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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 70,836 bioRxiv papers from 309,131 authors.

Most downloaded bioRxiv papers, all time

in category developmental biology

2,085 results found. For more information, click each entry to expand.

41: Brd4 and P300 regulate zygotic genome activation through histone acetylation
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Posted to bioRxiv 13 Jul 2018

Brd4 and P300 regulate zygotic genome activation through histone acetylation
1,897 downloads developmental biology

Shun Hang Chan, Yin Tang, Liyun Miao, Hiba Darwich-Codore, Charles E. Vejnar, Jean-Denis Beaudoin, Damir Musaev, Juan P. Fernandez, Miguel A. Moreno-Mateos, Antonio J. Giraldez

The awakening of the zygote genome, signaling the transition from maternal transcriptional control to zygotic control, is a watershed in embryonic development, but the factors and mechanisms controlling this transition are still poorly understood. By combining CRISPR-Cas9-mediated live imaging of the first transcribed genes (miR-430), chromatin and transcription analysis during zebrafish embryogenesis, we observed that genome activation is gradual and stochastic, and the active state is inherited in daughter cells. We discovered that genome activation is regulated through both translation of maternal mRNAs and the effects of these factors on the chromatin. We show that chemical inhibition of H3K27Ac writer (P300) and reader (Brd4) block genome activation, while induction of a histone acetylation prematurely activates transcription, and restore genome activation in embryos where translation of maternal mRNAs is impaired, demonstrating that they are limiting factors for the activation of the genome. In contrast to current models, we do not observe triggering of genome activation by a reduction of the nuclear-cytoplasmic(N/C) ratio or slower cell division. We conclude that genome activation is controlled by a time-dependent mechanism involving the translation of maternal mRNAs and the regulation of histone acetylation through P300 and Brd4. This mechanism is critical to initiating zygotic development and developmental reprogramming.

42: Single-cell RNA-Seq Resolves Cellular Heterogeneity and Transcriptional Dynamics in Spermatogonial Stem Cells Establishment
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Posted to bioRxiv 30 Dec 2017

Single-cell RNA-Seq Resolves Cellular Heterogeneity and Transcriptional Dynamics in Spermatogonial Stem Cells Establishment
1,856 downloads developmental biology

Jinyue Liao, Shuk Han Ng, Jiajie Tu, Alfred Chun Shui Luk, Yan Qian, Jacqueline Fung, Nelson Leung Sang Tang, Bo Feng, Wai-Yee Chan, Pierre Fouchet, Tin-Lap Lee

The transition of gonocytes to spermatogonia and subsequent differentiation provide the foundation of spermatogenesis. However, systematic understanding on the cellular and molecular basis of this process is still limited, mainly impeded by the asynchrony in development and the lack of stage-specific markers. Using single-cell RNA sequencing on Oct4-GFP+/KIT- cells isolated from PND5.5 mice, we dissected the cellular heterogeneity and established molecular regulations. We demonstrated that gonocyte-spermatogonial transition was characterized by gene expression change related to apoptosis, cell cycle progression, and regulation of migration processes. Pseudotime analysis reconstructed developmental dynamics of the spermatogonial populations and unraveled sequential cellular and molecular transitions. We also identified CD87 as a neonatal stem cell marker which are potentially involved in the intial establishment of SSC pool. Lastly, we uncovered an unexpected subpopulation of spermatogonia primed to differentiation within the undifferentiated compartment, which is characterized by the lack of self-renewal genes and enhanced Oct4 expression and retinoic acid signaling response. Our study thus provides a novel understanding of cellular and molecular changes during spermatogonial establishment.

43: A Single-Cell Transcriptome Atlas for Zebrafish Development
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Posted to bioRxiv 19 Aug 2019

A Single-Cell Transcriptome Atlas for Zebrafish Development
1,851 downloads developmental biology

