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Developmental Biology
Dynamic Control of dNTP Synthesis in Early Embryos
Jul 24, 2017   Developmental Cell
Song Y, Marmion RA, Park JO, Biswas D, Rabinowitz JD, Shvartsman SY
Dynamic Control of dNTP Synthesis in Early Embryos
Jul 24, 2017
Developmental Cell
Exponential increase of cell numbers in early embryos requires large amounts of DNA precursors (deoxyribonucleoside triphosphates (dNTPs)). Little is understood about how embryos satisfy this demand. We examined dNTP metabolism in the early Drosophila embryo, in which gastrulation is preceded by 13 sequential nuclear cleavages within only 2 hr of fertilization. Surprisingly, despite the breakneck speed at which Drosophila embryos synthesize DNA, maternally deposited dNTPs can generate less than half of the genomes needed to reach gastrulation. The rest of the dNTPs are synthesized "on the go." The rate-limiting enzyme of dNTP synthesis, ribonucleotide reductase, is inhibited by endogenous levels of deoxyATP (dATP) present at fertilization and is activated as dATP is depleted via DNA polymerization. This feedback inhibition renders the concentration of dNTPs at gastrulation robust, with respect to large variations in maternal supplies, and is essential for normal progression of embryogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.
N-myc regulates growth and fiber cell differentiation in lens development
Jul 18, 2017   Developmental Biology
Cavalheiro GR, Matos-Rodrigues GE, Zhao Y, Gomes AL, Anand D, Predes D, de Lima S, Abreu JG, Zheng D, Lachke SA, Cvekl A, Martins RAP
N-myc regulates growth and fiber cell differentiation in lens development
Jul 18, 2017
Developmental Biology
Myc proto-oncogenes regulate diverse cellular processes during development, but their roles during morphogenesis of specific tissues are not fully understood. We found that c-myc regulates cell proliferation in mouse lens development and previous genome-wide studies suggested functional roles for N-myc in developing lens. Here, we examined the role of N-myc in mouse lens development. Genetic inactivation of N-myc in the surface ectoderm or lens vesicle impaired eye and lens growth, while "late" inactivation in lens fibers had no effect. Unexpectedly, defective growth of N-myc-deficient lenses was not associated with alterations in lens progenitor cell proliferation or survival. Notably, N-myc-deficient lens exhibited a delay in degradation of DNA in terminally differentiating lens fiber cells. RNA-sequencing analysis of N-myc-deficient lenses identified a cohort of down-regulated genes associated with fiber cell differentiation that included DNaseIIβ. Further, an integrated analysis of differentially expressed genes in N-myc-deficient lens using normal lens expression patterns of iSyTE, N-myc-binding motif analysis and molecular interaction data from the String database led to the derivation of an N-myc-based gene regulatory network in the lens. Finally, analysis of N-myc and c-myc double-deficient lens demonstrated that these Myc genes cooperate to drive lens growth prior to lens vesicle stage. Together, these findings provide evidence for exclusive and cooperative functions of Myc transcription factors in mouse lens development and identify novel mechanisms by which N-myc regulates cell differentiation during eye morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.
Lipidomic profiling of patient-specific induced pluripotent stem cell-derived hepatocyte-like cells
Jul 22, 2017   Disease Models & Mechanisms
Kiamehr M, Viiri LE, Vihervaara T, Koistinen KM, Hilvo M, Ekroos K, Käkelä R, Aalto-Setälä K
Lipidomic profiling of patient-specific induced pluripotent stem cell-derived hepatocyte-like cells
Jul 22, 2017
Disease Models & Mechanisms
Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem cells (iPSCs) offer an alternative model for primary human hepatocytes to study lipid aberrations. However, the detailed lipid profile of HLCs is yet unknown. In the current study, functional HLCs were differentiated from iPSCs generated from dermal fibroblasts of three individuals by a 3-step protocol through definitive endoderm (DE) stage. In parallel, detailed lipidomic analyses as well as gene expression profiling of a set of lipid metabolism-related genes were performed during the entire differentiation process from iPSC to HLCs. Additionally, fatty acid (FA) composition of the cell culture media at different stages was determined. Our results show that major alterations in the molecular species of lipids occurring during DE and early hepatic differentiation stages mainly mirror the quality and quantity of the FAs supplied in culture medium at each stage. Polyunsaturated phospholipids and sphingolipids with a very-long FA were produced in the cells at later stage of differentiation. This work uncovers previously unknown lipid composition of iPSC-HLCs and its alterations during the differentiation in conjunction with the expression of key lipid associated genes. Together with biochemical, functional and gene expression measurements the lipidomic analyses allowed us to improve our understanding of the concerted influence of the exogenous metabolite supply and cellular biosynthesis essential for iPSC-HLCs differentiation and function. Importantly, the study describes in detail a cell model that can be applied in exploring e.g. the lipid metabolism involved in the development of fatty liver disease or atherosclerosis. © 2017. Published by The Company of Biologists Ltd.
