulated prematurely in STAT3K392R cells together with insulin (INS) and glucagon (GCG). RNA sequencing (RNA-seq) showed robust NEUROG3 downstream targets upregulation. STAT3 mutation correction with CRISPR/Cas9 reversed completely the disease phenotype. STAT3K392R-activating properties were not explained fully by altered DNA-binding affinity or increased phosphorylation. Instead, reporter assays demonstrated NEUROG3 promoter activation by STAT3 in pancreatic cells. Furthermore, proteomic and immunocytochemical analyses revealed increased nuclear translocation of STAT3K392R. Collectively, our results demonstrate that the STAT3K392R mutation causes premature endocrine differentiation through direct induction of NEUROG3 expression. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
EED orchestration of heart maturation through interaction with HDACs is H3K27me3-independent
Apr 10, 2017   ELife
Ai S, Peng Y, Li C, Gu F, Yu X,   . . . . . .   , Orkin SH, Wang DZ, Yu J, Pu WT, He A
EED orchestration of heart maturation through interaction with HDACs is H3K27me3-independent
Apr 10, 2017
ELife
In proliferating cells, where most Polycomb repressive complex 2 (PRC2) studies have been performed, gene repression is associated with PRC2 trimethylation of H3K27 (H3K27me3). However, it is uncertain whether PRC2 writing of H3K27me3 is mechanistically required for gene silencing. Here, we studied PRC2 function in postnatal mouse cardiomyocytes, where the paucity of cell division obviates bulk H3K27me3 rewriting after each cell cycle. EED (embryonic ectoderm development) inactivation in the postnatal heart (EedCKO) caused lethal dilated cardiomyopathy. Surprisingly, gene upregulation in EedCKO was not coupled with loss of H3K27me3. Rather, the activating histone mark H3K27ac increased. EED interacted with histone deacetylases (HDACs) and enhanced their catalytic activity. HDAC overexpression normalized EedCKO heart function and expression of derepressed genes. Our results uncovered a non-canonical, H3K27me3-independent EED repressive mechanism that is essential for normal heart function. Our results further illustrate that organ dysfunction due to epigenetic dysregulation can be corrected by epigenetic rewiring.
Loss of Polycomb Group Protein Pcgf1 Severely Compromises Proper Differentiation of Embryonic Stem Cells
Apr 10, 2017   Scientific Reports
Yan Y, Zhao W, Huang Y, Tong H, Xia Y, Jiang Q, Qin J
Loss of Polycomb Group Protein Pcgf1 Severely Compromises Proper Differentiation of Embryonic Stem Cells
Apr 10, 2017
Scientific Reports
The Polycomb repressive complex 1 (PRC1) is essential for fate decisions of embryonic stem (ES) cells. Emerging evidence suggests that six major variants of PRC1 complex, defined by the mutually exclusive presence of Pcgf subunit, regulate distinct biological processes, yet very little is known about the mechanism by which each version of PRC1 instructs and maintains cell fate. Here, we disrupted the Pcgf1, also known as Nspc1 and one of six Pcgf paralogs, in mouse ES cells by the CRISPR/Cas9 technology. We showed that although these mutant cells were viable and retained normal self-renewal, they displayed severe defects in differentiation in vitro. To gain a better understanding of the role of Pcgf1 in transcriptional control of differentiation, we analysed mRNA profiles from Pcgf1 deficient cells using RNA-seq. Interestingly, we found that Pcgf1 positively regulated expression of essential transcription factors involved in ectoderm and mesoderm differentiation, revealing an unexpected function of Pcgf1 in gene activation during ES cell lineage specification. Chromatin immunoprecipitation experiments demonstrated that Pcgf1 deletion caused a decrease in Ring1B and its associated H2AK119ub1 mark binding to target genes. Altogether, our results suggested an unexpected function of Pcgf1 in gene activation during ES cell maintenance.