Dylan R. Farnsworth, Lauren Saunders, Adam C Miller

The ability to define cell types and how they change during organogenesis is central to our understanding of animal development and human disease. Despite the crucial nature of this knowledge, we have yet to fully characterize all distinct cell types and the gene expression differences that generate cell types during development. To address this knowledge gap, we produced an Atlas using single-cell RNA-sequencing methods to investigate gene expression from the pharyngula to early larval stages in developing zebrafish. Our single-cell transcriptome Atlas encompasses transcriptional profiles from 44,102 cells across four days of development using duplicate experiments that confirmed high reproducibility. We annotated 220 identified clusters and highlighted several strategies for interrogating changes in gene expression associated with the development of zebrafish embryos at single-cell resolution. Furthermore, we highlight the power of this analysis to assign new cell-type or developmental stage-specific expression information to many genes, including those that are currently known only by sequence and/or that lack expression information altogether. The resulting Atlas is a resource of biologists to generate hypotheses for genetic (mutant) or functional analysis, to launch an effort to define the diversity of cell-types during zebrafish organogenesis, and to examine the transcriptional profiles that produce each cell type over developmental time.

44: Molecular mechanism of symmetry breaking in a 3D model of a human epiblast
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Posted to bioRxiv 29 May 2018

Molecular mechanism of symmetry breaking in a 3D model of a human epiblast
1,849 downloads developmental biology

Mijo Simunovic, Jakob J Metzger, Fred Etoc, Anna Yoney, Albert Ruzo, Iain Martyn, Gist Croft, Ali H. Brivanlou, E.D. Siggia

Breaking the anterior-posterior (AP) symmetry in mammals takes place at gastrulation. Much of the signaling network underlying this process has been elucidated in the mouse, however there is no direct molecular evidence of events driving axis formation in humans. Here, we use human embryonic stem cells to generate an in vitro 3D model of a human epiblast whose size, cell polarity, and gene expression are similar to a 10-day human epiblast. A defined dose of bone morphogenetic protein 4 (BMP4) spontaneously breaks axial symmetry, and induces markers of the primitive streak and epithelial to mesenchymal transition. By gene knockouts and live-cell imaging we show that, downstream of BMP4, WNT3 and its inhibitor DKK1 play key roles in this process. Our work demonstrates that a model human epiblast can break axial symmetry despite no asymmetry in the initial signal and in the absence of extraembryonic tissues or maternal cues. Our 3D model opens routes to capturing molecular events underlying axial symmetry breaking phenomena, which have largely been unexplored in model human systems.

45: Generation of Aggregates of Mouse ES Cells that Show Symmetry Breaking, Polarisation and Emergent Collective Behaviour in vitro.
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Posted to bioRxiv 15 May 2014

Generation of Aggregates of Mouse ES Cells that Show Symmetry Breaking, Polarisation and Emergent Collective Behaviour in vitro.
1,805 downloads developmental biology

P. Baillie-Johnson, Susanne C van den Brink, Tina Balayo, David A Turner, Alfonso Martinez-Arias

Dissociated mouse embryonic stem (ES) cells were cultured to form aggregates in small volumes of basal medium in U-bottomed, non tissue-culture-treated 96-well plates and subsequently maintained in suspension culture. After growth for 48 hours, the aggregates are competent to respond to ubiquitous experimental signals which result in their symmetry-breaking and generation of defined polarised structures by 96 hours. It is envisaged that this system can be applied both to the study of early developmental events and more broadly to the processes of self-organisation and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo but unobtainable from conventional adherent culture.

46: Long non-coding RNAs are largely dispensable for zebrafish embryogenesis, viability and fertility
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Posted to bioRxiv 23 Jul 2018

Long non-coding RNAs are largely dispensable for zebrafish embryogenesis, viability and fertility
1,791 downloads developmental biology

Mehdi Goudarzi, Kathryn Berg, Lindsey M Pieper, Alexander F. Schier

Hundreds of long non-coding RNAs (lncRNAs) have been identified as potential regulators of gene expression, but their functions remain largely unknown. To study the role of lncRNAs during vertebrate development, we selected 25 zebrafish lncRNAs based on their conservation, expression profile or proximity to developmental regulators, and used CRISPR-Cas9 to generate 32 deletion alleles. We observed altered transcription of neighboring genes in some mutants, but none of the lncRNAs were required for embryogenesis, viability or fertility. Even RNAs with previously proposed non-coding functions (cyrano and squint) and other conserved lncRNAs (gas5 and lnc-setd1ba) were dispensable. In one case (lnc-phox2bb), absence of putative DNA regulatory-elements, but not of the lncRNA transcript itself, resulted in abnormal development. LncRNAs might have redundant, subtle, or context-dependent roles, but extrapolation from our results suggests that the majority of individual zebrafish lncRNAs are dispensable for embryogenesis, viability and fertility.