Systematic identification and characterization of regulatory elements derived from human endogenous retroviruses
Jul 12, 2017   PLoS Genetics
Ito J, Sugimoto R, Nakaoka H, Yamada S, Kimura T, Hayano T, Inoue I
Systematic identification and characterization of regulatory elements derived from human endogenous retroviruses
Jul 12, 2017
PLoS Genetics
Human endogenous retroviruses (HERVs) and other long terminal repeat (LTR)-type retrotransposons (HERV/LTRs) have regulatory elements that possibly influence the transcription of host genes. We systematically identified and characterized these regulatory elements based on publicly available datasets of ChIP-Seq of 97 transcription factors (TFs) provided by ENCODE and Roadmap Epigenomics projects. We determined transcription factor-binding sites (TFBSs) using the ChIP-Seq datasets and identified TFBSs observed on HERV/LTR sequences (HERV-TFBSs). Overall, 794,972 HERV-TFBSs were identified. Subsequently, we identified "HERV/LTR-shared regulatory element (HSRE)," defined as a TF-binding motif in HERV-TFBSs, shared within a substantial fraction of a HERV/LTR type. HSREs could be an indication that the regulatory elements of HERV/LTRs are present before their insertions. We identified 2,201 HSREs, comprising specific associations of 354 HERV/LTRs and 84 TFs. Clustering analysis showed that HERV/LTRs can be grouped according to the TF binding patterns; HERV/LTR groups bounded to pluripotent TFs (e.g., SOX2, POU5F1, and NANOG), embryonic endoderm/mesendoderm TFs (e.g., GATA4/6, SOX17, and FOXA1/2), hematopoietic TFs (e.g., SPI1 (PU1), GATA1/2, and TAL1), and CTCF were identified. Regulatory elements of HERV/LTRs tended to locate nearby and/or interact three-dimensionally with the genes involved in immune responses, indicating that the regulatory elements play an important role in controlling the immune regulatory network. Further, we demonstrated subgroup-specific TF binding within LTR7, LTR5B, and LTR5_Hs, indicating that gains or losses of the regulatory elements occurred during genomic invasions of the HERV/LTRs. Finally, we constructed dbHERV-REs, an interactive database of HERV/LTR regulatory elements (http://herv-tfbs.com/). This study provides fundamental information in understanding the impact of HERV/LTRs on host transcription, and offers insights into the transcriptional modulation systems of HERV/LTRs and ancestral HERVs.
A high-content small molecule screen identifies novel inducers of definitive endoderm
Jul 13, 2017   Molecular Metabolism
Korostylev A, Mahaddalkar PU, Keminer O, Hadian K, Schorpp K, Gribbon P, Lickert H
A high-content small molecule screen identifies novel inducers of definitive endoderm
Jul 13, 2017
Molecular Metabolism
Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can generate any given cell type in the human body. One challenge for cell-replacement therapy is the efficient differentiation and expansion of large quantities of progenitor cells from pluripotent stem cells produced under good manufacturing practice (GMP). FOXA2 and SOX17 double positive definitive endoderm (DE) progenitor cells can give rise to all endoderm-derived cell types in the thymus, thyroid, lung, pancreas, liver, and gastrointestinal tract. FOXA2 is a pioneer transcription factor in DE differentiation that is also expressed and functionally required during pancreas development and islet cell homeostasis. Current differentiation protocols can successfully generate endoderm; however, generation of mature glucose-sensitive and insulin-secreting β-cells is still a challenge. As a result, it is of utmost importance to screen for small molecules that can improve DE and islet cell differentiation for cell-replacement therapy for diabetic patients. The aim of this study was to identify and validate small molecules that can induce DE differentiation and further enhance pancreatic progenitor differentiation. Therefore, we developed a large scale, high-content screen for testing a chemical library of 23,406 small molecules to identify compounds that induce FoxA2 in mouse embryonic stem cells (mESCs). Based on our high-content screen algorithm, we selected 84 compounds that directed differentiation of mESCs towards the FoxA2 lineage. Strikingly, we identified ROCK inhibition (ROCKi) as a novel mechanism of endoderm induction in mESCs and hESCs. DE induced by the ROCK inhibitor Fasudil efficiently gives rise to PDX1+ pancreatic progenitors from hESCs. Taken together, DE induction by ROCKi can simplify and improve current endoderm and pancreatic differentiation protocols towards a GMP-grade cell product for β-cell replacement.