Transcriptome dynamics in early zebrafish embryogenesis determined by high-resolution time course analysis of 180 successive, individual zebrafish embryos
Apr 12, 2017   BMC Genomics
Rauwerda H, Pagano JF, de Leeuw WC, Ensink W, Nehrdich U, de Jong M, Jonker M, Spaink HP, Breit TM
Transcriptome dynamics in early zebrafish embryogenesis determined by high-resolution time course analysis of 180 successive, individual zebrafish embryos
Apr 12, 2017
BMC Genomics
Recently, much progress has been made in the field of gene-expression in early embryogenesis. However, the dynamic behaviour of transcriptomes in individual embryos has hardly been studied yet and the time points at which pools of embryos are collected are usually still quite far apart. Here, we present a high-resolution gene-expression time series with 180 individual zebrafish embryos, obtained from nine different spawns, developmentally ordered and profiled from late blastula to mid-gastrula stage. On average one embryo per minute was analysed. The focus was on identification and description of the transcriptome dynamics of the expressed genes in this embryonic stage, rather than to biologically interpret profiles in cellular processes and pathways. In the late blastula to mid-gastrula stage, we found 6,734 genes being expressed with low variability and rather gradual changes. Ten types of dynamic behaviour were defined, such as genes with continuously increasing or decreasing expression, and all expressed genes were grouped into these types. Also, the exact expression starting and stopping points of several hundred genes during this developmental period could be pinpointed. Although the resolution of the experiment was so high, that we were able to clearly identify four known oscillating genes, no genes were observed with a peaking expression. Additionally, several genes showed expression at two or three distinct levels that strongly related to the spawn an embryo originated from. Our unique experimental set-up of whole-transcriptome analysis of 180 individual embryos, provided an unparalleled in-depth insight into the dynamics of early zebrafish embryogenesis. The existence of a tightly regulated embryonic transcriptome program, even between individuals from different spawns is shown. We have made the expression profile of all genes available for domain experts. The fact that we were able to separate the different spawns by their gene-expression variance over all expressed genes, underlines the importance of spawn specificity, as well as the unexpectedly tight gene-expression regulation in early zebrafish embryogenesis.
Human definitive hematopoietic specification from pluripotent stem cells is regulated by mesodermal expression of CDX4
Apr 14, 2017   Blood
Creamer JP, Dege C, Ren Q, Ho JT, Valentine MC, Druley TE, Sturgeon CM
Human definitive hematopoietic specification from pluripotent stem cells is regulated by mesodermal expression of CDX4
Apr 14, 2017
Blood
The generation of hematopoietic stem cells from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. Achieving this goal is complicated by our incomplete understanding of the mechanism regulating definitive hematopoietic specification. We used our stage-specific hPSC differentiation method to obtain and identify, via CD235a expression, mesoderm harboring exclusively primitive or definitive hematopoietic potential to understand the genetic regulation of definitive hematopoietic specification. Whole-transcriptome gene expression analyses on WNT-dependent KDR+CD235a- definitive hematopoietic mesoderm and WNT-independent KDR+CD235a+ primitive hematopoietic mesoderm revealed strong CDX gene expression within definitive hematopoietic mesoderm. Temporal expression analyses revealed that CDX4 was expressed exclusively within definitive hematopoietic KDR+CD235a- mesoderm in a WNT- and FGF-dependent manner. We found that exogenous CDX4 expression exclusively during mesoderm specification resulted in a >90% repression in primitive hematopoietic potential, but conferred 5-fold greater definitive hematopoietic potential, similar to that observed following WNT stimulation. In contrast, CDX4 knockout hPSCs had intact primitive hematopoietic potential, but exhibited a 5-fold decrease in multilineage definitive hematopoietic potential. Taken together, these findings indicate that CDX4 is a critical transcription factor in the regulation of human definitive hematopoietic specification, and provides a mechanistic basis for WNT-mediated definitive hematopoietic specification from hPSCs. Copyright © 2017 American Society of Hematology.