47: Genome-wide analysis of H3K4me3 and H3K27me3 modifications throughout the mouse urogenital ridge at E11.5.
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Posted to bioRxiv 15 Jun 2018

Genome-wide analysis of H3K4me3 and H3K27me3 modifications throughout the mouse urogenital ridge at E11.5.
1,771 downloads developmental biology

Yisheng Yang, Megan J Wilson

In mammals, the adrenal gland, testis and ovary arise from a common progenitor tissue known as the urogenital ridge (UGR). This small population of cells will adopt a number of different cell fates following sex determination, including forming the precursors of somatic cells (such as Sertoli and granulosa cells) and steroidogenic cells. In addition, this tissues also contains the Wolffian and Müllerian ducts that later form components of the reproductive tracts. A potential mechanism to maintain developmental plasticity of the UGR until gonad formation is through the epigenetic modification of histone proteins. In order to provide a resource for future studies, we used chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) for two histone modifications, H3K4me3 and H3K27me3, in the E11.5 mouse UGR. These marks are both known to reflect the active, repressive or a poised chromatin state. We found that enrichment for each histone mark reflected transcriptional activity in precursor cells of the developing gonad. From the analysis of potential enhancer/regulator peak regions for DNA binding motifs, we identified several candidate transcription factors that may contribute to gonadal cell lineage specification. We additionally identified signaling pathway genes that are targeted by both chromatin modifications. Together, these datasets provide a useful resource for investigating gene regulatory networks functioning during UGR development at E11.5.

48: Embryonic signals perpetuate polar-like trophoblast stem cells and pattern the blastocyst axis.
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Posted to bioRxiv 03 Jan 2019

Embryonic signals perpetuate polar-like trophoblast stem cells and pattern the blastocyst axis.
1,766 downloads developmental biology

Javier Frias Aldeguer, Maarten Kip, Judith Vivie, Linfeng Li, Anna Alemany, Jeroen Korving, Frank Darmis, Alexander van Oudenaarden, Niels Geijsen, Nicolas C Rivron

The early mammalian conceptus (blastocyst) comprises an outer trophoblast globe that forms an axis originating from the inner embryonic cells. From the mouse conceptus, Trophoblast stem cells (TSCs) are derived, which are in vitro analogues of early trophoblasts. Here, we show that TSCs contain plastic subpopulations reflecting developmental states ranging from pre- to post-implantation trophoblasts. However, upon exposure to a specific combination of embryonic inductive signals, TSCs globally acquire properties of pre-implantation polar trophoblasts (gene expression, self-renewal) juxtaposing the inner embryonic cells, and an enhanced, homogeneous epithelial phenotype. These lines of polar-like TSCs (pTSCs) represent a transcriptionally earlier state that more efficiently forms blastoids, whose inner embryonic cells then induce the patterning of gene expression along the embryonic-abembryonic axis. Altogether, delineating the requirements and properties of polar trophoblasts and blastocyst axis formation in vitro provides a foundation for the precise description and dissection of early development.

49: Rainbow-seq: combining cell lineage tracking with single-cell RNA sequencing in preimplantation embryos
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Posted to bioRxiv 02 Apr 2018

Rainbow-seq: combining cell lineage tracking with single-cell RNA sequencing in preimplantation embryos
1,763 downloads developmental biology

Fernando Biase, Qiuyang Wu, Riccardo Calandrelli, Marcelo Rivas-Astroza, Shuigeng Zhou, Sheng Zhong

Single-cell RNA-seq experiments cannot record cell division history and therefore cannot directly connect intercellular differences at a later developmental stage to their progenitor cells. We developed Rainbow-seq to combine cell division lineage tracing with single-cell RNA-seq. With distinct fluorescent protein genes as lineage markers, Rainbow-seq enables each single-cell RNA-seq experiment to simultaneously read single-cell transcriptomes and decode the lineage marker genes. We traced the lineages deriving from each blastomere in two-cell mouse embryos and observed inequivalent contributions to the embryonic and abembryonic poles in 72% of the blastocysts evaluated. Rainbow-seq on four- and eight-cell embryos with lineage tracing triggered at two-cell stage exhibited remarkable transcriptome-wide differences between the two cell lineages at both stages, including genes involved in negative regulation of transcription and signaling. These data provide critical insights on cell fate choices in cleavage embryos. Rainbow-seq bridged a critical gap between cellular division history and single-cell RNA-seq assays.