Bmp signaling maintains a mesoderm progenitor cell state in the mouse tailbud
Jul 14, 2017   Development (Cambridge, England)
Sharma R, Shafer MER, Bareke E, Tremblay M, Majewski J, Bouchard M
Bmp signaling maintains a mesoderm progenitor cell state in the mouse tailbud
Jul 14, 2017
Development (Cambridge, England)
Caudal somites are generated from a pool of progenitor cells located in the tailbud region. These progenitor cells form the presomitic mesoderm that gradually differentiates into somites under the action of the segmentation clock. The signals responsible for tailbud mesoderm progenitor pool maintenance during axial elongation are still elusive. Here, we show that Bmp signaling is sufficient to activate the entire mesoderm progenitor gene signature in primary cultures of caudal mesoderm cells. Bmp signaling acts through the key regulatory genes Brachyury (T) and Nkx1-2 and contributes to the activation of several other regulators of the mesoderm progenitor gene network. In the absence of Bmp signaling, tailbud mesoderm progenitor cells acquire aberrant gene expression signatures of the heart, blood, muscle and skeletal embryonic lineages. Treatment of embryos with the Bmp inhibitor Noggin confirmed the requirement for Bmp signaling for normal Brachyury expression and the prevention of abnormal lineage marker activation. Together, these results identify Bmp signaling as a non-cell autonomous signal necessary for mesoderm progenitor cell homeostasis. © 2017. Published by The Company of Biologists Ltd.
Cas9-mediated excision of Nematostella brachyury disrupts endoderm development, pharynx formation, and oral-aboral patterning
Jul 14, 2017   Development (Cambridge, England)
Servetnick MD, Steinworth B, Babonis LS, Simmons D, Salinas-Saavedra M, Martindale MQ
Cas9-mediated excision of Nematostella brachyury disrupts endoderm development, pharynx formation, and oral-aboral patterning
Jul 14, 2017
Development (Cambridge, England)
The mesoderm was a key novelty in animal evolution, though we understand little of how mesoderm arose. brachyury, the founding member of the T-box gene family, is a key gene in chordate mesoderm development. However, the brachyury gene was present in the common ancestor of fungi and animals, long before mesoderm appeared. To explore ancestral roles of brachyury prior to the evolution of definitive mesoderm, we excised the gene using CRISPR/Cas9 in the diploblastic cnidarian Nematostella vectensisNvbrachyury is normally expressed in precursors of the pharynx, which separates endoderm from ectoderm. In knockout embryos, the pharynx does not form, embryos fail to elongate, and endoderm organization, ectodermal cell polarity and patterning along the oral-aboral axis are disrupted. Expression of many genes both inside and outside the Nvbrachyury expression domain is affected, including downregulation of Wnt genes at the oral pole. Our results point to an ancient role for brachyury in morphogenesis, cell polarity, and patterning both ectodermal and endodermal derivatives along the primary body axis. © 2017. Published by The Company of Biologists Ltd.