Ploidy has little effect on timing early embryonic events in the haplo-diploid wasp Nasonia
Apr 22, 2017   Genesis (New York, N.Y. : 2000)
Arsala D, Lynch JA
Ploidy has little effect on timing early embryonic events in the haplo-diploid wasp Nasonia
Apr 22, 2017
Genesis (New York, N.Y. : 2000)
The nucleocytoplasmic (N/C) ratio plays a prominent role in the maternal-to-zygotic transition (MZT) in many animals. The effect of the N/C ratio on cell-cycle lengthening and zygotic genome activation (ZGA) has been studied extensively in Drosophila, where haploid embryos experience an additional division prior to completing cellularization and triploid embryos cellularize precociously by one division. In this study, we set out to understand how the obligate difference in ploidy in the haplodiploid wasp, Nasonia, affects the MZT and which aspects of the Drosophila MZT are conserved. While subtle differences in early embryonic development were observed in comparisons among haploid, diploid, and triploid embryos, in all cases embryos cellularize at cell cycle 12. When ZGA was inhibited, both diploid female, and haploid male, embryos went through 12 syncytial divisions and failed to cellularize before dying without further divisions. We also found that key players of the Drosophila MZT are conserved in Nasonia but have novel expression patterns. Our results suggest that zygotically expressed genes have a reduced role in determining the timing of cellularization in Nasonia relative to Drosophila, and that a stronger reliance on a maternal timer is more compatible with species where variations in embryonic ploidy are obligatory. © 2017 Wiley Periodicals, Inc.
Identification of morphogenetic capability limitations via a single starfish embryo/larva reconstruction method
Apr 21, 2017   Development, Growth & Differentiation
Kawai N, Omori I, Kuraishi R, Kaneko H
Identification of morphogenetic capability limitations via a single starfish embryo/larva reconstruction method
Apr 21, 2017
Development, Growth & Differentiation
Reconstruction of a starfish embryo provides unique morphogenesis during the developmental process that is not observed in normal development. Here, we established a novel method for reconstruction from single embryos/larvae. By using this method, we investigated the morphogenetic capabilities in critical steps during the reconstruction process as showed by the reconstructed embryos generated from embryos/larvae at the six developmental stages, or from segregated ectodermal and/or endomesodermal cells. Additionally, the novel method addressed several problems found in prior methods related to reproducibly generating reconstructed embryos. In the reconstructions from the various stage embryos/larvae, the morphogenetic capabilities were substantively reduced in the reconstructed embryos generated from 3-day bipinnaria (3dBp). The combination experiments using ectodermal or endomesodermal cells segregated from 2dBp or 3dBp showed a reduction of the morphogenetic capabilities in both cells types in 3dBp. The reconstructed embryos generated from ectodermal or endomesodermal cells segregated from 2dBp possessed partial morphological features, such as formation of the epithelium or blastopore, but all failed to develop into bipinnariae. These results indicate two limitations of the morphogenetic capabilities during the reconstruction process. Firstly, the morphogenetic capabilities to reconstruct an embryo are considerably reduced between 2dBp and 3dBp. Secondly, cells specified as ectoderm or endomesoderm possess limited morphogenetic capabilities to reconstruct bipinnaria. Furthermore, our results demonstrate that the interaction between these specified cell types is required for reconstruction. © 2017 Japanese Society of Developmental Biologists.
CRISPR/Cas9-mediated deletion of lncRNA Gm26878 in the distant Foxf1 enhancer region
Apr 13, 2017   Mammalian Genome : Official Journal Of The International Mammalian Genome Society
Szafranski P, Karolak JA, Lanza D, Gajęcka M, Heaney J, Stankiewicz P
CRISPR/Cas9-mediated deletion of lncRNA Gm26878 in the distant Foxf1 enhancer region
Apr 13, 2017
Mammalian Genome : Official Journal Of The International Mammalian Genome Society
Recent genome editing techniques, including CRISPR mutagenesis screens, offer unparalleled opportunities to study the regulatory non-coding genomic regions, enhancers, promoters, and functional non-coding RNAs. Heterozygous point mutations in FOXF1 and genomic deletion copy-number variants at chromosomal region 16q24.1 involving FOXF1 or its regulatory region mapping ~300 kb upstream of FOXF1 and leaving it intact have been identified in the vast majority of patients with a lethal neonatal lung disease, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Homozygous Foxf1 -/- mice have been shown to die by embryonic day 8.5 because of defects in the development of extraembryonic and lateral mesoderm-derived tissues, whereas heterozygous Foxf1 +/- mice exhibit features resembling ACDMPV. We have previously defined a human lung-specific enhancer region encoding two long non-coding RNAs, LINC01081 and LINC01082, expressed in the lungs. To investigate the biological significance of lncRNAs in the Foxf1 enhancer region, we have generated a CRISPR/Cas9-mediated ~2.4 kb deletion involving the entire lncRNA-encoding gene Gm26878, located in the mouse region syntenic with the human Foxf1 upstream enhancer. Very recently, this mouse genomic region has been shown to function as a Foxf1 enhancer. Our results indicate that homozygous loss of Gm26878 is neonatal lethal with low penetrance. No changes in Foxf1 expression were observed, suggesting that the regulation of Foxf1 expression differs between mouse and human.