50: Single-Cell RNA-seq Reveals a Subpopulation of Cells Underlying β Cell Expansion in the Postnatal Islets
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Posted to bioRxiv 17 Apr 2018

Single-Cell RNA-seq Reveals a Subpopulation of Cells Underlying β Cell Expansion in the Postnatal Islets
1,758 downloads developmental biology

Jingli A Zhang, Chunyan Gu, Derek K Smith, Monica K. Beltran, Noelyn Kljavin, Hai Ngu, Rowena Suriben, Jeremy Stinson, Zora Modrusan, Andrew S. Peterson

Pancreatic β cells undergo significant expansion and maturation during human and rodent postnatal development. Here, we used single-cell RNA-seq to characterize gene expression patterns at various stages of mouse islet cell development and uncovered a population of cells that is most abundant during the early postnatal period. This cell population lacks expression of FLTP and expresses PDGF receptors. Each of these conditions have previously been associated with proliferative capacity in β cells suggesting that we have identified the proliferative competent of β cell mass expansion. The subpopulation co-express many endocrine lineage-specific genes and exhibits a downregulation of genes associated with mitochondrial oxidative phosphorylation and global protein synthesis. It has upregulated activity of genes in the Wnt, Hippo, PDGF, and Notch pathways and has a significantly higher proliferation potential than the more mature β population. We show that activity of the Notch pathway is required in postnatal β cell expansion where it serves to maintain an undifferentiated endocrine state in the polyhormonal cell population. Collectively, our study identifies a proliferative, progenitor-like cell subpopulation in the postnatal islet as the source of postnatal β cell expansion.

51: Gastruloids develop the three body axes in the absence of extraembryonic tissues and spatially localised signalling
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Posted to bioRxiv 31 Jan 2017

Gastruloids develop the three body axes in the absence of extraembryonic tissues and spatially localised signalling
1,730 downloads developmental biology

D.A. Turner, L. Alonso-Crisostomo, M. Girgin, P. Baillie-Johnson, C. R. Glodowski, P. C. Hayward, J. Collignon, C. Gustavsen, P. Serup, Benjamin Steventon, M. Lutolf, A. Martinez Arias

Establishment of the three body axes is a critical step during animal development. In mammals, genetic studies have shown that a combination of precisely deployed signals from extraembryonic tissues position the anteroposterior axis (AP) within the embryo and lead to the emergence of the dorsoventral (DV) and left-right (LR) axes. We have used Gastruloids, embryonic organoids, as a model system to understand this process and find that they are able to develop AP, DV and LR axes as well as to undergo axial elongation in a manner that mirror embryos. The Gastruloids can be grown for 160 hours and form derivatives from ectoderm, mesoderm and endoderm. We focus on the AP axis and show that in the Gastruloids this axis is registered in the expression of T/Bra at one pole that corresponds to the tip of the elongation. We find that localisation of T/Bra expression depends on the combined activities of Wnt/β-Catenin and Nodal/Smad2,3 signalling, and that BMP signalling is dispensable for this process. Furthermore, AP axis specification occurs in the absence of both extraembryonic tissues and of localised sources of signalling. Our experiments show that Nodal, together with Wnt/β-Catenin signalling, is essential for the expression of T/Bra but that Wnt signalling has a separable activity in the elongation of the axis. The results lead us to suggest that, in the embryo, the role of the extraembryonic tissues might not be to induce the axes but to bias an intrinsic ability of the embryo to break its initial symmetry and organise its axes.