Recent advances in understanding the role of protein-tyrosine phosphatases in development and disease
Jul 21, 2017   Developmental Biology
Hale AJ, Ter Steege E, den Hertog J
Recent advances in understanding the role of protein-tyrosine phosphatases in development and disease
Jul 21, 2017
Developmental Biology
Protein-tyrosine phosphatases (PTPs) remove phosphate groups from tyrosine residues, and thereby propagate or inhibit signal transduction, and hence influence cellular processes such as cell proliferation and differentiation. The importance of tightly controlled PTP activity is reflected by the numerous mechanisms employed by the cell to control PTP activity, including a variety of post-translational modifications, and restricted subcellular localization. This review highlights the strides made in the last decade and discusses the important role of PTPs in key aspects of embryonic development: the regulation of stem cell self-renewal and differentiation, gastrulation and somitogenesis during early embryonic development, osteogenesis, and angiogenesis. The tentative importance of PTPs in these processes is highlighted by the diseases that present upon aberrant activity. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Apical constriction in distal visceral endoderm cells initiates global, collective cell rearrangement in embryonic visceral endoderm to form anterior visceral endoderm
Jul 17, 2017   Developmental Biology
Shioi G, Hoshino H, Abe T, Kiyonari H, Nakao K, Meng W, Furuta Y, Fujimori T, Aizawa S
Apical constriction in distal visceral endoderm cells initiates global, collective cell rearrangement in embryonic visceral endoderm to form anterior visceral endoderm
Jul 17, 2017
Developmental Biology
The behavior of visceral endoderm cells was examined as the anterior visceral endoderm (AVE) formed from the distal visceral endoderm (DVE) using the mouse lines R26-H2B-EGFP and R26-PHA7-EGFP to visualize cell nuclei and adherens junction, respectively. The analysis using R26-H2B-EGFP demonstrated global cell rearrangement that was not specific to the DVE cells in the monolayer embryonic visceral endoderm sheet; each population of the endoderm cells moved collectively in a swirling movement as a whole. Most of the AVE cells at E6.5 were not E5.5 DVE cells but were E5.5 cells that were located caudally behind them, as previously reported (Hoshino et al., 2015; Takaoka et al., 2011). In the rearrangement, the posterior embryonic visceral endoderm cells did not move, as extraembryonic visceral endoderm cells did not, and they constituted a distinct population during the process of anterior-posterior axis formation. The analysis using R26-PHA7-EGFP suggested that constriction of the apical surfaces of the cells in prospective anterior portion of the DVE initiated the global cellular movement of the embryonic visceral endoderm to drive AVE formation. Copyright © 2017 Elsevier Inc. All rights reserved.
Male Germline Recombination of a Conditional Allele by the Widely Used Dermo1-Cre (Twist2-Cre) Transgene
Jul 19, 2017   Genesis (New York, N.Y. : 2000)
He Y, Sun X, Wang L, Mishina Y, Guan JL, Liu F
Male Germline Recombination of a Conditional Allele by the Widely Used Dermo1-Cre (Twist2-Cre) Transgene
Jul 19, 2017
Genesis (New York, N.Y. : 2000)
Conditional gene knockout using the Cre/loxP system is instrumental in advancing our understanding of the function of genes in a wide range of disciplines. It is becoming increasingly apparent in the literature that some Cre transgenes mediated recombination occurs in unexpected tissues. Dermo1-Cre (Twist2-Cre) has been widely used to target skeletal lineage cells as well as other mesoderm-derived cells. Here we report that Dermo1-Cre exhibits spontaneous male germline recombination activity leading to a Cre-mediated recombination of a floxed Ptk2 (Protein tyrosine kinase 2, also known as Fak [Focal adhesion kinase]) allele but not a floxed Rb1cc1 (RB1 inducible coiled-coil 1, also known as Fip200 [FAK-family Interacting Protein of 200 kDa]) allele at high frequency. This ectopic germline activity of Dermo1-Cre occurred in all or none manner in a given litter. We demonstrated that the occurrence of germline recombination activity of Dermo1-Cre transgene can be avoided by using female mice as parental Dermo1-Cre carriers. This article is protected by copyright. All rights reserved. © 2017 Wiley Periodicals, Inc.