The lens growth process
Apr 15, 2017   Progress In Retinal And Eye Research
Bassnett S, Šikić H
The lens growth process
Apr 15, 2017
Progress In Retinal And Eye Research
The factors that regulate the size of organs to ensure that they fit within an organism are not well understood. A simple organ, the ocular lens serves as a useful model with which to tackle this problem. In many systems, considerable variance in the organ growth process is tolerable. This is almost certainly not the case in the lens, which in addition to fitting comfortably within the eyeball, must also be of the correct size and shape to focus light sharply onto the retina. Furthermore, the lens does not perform its optical function in isolation. Its growth, which continues throughout life, must therefore be coordinated with that of other tissues in the optical train. Here, we review the lens growth process in detail, from pioneering clinical investigations in the late nineteenth century to insights gleaned more recently in the course of cell and molecular studies. During embryonic development, the lens forms from an invagination of surface ectoderm. Consequently, the progenitor cell population is located at its surface and differentiated cells are confined to the interior. The interactions that regulate cell fate thus occur within the obligate ellipsoidal geometry of the lens. In this context, mathematical models are particularly appropriate tools with which to examine the growth process. In addition to identifying key growth determinants, such models constitute a framework for integrating cell biological and optical data, helping clarify the relationship between gene expression in the lens and image quality at the retinal plane. Copyright © 2017. Published by Elsevier Ltd.
Generation and characterization of human iPSC line from CD34+ cells isolated from umbilical cord blood belonging to Indian origin
Apr 11, 2017   Stem Cell Research
Fernandes S, Talwadekar M, Agarwal R, Nair V, Kale V, Limaye L
Generation and characterization of human iPSC line from CD34+ cells isolated from umbilical cord blood belonging to Indian origin
Apr 11, 2017
Stem Cell Research
We describe here the reprogramming of CD34+ cells isolated from umbilical cord blood obtained after full term delivery of a healthy female child of Indian origin. The cells were nucleofected by episomal vectors expressing Oct4, Sox2, L-Myc, Klf4, Lin28 and p53DD (negative mutation in p53). Colonies were identified by alkaline phosphatase staining and characterized for expression of pluripotency markers at protein level by immunofluorescence, flow cytometry and at transcript level by PCR. Genomic stability of the cell line was checked by G-banded karyotype. The ability to differentiate to endoderm, mesoderm and ectoderm in vitro was confirmed by immunofluorescence staining. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
Modeling human infertility with pluripotent stem cells
Apr 22, 2017   Stem Cell Research
Chen D, Gell JJ, Tao Y, Sosa E, Clark AT
Modeling human infertility with pluripotent stem cells
Apr 22, 2017
Stem Cell Research
Human fertility is dependent upon the correct establishment and differentiation of the germline. This is because no other cell type in the body is capable of passing a genome and epigenome from parent to child. Terminally differentiated germline cells in the adult testis and ovary are called gametes. However, the initial specification of germline cells occurs in the embryo around the time of gastrulation. Most of our knowledge regarding the cell and molecular events that govern human germline specification involves extrapolating scientific principles from model organisms, most notably the mouse. However, recent work using next generation sequencing, gene editing and differentiation of germline cells from pluripotent stem cells has revealed that the core molecular mechanisms that regulate human germline development are different from rodents. Here, we will discuss the major molecular pathways required for human germline differentiation and how pluripotent stem cells have revolutionized our ability to study the earliest steps in human embryonic lineage specification in order to understand human fertility. Copyright © 2017. Published by Elsevier B.V.

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