52: Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution
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Posted to bioRxiv 19 Dec 2018

Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution
1,710 downloads developmental biology

Alex A. Pollen, Aparna Bhaduri, Madeline G Andrews, Tomasz J. Nowakowski, Olivia S Meyerson, Mohammed A. Mostajo-Radji, Elizabeth Di Lullo, Beatriz Alvarado, Melanie Bedolli, Max L Dougherty, Ian T. Fiddes, Zev N. Kronenberg, Joe Shuga, Anne A Leyrat, Jay A West, Marina Bershteyn, Craig B Lowe, Bryan Pavlovic, Sofie R. Salama, David Haussler, Evan E. Eichler, Arnold R. Kriegstein

Direct comparisons of human and non-human primate brain tissue have the potential to reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is largely inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. First, we systematically evaluated the fidelity of organoid models to primary human and macaque cortex, finding organoid models preserve gene regulatory networks related to cell types and developmental processes but exhibit increased metabolic stress. Second, we identified 261 genes differentially expressed in human compared to chimpanzee organoids and macaque cortex. Many of these genes overlap with human-specific segmental duplications and a subset suggest increased PI3K/AKT/mTOR activation in human outer radial glia. Together, our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.

53: Species-specific oscillation periods of human and mouse segmentation clocks are due to cell autonomous differences in biochemical reaction parameters
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Posted to bioRxiv 26 May 2019

Species-specific oscillation periods of human and mouse segmentation clocks are due to cell autonomous differences in biochemical reaction parameters
1,696 downloads developmental biology

Mitsuhiro Matsuda, Hanako Hayashi, Jordi Garcia-Ojalvo, Kumiko Yoshioka-Kobayashi, Ryoichiro Kageyama, Yoshihiro Yamanaka, Makoto Ikeya, Junya Toguchida, Cantas Alev, Miki Ebisuya

While the mechanisms of embryonic development are similar between mouse and human, the tempo is in general slower in human. The cause of interspecies differences in developmental time remains a mystery partly due to lack of an appropriate model system. Since murine and human embryos differ in their sizes, geometries, and nutrients, we use in vitro differentiation of pluripotent stem cells (PSCs) to compare the same type of cells between the species in similar culture conditions. As an example of well-defined developmental time, we focus on the segmentation clock, oscillatory gene expression that regulates the timing of sequential formation of body segments. In this way we recapitulate the murine and human segmentation clocks in vitro, showing that the species-specific oscillation periods are derived from cell autonomous differences in the speeds of biochemical reactions. Presomitic mesoderm (PSM)-like cells induced from murine and human PSCs displayed the oscillatory expression of HES7, the core gene of the segmentation clock, with oscillation periods of 2-3 hours (mouse PSM) and 5-6 hours (human PSM). Swapping HES7 loci between murine and human genomes did not change the oscillation periods dramatically, denying the possibility that interspecies differences in the sequences of HES7 loci might be the cause of the observed period difference. Instead, we found that the biochemical reactions that determine the oscillation period, such as the degradation of HES7 and delays in its expression, are slower in human PSM compared with those in mouse PSM. With the measured biochemical parameters, our mathematical model successfully accounted for the 2-3-fold period difference between mouse and human. We further demonstrate that the concept of slower biochemical reactions in human cells is generalizable to several other genes, as well as to another cell type. These results collectively indicate that differences in the speeds of biochemical reactions between murine and human cells give rise to the interspecies period difference of the segmentation clock and may contribute to other interspecies differences in developmental time.

54: Mouse embryonic stem cells can differentiate via multiple paths to the same state
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Posted to bioRxiv 05 Apr 2017

Mouse embryonic stem cells can differentiate via multiple paths to the same state
1,691 downloads developmental biology

James A. Briggs, Victor C Li, Seungkyu Lee, Clifford J Woolf, Allon M. Klein, Marc W. Kirschner

In embryonic development, cells must differentiate through stereotypical sequences of intermediate states to generate mature states of a particular fate. By contrast, direct programming can generate similar fates through alternative routes, by directly expressing terminal transcription factors. Yet the cell state transitions defining these new routes are unclear. We applied single-cell RNA sequencing to compare two mouse motor neuron differentiation protocols: a standard protocol approximating the embryonic lineage, and a direct programming method. Both undergo similar early neural commitment. Then, rather than transitioning through spinal intermediates like the standard protocol, the direct programming path diverges into a novel transitional state. This state has specific and abnormal gene expression. It opens a ‘loop’ or ‘worm hole’ in gene expression that converges separately onto the final motor neuron state of the standard path. Despite their different developmental histories, motor neurons from both protocols structurally, functionally, and transcriptionally resemble motor neurons from embryos.