Putative binding sites for mir-125 family miRNAs in the mouse Lfng 3'UTR affect transcript expression in the segmentation clock, but mir-125a-5p is dispensable for normal somitogenesis
Jul 15, 2017   Developmental Dynamics : An Official Publication Of The American Association Of Anatomists
Wahi K, Friesen S, Coppola V, Cole SE
Putative binding sites for mir-125 family miRNAs in the mouse Lfng 3'UTR affect transcript expression in the segmentation clock, but mir-125a-5p is dispensable for normal somitogenesis
Jul 15, 2017
Developmental Dynamics : An Official Publication Of The American Association Of Anatomists
In vertebrate embryos, a "segmentation clock" times somitogenesis. Clock-linked genes, including Lunatic fringe (Lfng), exhibit cyclic expression in the presomitic mesoderm (PSM), with a period matching the rate of somite formation. The clock period varies widely across species, but the mechanisms that underlie this variability are not clear. The half-lives of clock components are proposed to influence the rate of clock oscillations, and are tightly regulated in the PSM. Interactions between Lfng and mir-125a-5p in the embryonic chicken PSM promote Lfng transcript instability, but the conservation of this mechanism in other vertebrates has not been tested. Here, we examine whether this interaction affects clock activity in a mammalian species. Mutation of mir-125 binding sites in the Lfng 3'UTR leads to persistent, non-oscillatory reporter transcript expression in the caudal-most mouse PSM, though dynamic transcript expression recovers in the central PSM. Despite this, expression of endogenous mir-125a-5p is dispensable for mouse somitogenesis. These results suggest that mir-125a sites in the Lfng 3'UTR influence transcript turnover in both mouse and chicken embryos, and support the existence of position-dependent regulatory mechanisms in the PSM. They further suggest the existence of compensatory mechanisms that can rescue the loss of mir-125a-5p in mice. This article is protected by copyright. All rights reserved. © 2017 Wiley Periodicals, Inc.
Mesoderm lineage 3D tissue constructs are produced at large-scale in a 3D stem cell bioprocess
Jul 21, 2017   Biotechnology Journal
Cha JM, Mantalaris A, Jung S, Ji Y, Bang OY, Bae H
Mesoderm lineage 3D tissue constructs are produced at large-scale in a 3D stem cell bioprocess
Jul 21, 2017
Biotechnology Journal
Various studies have presented different approaches to direct pluripotent stem cell differentiation such as applying defined sets of exogenous biochemical signals and genetic/epigenetic modifications. Although differentiation to target lineages was successfully regulated, such conventional methods are often complicated, laborious, and not cost-effective to be employed to the large-scale production of 3D stem cell-based tissue constructs. In this study, we developed a 3D-culture platform that could realize the large-scale production of mesoderm lineage tissue constructs from embryonic stem cells (ESCs). ESCs were cultured using our previously established 3D-bioprocess platform which was amenable to mass-production of 3D ESC-based tissue constructs. Hepatocarcinoma cell line conditioned medium was introduced to the large-scale 3D culture to provide a specific biotolecular microenvironment to mimic in vivo mesoderm formation process. After 5 days of spontaneous differentiation period, the resulting 3D tissue constructs were composed of multipotent mesodermal progenitor cells verified by gene and molecular expression profiles. We subsequently found the optimal time points to trigger terminal differentiation towards cardiomyogenesis or osteogenesis from the mesodermal tissue constructs. Our study demonstrated a simple and affordable 3D ESC-bioprocess that could reach the scalable production of mesoderm origin tissues with significantly improved correspondent tissue properties. This article is protected by copyright. All rights reserved.
Comparative transcriptomics of hepatic differentiation of human pluripotent stem cells and adult human liver tissue
Jul 12, 2017   Physiological Genomics
Ghosheh N, Küppers-Munther B, Asplund A, Edsbagge J, Ulfenborg B, Andersson TB, Björquist P, Andersson CX, Carén H, Simonsson S, Sartipy P, Synnergren J
Comparative transcriptomics of hepatic differentiation of human pluripotent stem cells and adult human liver tissue
Jul 12, 2017
Physiological Genomics
Hepatocyte derived from human pluripotent stem cells (hPSC-HEP) have the potential to replace presently used hepatocyte sources applied in liver disease treatment and models of drug discovery and development. Established hepatocyte differentiation protocols are effective and generate hepatocytes, which recapitulate some key features of their in vivo counterparts. However, generating mature hPSC-HEP remains a challenge. In this study, we applied transcriptomics to investigate the progress of in vitro hepatic differentiation of hPSCs at the developmental stages, definitive endoderm (DE), hepatoblasts, early hPSC-HEP, and mature hPSC-HEP, to identify functional targets that enhance efficient hepatocyte differentiation. Using functional annotation, pathway- and protein interaction network analyses, we observed the grouping of differentially expressed genes in specific clusters representing typical developmental stages of hepatic differentiation. In addition, we identified hub proteins and modules that were involved in the cell cycle process at early differentiation stages. We also identified hub proteins which differed in expression levels between hPSC-HEP and the liver tissue controls. Moreover, a module of genes, which were expressed at higher levels in the liver tissue samples than in the hPSC-HEP, was identified. Considering that hub proteins and modules generally are essential and have important roles in the protein- protein interactions, further investigation of these genes and their regulators may contribute to a better understanding of the differentiation process. This may suggest novel target pathways and molecules for improvement of hPSC-HEP functionality, having the potential to finally bringing this technology to a wider use. Copyright © 2017, Physiological Genomics.
Spatiotemporal expression of a twist homolog in the leech Helobdella austinensis
Jul 12, 2017   Development Genes And Evolution
Kim JS, Jiménez BIM, Kwak HJ, Park SC, Xiao P, Weisblat DA, Cho SJ
Spatiotemporal expression of a twist homolog in the leech Helobdella austinensis
Jul 12, 2017
Development Genes And Evolution
Genes of the twist family encode bHLH transcription factors known to be involved in the regulation and differentiation of early mesoderm. Here, we report our characterization of Hau-twist, a twist homolog from the leech Helobdella austinensis, a tractable lophotrochozoan representative. Hau-twist was expressed in segmental founder cells of the mesodermal lineage, in subsets of cells within the mesodermal lineage of the germinal plate, in circumferential muscle fibers of a provisional integument during segmentation and organogenesis stages and on the ventral side of the developing proboscis. Thus, consistent with other systems, our results suggest that twist gene of the leech Helobdella might function in mesoderm differentiation.
De Novo Tumors of Teratoma: Ganglioneuroma Arising From a Mature Cystic Teratoma of the Ovary
Jul 12, 2017   International Journal Of Gynecological Pathology : Official Journal Of The International Society Of Gynecological Pathologists
Coy S, Meserve E, Berkowitz R, S Hirsch M
De Novo Tumors of Teratoma: Ganglioneuroma Arising From a Mature Cystic Teratoma of the Ovary
Jul 12, 2017
International Journal Of Gynecological Pathology : Official Journal Of The International Society Of Gynecological Pathologists
Mature teratomas are the most common ovarian neoplasms, accounting for 40% to 50% of ovarian tumors, and are histologically defined by the presence of multiple lineages of mature differentiated cells derived from one or more of the 3 embryonic germ layers; ectoderm, mesoderm, and endoderm. Neuroectodermal and neural crest differentiation can be observed in mature teratomas, but it is uncommon to find secondary tumors that arise from the neural crest lineage. Herein we report the uncommon finding of a ganglioneuroma arising in a mature cystic teratoma in a 26-yr-old woman.
Klf8 regulates left-right asymmetric patterning through modulation of Kupffer's vesicle morphogenesis and spaw expression
Jul 18, 2017   Journal Of Biomedical Science
Lin CY, Tsai MY, Liu YH, Lu YF, Chen YC, Lai YR, Liao HC, Lien HW, Yang CH, Huang CJ, Hwang SL
Klf8 regulates left-right asymmetric patterning through modulation of Kupffer's vesicle morphogenesis and spaw expression
Jul 18, 2017
Journal Of Biomedical Science
Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos. Zebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer's vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody. Heart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18-22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8△zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer's vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed. Interestingly, klf8 mutant embryos, generated by a CRISPR-Cas9 system, showed bilateral spaw expression in the LPM at late somite stages. This observation may be partly attributed to compensatory upregulation of klf12b, because klf12b knockdown reduced the percentage of klf8 mutants exhibiting bilateral spaw expression. Our results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer's vesicle morphogenesis and spaw expression in the left LPM.