55: High-Resolution Dissection of Conducive Reprogramming Trajectory to Ground State Pluripotency
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Posted to bioRxiv 07 Sep 2017

High-Resolution Dissection of Conducive Reprogramming Trajectory to Ground State Pluripotency
1,676 downloads developmental biology

Asaf Zviran, Nofar Mor, Yoach Rais, Hila Gingold, Shani Peles, Elad Chomsky, Sergey Viukov, Jason D. Buenrostro, Leehee Weinberger, Yair S. Manor, Vladislav Krupalnik, Mirie Zerbib, Hadas Hezroni, Diego Adhemar Jaitin, David Larastiaso, Shlomit Gilad, Sima Benjamin, Awni Mousa, Muneef Ayyash, Daoud Sheban, Jonathan Bayerl, Alejandro Aguilera Castrejon, Rada Massarwa, Itay Maza, Suhair Hanna, Ido Amit, Yonatan Stelzer, Igor Ulitsky, William J Greenleaf, Yitzhak Pilpel, Noa Novershtern, Jacob H. Hanna

The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) with four transcription factors Oct4, Sox2, Klf4 and cMyc (abbreviated as OSKM) has provoked interest to define the molecular characteristics of the process. Despite important progress, the dynamics of epigenetic reprogramming at high resolution in correctly reprogrammed iPSCs and throughout the entire process remain largely undefined. This gap in understanding results from the inefficiency of conventional reprogramming methods coupled with the difficulty of prospectively isolating the rare cells that eventually correctly reprogram into iPSCs. Here we characterize cell fate conversion from fibroblast to iPSC using a highly efficient deterministic murine reprogramming system engineered through optimized inhibition of Gatad2a-Mbd3/NuRD repressive sub-complex. This comprehensive characterization provides single-day resolution of dynamic changes in levels of gene expression, chromatin modifications, TF binding, DNA accessibility and DNA methylation. The integrative analysis identified two transcriptional modules that dominate successful reprogramming. One consists of genes whose transcription is regulated by on/off epigenetic switching of modifications in their promoters (abbreviated as ESPGs), and the second consists of genes with promoters in a constitutively active chromatin state, but a dynamic expression pattern (abbreviated as CAPGs). ESPGs are mainly regulated by OSK, rather than Myc, and are enriched for cell fate determinants and pluripotency factors. CAPGs are predominantly regulated by Myc, and are enriched for cell biosynthetic regulatory functions. We used the ESPG module to study the identity and temporal occurrence of activating and repressing epigenetic switching during reprogramming. Removal of repressive chromatin modifications precedes chromatin opening and binding of RNA polymerase II at enhancers and promoters, and the opposite dynamics occur during repression of enhancers and promoters. Genome wide DNA methylation analysis demonstrated that de novo DNA methylation is not required for highly efficient conducive iPSC reprogramming, and identified a group of super-enhancers targeted by OSK, whose early demethylation marks commitment to a successful reprogramming trajectory also in inefficient conventional reprogramming systems. CAPGs are distinctively regulated by multiple synergetic ways: 1) Myc activity, delivered either endogenously or exogenously, dominates CAPG expression changes and is indispensable for induction of pluripotency in somatic cells; 2) A change in tRNA codon usage which is specific to CAPGs, but not ESPGs, and favors their translation. In summary, our unbiased high-resolution mapping of epigenetic changes on somatic cells that are committed to undergo successful reprogramming reveals interleaved epigenetic and biosynthetic reconfigurations that rapidly commission and propel conducive reprogramming toward naive pluripotency.