Oxidative stress and cellular and tissue damage in organogenic outbred mouse embryos after moderate perigestational alcohol intake
Jul 14, 2017   Molecular Reproduction And Development
Coll TA, Chaufan G, Pérez-Tito L, Ventureira MR, Sobarzo CMA, Del Carmen Ríos de Molina M, Cebral E
Oxidative stress and cellular and tissue damage in organogenic outbred mouse embryos after moderate perigestational alcohol intake
Jul 14, 2017
Molecular Reproduction And Development
Perigestational alcohol consumption by CF-1 mouse, from before mating up to the period of embryo organogenesis, leads to retarded early embryo development and neural tube defects. Here, we addressed if perigestational alcohol ingestion up to Day 10 of pregnancy induces oxidative stress and changes in macromolecules and organ tissues of early organogenic embryos. Adult CF-1 female mice were administered 10% ethanol in their drinking water for 17 days prior to mating and until Day 10 of gestation, whereas control females were administered ethanol-free water. Our results demonstrated significantly reduced Catalase abundance and activity and increased glutathione content in the embryos of ethanol-treated females. The nitrite level was significantly reduced, but TBARS (thiobarbituric acid reactive substances) content, an index of lipid peroxidation, did not change. Embryos derived from ethanol-treated females also showed higher abundance of 3-nitrotyrosine (3-NT)-containing proteins in all tissues, compared to the control group. Apoptosis was significantly increased in the ectoderm and mesoderm, but not in the heart - although this organ did contain more cleaved caspase-3-positive cardiomyocytes per area of ventricular myocardium than controls. In sum, moderate perigestational alcohol ingestion up to Day 10 of gestation in mice induces oxidative stress by altering radical nitrogen species and antioxidant enzymatic and non-enzymatic mechanisms in embryos. Further, generalized protein nitration, due to unbalanced nitric oxide levels associated with tissue-specific apoptosis, was detected in embryos, suggesting that oxidative mechanisms may play an important role in the perigestational alcohol-induced malformation of organogenic embryos exposed to ethanol. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Mammalian blastocyst mimicry
Jul 12, 2017   Molecular Reproduction And Development
Bianchi N, Longo M, Redi C, Monti M
Mammalian blastocyst mimicry
Jul 12, 2017
Molecular Reproduction And Development
A gradual restriction in stem cells potency is observed during the different stages of mammalian embryonic development, starting with the zygote, the totipotent stem cell par excellence, up to the blastocyst stage. Stem cells of the inner cell mass are endowed with pluripotent characteristic, being able to form cells of the three germ layers (ectoderm, endoderm, and mesoderm). Pluripotent stem cells retain proliferative capacity throughout the life of an individual, and divide asymmetrically, wherein one cell is the copy of the original stem cell while the other is programmed to begin the differentiation process. In mammals, pluripotent stem cells can also be found in the fetus and in some adult tissues, including the umbilical cord blood. This article is protected by copyright. All rights reserved.
Developmental mechanisms of intervertebral disc and vertebral column formation
Jul 18, 2017   Wiley Interdisciplinary Reviews. Developmental Biology
Lawson LY, Harfe BD
Developmental mechanisms of intervertebral disc and vertebral column formation
Jul 18, 2017
Wiley Interdisciplinary Reviews. Developmental Biology
The vertebral column consists of repeating units of ossified vertebrae that are adjoined by fibrocartilagenous intervertebral discs. These structures form from the embryonic notochord and somitic mesoderm. In humans, congenital malformations of the vertebral column include scoliosis, kyphosis, spina bifida, and Klippel Feil syndrome. In adulthood, a common malady affecting the vertebral column includes disc degeneration and associated back pain. Indeed, recent reports estimate that low back pain is the number one cause of disability worldwide. Our review provides an overview of the molecular mechanisms underlying vertebral column morphogenesis and intervertebral disc development and maintenance, with an emphasis on what has been gleaned from recent genetic studies in mice. The aim of this review is to provide a developmental framework through which vertebral column formation can be understood so that ultimately, research scientists and clinicians alike can restore disc health with appropriately designed gene and cell-based therapies. For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.
Wnt and Nodal signaling simultaneously induces definitive endoderm differentiation of mouse embryonic stem cells
Jul 21, 2017   Romanian Journal Of Morphology And Embryology = Revue Roumaine De Morphologie Et Embryologie
Zhong W, Lai Y, Yu T, Xia ZS, Yuan YH, Ouyang H, Shan TD, Chen QK
Wnt and Nodal signaling simultaneously induces definitive endoderm differentiation of mouse embryonic stem cells
Jul 21, 2017
Romanian Journal Of Morphology And Embryology = Revue Roumaine De Morphologie Et Embryologie
Induced differentiation of definitive endoderm (DE) from embryonic stem cells (ESCs) has been the recent focus of studies investigating regeneration and transplantation of organs of the digestive system. Poor cell survival is the most important challenge to DE differentiation from ESCs. This study aimed to optimize culture conditions to promote the differentiation of mouse ESCs into DE, and to investigate the roles of the Wnt and Nodal signaling pathways in the DE differentiation. The mouse ESCs were treated with or without leukemia inhibitory factor, Wnt3a and Activin A alone or together, and examined the DE differentiation by the DE marker CXCR4 and the ESC marker Oct4. The result showed the optimal induction of differentiation was achieved in cells simultaneously treated with Wnt3a and Activin A. Induction of CXCR4 was also earlier when there was simultaneous activation of Wnt and Nodal signaling compared to the groups treated with only Wnt3a or Activin A alone. These findings provide the basis for the induced differentiation of ESCs for the generation of functional, mature cells of gastrointestinal lineage, which can be potentially used for cell replacement therapy, disease modeling, as well as drug discovery studies.
A differential network analysis approach for lineage specifier prediction in stem cell subpopulations
Jul 20, 2017   NPJ Systems Biology And Applications
Okawa S, Angarica VE, Lemischka I, Moore K, Del Sol A
A differential network analysis approach for lineage specifier prediction in stem cell subpopulations
Jul 20, 2017
NPJ Systems Biology And Applications
Stem cell differentiation is a complex biological process. Cellular heterogeneity, such as the co-existence of different cell subpopulations within a population, partly hampers our understanding of this process. The modern single-cell gene expression technologies, such as single-cell RT-PCR and RNA-seq, have enabled us to elucidate such heterogeneous cell subpopulations. However, the identification of a transcriptional regulatory network (TRN) for each cell subpopulation within a population and genes determining specific cell fates (lineage specifiers) remains a challenge due to the slower development of appropriate computational and experimental workflows. Here, we propose a computational differential network analysis approach for predicting lineage specifiers in binary-fate differentiation events. The proposed method is based on a model that considers each stem cell subpopulation being in a stable state maintained by its specific TRN stability core, and cell differentiation involves changes in these stability cores between parental and daughter cell subpopulations. The method first reconstructs topologically different cell-subpopulation specific TRNs from single-cell gene expression data, literature knowledge and transcription factor (TF)-DNA binding-site prediction. Then, it systematically predicts lineage specifiers by identifying genes in the TRN stability cores in both parental and daughter cell subpopulations. Application of this method to different stem cell differentiation systems was able to predict known and putative novel lineage specifiers. These examples include the differentiation of inner cell mass into either primitive endoderm or epiblast, different progenitor cells in the hematopoietic system, and the lung alveolar bipotential progenitor into either alveolar type 1 or alveolar type 2. The method is generally applicable to any binary-fate differentiation system, for which single-cell gene expression data are available. Therefore, it should aid in understanding stem cell lineage specification, and in the development of experimental strategies for regenerative medicine.
The use of human mature cystic ovarian teratoma as a model to study the development of human lymphatics
Jul 17, 2017   Anatomy & Cell Biology
Al-Jomard R, Amarin Z
The use of human mature cystic ovarian teratoma as a model to study the development of human lymphatics
Jul 17, 2017
Anatomy & Cell Biology
The angiogenic theory to the development of human lymphatics is not clear. The objective of this study was to investigate the development of human lymphatics. Semi-thin and thin paraffin sections from human mature cystic ovarian teratoma tissues were studied using light and electron microscopy. Lymphatics were formed by the differentiation of mesenchymal cells that gradually acquired morphological features of endothelial cells. It is suggested that in human mature cystic ovarian teratoma the lymphatic endothelium develops from mesenchymal cells, and not from cells derived from mature endothelium of a preexisting vein or lymphatic.

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