56: Single-Cell Sequencing of Primate Preimplantation Embryos Reveals Chromosome Elimination Via Cellular Fragmentation and Blastomere Exclusion
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Posted to bioRxiv 02 Jan 2018

Single-Cell Sequencing of Primate Preimplantation Embryos Reveals Chromosome Elimination Via Cellular Fragmentation and Blastomere Exclusion
1,675 downloads developmental biology

Brittany L. Daughtry, Jimi L. Rosenkrantz, Nathan H. Lazar, Suzanne S. Fei, Nash Redmayne, Kristof A. Torkenczy, Andrew C. Adey, Lina Gao, Byung Park, Kimberly A. Nevonen, Lucia Carbone, Shawn L. Chavez

Aneuploidy that arises during meiosis and/or mitosis is a major contributor to early embryo loss. We previously demonstrated that human preimplantation embryos encapsulate mis-segregated chromosomes into micronuclei while undergoing cellular fragmentation and that fragments can contain chromosomal material, but the source of this DNA was unknown. Here, we leveraged the use of a non-human primate model and single-cell DNA-sequencing (scDNA-seq) to examine the chromosomal content of 471 individual samples comprising 254 blastomeres, 42 polar bodies, and 175 cellular fragments from a large number (N=50) of disassembled rhesus cleavage-stage embryos. Our analysis revealed that the frequency of aneuploidy and micronucleation is conserved between humans and macaques and that cellular fragments encapsulate whole and/or partial chromosomes lost from blastomeres. Single-cell/fragment genotyping demonstrated that these chromosome-containing cellular fragments (CCFs) can be either maternal or paternal in origin and display DNA damage via double-stranded breaks. Chromosome breakage and abnormal cytokinesis resulted in reciprocal losses/gains at the terminal ends of chromosome arms, uniparental genome segregation, and mixoploidy between blastomeres. Combining time-lapse imaging with scDNA-seq, we also determined that multipolar divisions at the zygote or 2-cell stage generated chaotic aneuploidy encompassing a complex mixture of maternal and paternal chromosomes. Despite frequent chromosomal mis-segregation at the cleavage-stage, we show that CCFs and non-dividing aneuploid blastomeres exhibiting extensive DNA damage are prevented from incorporation at the blastocyst stage. These findings suggest that embryos respond to chromosomal errors by encapsulation into micronuclei, elimination by cellular fragmentation, and selection against highly aneuploid blastomeres to overcome chromosome instability during preimplantation development.

57: NAD+ repletion rescues female fertility during reproductive ageing
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Posted to bioRxiv 02 Aug 2019

NAD+ repletion rescues female fertility during reproductive ageing
1,657 downloads developmental biology

Michael J. Bertoldo, Dave R Listijono, Wing-Hong Jonathan Ho, Angelique H. Riepsamen, Xing L. Jin, Kaisa Selesniemi, Dale M. Goss, Saabah Mahbub, Jared M. Campbell, Abbas Habibalahi, Wei-Guo Nicholas Loh, Neil A. Youngson, Jayanthi Maniam, Ashley S.A. Wong, Dulama Richani, Catherine Li, Yiqing Zhao, Maria Marinova, Lynn-Jee Kim, Laurin Lau, Rachael M. Wu, A. Stefanie Mikolaizak, Toshiyuki Araki, David G. Le Couteur, Nigel Turner, Margaret J. Morris, Kirsty A. Walters, Ewa Goldys, Christopher O’Neill, Robert B. Gilchrist, David A. Sinclair, Hayden A. Homer, Lindsay E. Wu

Female infertility is a common and devastating condition with life-long health, emotional and social consequences. There is currently no pharmacological therapy for preserving oocyte quality during aging, which is the strongest risk factor for infertility. This leads to an age dependent decline in natural conception and IVF success rates. Here, we show that this is due in part to declining levels of the metabolic cofactor nicotinamide adenine dinucleotide (NAD+), and that restoring NAD+ levels with its metabolic precursor nicotinamide mononucleotide (NMN) rejuvenates oocyte quality and quantity in aged animals, leading to improved fertility. These benefits extend to the developing embryo, where NMN supplementation in embryo culture media following IVF enhances blastocyst formation in older mice. The NAD+ dependent deacylase SIRT2 is sufficient, but not essential, to recapitulate the benefits of in vivo NMN treatment, and transgenic overexpression of SIRT2 maintains oocyte spindle assembly, accurate chromosome segregation, decreased oxidative stress and overall fertility with ageing. Pharmacological elevation of NAD+ may be an effective, non-invasive strategy for restoring and maintaining female fertility during ageing, and for improving the success of IVF.

58: Multimodal transcriptional control of pattern formation in embryonic development
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Posted to bioRxiv 31 May 2018

Multimodal transcriptional control of pattern formation in embryonic development
1,653 downloads developmental biology

Nicholas Lammers, Vahe Galstyan, Armando Reimer, Sean A Medin, Chris H Wiggins, Hernan G. Garcia

Predicting developmental outcomes from regulatory DNA sequence and transcription factor patterns remains an open challenge in physical biology. Using stripe 2 of the even-skipped gene in Drosophila embryos as a case study, we dissect the regulatory forces underpinning a key step along the developmental decision-making cascade: the generation of cytoplasmic mRNA patterns via the control of transcription in individual cells. Using live imaging and computational approaches, we found that the transcriptional burst frequency is modulated across the stripe to control the mRNA production rate. However, we discovered that bursting alone cannot quantitatively recapitulate the formation of the stripe, and that control of the window of time over which each nucleus transcribes even-skipped plays a critical role in stripe formation. Theoretical modeling revealed that these two regulatory strategies--bursting and the time window--obey different kinds of regulatory logic, suggesting that the stripe is shaped by the interplay of two distinct underlying molecular processes.

59: Single-cell transcriptomics identifies CD44 as a new marker and regulator of haematopoietic stem cells development
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Posted to bioRxiv 06 Jun 2018

Single-cell transcriptomics identifies CD44 as a new marker and regulator of haematopoietic stem cells development
1,641 downloads developmental biology

Morgan Oatley, Özge Vargel Bölükbasi, Valentine Svensson, Maya Shvartsman, Kerstin Ganter, Katharina Zirngibl, Polina V. Pavlovich, Vladislava Milchevskaya, Vladimira Foteva, Kedar N Natarajan, Bianka Baying, Vladimir Benes, Kiran R. Patil, Sarah A. Teichmann, Christophe Lancrin

The endothelial to haematopoietic transition (EHT) is the process whereby haemogenic endothelium differentiates into haematopoietic stem and progenitor cells (HSPCs). The intermediary steps of this process are unclear, in particular the identity of endothelial cells that give rise to HSPCs is unknown. Using single-cell transcriptome analysis and antibody screening we identified CD44 as a new marker of EHT enabling us to isolate robustly the different stages of EHT in the aorta gonad mesonephros (AGM) region. This allowed us to provide a very detailed phenotypical and transcriptional profile for haemogenic endothelial cells, characterising them with high expression of genes related to Notch signalling, TGFbeta/BMP antagonists (Smad6, Smad7 and Bmper) and a downregulation of genes related to glycolysis and the TCA cycle. Moreover, we demonstrated that by inhibiting the interaction between CD44 and its ligand hyaluronan we could block EHT, identifying a new regulator of HSPC development.

60: Highly efficient CRISPR/Cas9-mediated tissue specific mutagenesis in Drosophila
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Posted to bioRxiv 20 Feb 2018

Highly efficient CRISPR/Cas9-mediated tissue specific mutagenesis in Drosophila
1,626 downloads developmental biology

Amy R Poe, Bei Wang, Maria L Sapar, Hui Ji, Kailyn Li, Tireniolu Onabajo, Rushaniya Fazliyeva, Mary Gibbs, Yue Qiu, Yuzhao Hu, Chun Han

Tissue-specific loss-of-function (LOF) analysis is an essential approach for characterizing gene function. Here we describe an efficient CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) method for ablating gene function in Drosophila. This binary system consists of a tissue-specific Cas9 and a ubiquitously expressed multi-guide RNA (gRNA) transgene. To facilitate the construction of these components, we developed convenient tools for generating and evaluating enhancer-driven Cas9 lines, identified a multi-gRNA design that is highly efficient in mutagenizing somatic cells, and established an assay for testing the efficiency of multi-gRNAs in creating double-stranded breaks. We found that excision of genomic DNA induced by two gRNAs is infrequent in somatic cells, while indels more reliably cause tissue-specific LOF. Furthermore, we show that enhancer-driven Cas9 is less cytotoxic yet results in more complete gene removal than Gal4-driven Cas9 in larval neurons. Finally, we demonstrate that CRISPR-TRiM efficiently unmasks redundant gene functions in neuronal morphogenesis. Importantly, two Cas9 transgenes that turn on with different timings in the neuronal lineage revealed the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISRPR tools can be applied to analyze tissue-specific gene function in many biological processes.

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