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in category developmental biology

2,586 results found. For more information, click each entry to expand.

21: Decoding the development of the blood and immune systems during human fetal liver haematopoiesis
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Posted to bioRxiv 31 May 2019

Decoding the development of the blood and immune systems during human fetal liver haematopoiesis
3,121 downloads developmental biology

Dorin-Mirel Popescu, Rachel A. Botting, Emily Stephenson, Kile Green, Laura Jardine, Emily F Calderbank, Mirjana Efremova, Meghan Acres, Daniel Maunder, Peter Vegh, Issac Goh, Yorick Gitton, Jongeun Park, Krzysztof Polanski, Roser Vento-Tormo, Zhichao Miao, Rachel Rowell, David McDonald, James Fletcher, David Dixon, Elizabeth Poyner, Gary Reynolds, Michael Mather, Corina Moldovan, Lira Mamanova, Frankie Greig, Matthew Young, Kerstin Meyer, Steven Lisgo, Jaume Bacardit, Andrew Fuller, Ben Millar, Barbara Innes, Susan Lindsay, Michael J. T. Stubbington, Monika S. Kowalczyk, Bo Li, Orr Ashenbrg, Marcin Tabaka, Danielle Dionne, Timothy L. Tickle, Michal Slyper, Orit Rozenblatt-Rosen, Andrew Filby, Alexandra-Chloe Villani, Anindita Roy, Aviv Regev, Alain Chedotal, Irene Roberts, Berthold Göttgens, Elisa Laurenti, Sam Behjati, Sarah A Teichmann, Muzlifah Haniffa

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells/multipotent progenitors (HSC/MPPs), yet remains poorly defined in humans. Using single cell transcriptome profiling of ~133,000 fetal liver and ~65,000 fetal skin and kidney cells, we identify the repertoire of blood and immune cells in first and early second trimesters of development. From this data, we infer differentiation trajectories from HSC/MPPs, and evaluate the impact of tissue microenvironment on blood and immune cell development. We predict coupling of mast cell differentiation with erythro-megakaryopoiesis and identify physiological erythropoiesis in fetal skin. We demonstrate a shift in fetal liver haematopoietic composition during gestation away from being erythroid-predominant, accompanied by a parallel change in HSC/MPP differentiation potential, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a valuable reference for understanding and harnessing the therapeutic potential of HSC/MPPs.

22: Insect wings and body wall evolved from ancient leg segments
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Posted to bioRxiv 09 Jan 2018

Insect wings and body wall evolved from ancient leg segments
2,962 downloads developmental biology

Heather S Bruce, Nipam H. Patel

The origin of insect wings has long been debated. Central to this debate is whether wings evolved from an epipod (outgrowth, e.g., a gill) on ancestral crustacean leg segments, or represent a novel outgrowth from the dorsal body wall that co-opted some of the genes used to pattern the epipods. To determine whether wings can be traced to ancestral, pre-insect structures, or arose by co-option, comparisons are necessary between insects and arthropods more representative of the ancestral state, where the hypothesized proximal leg region is not fused to the body wall. To do so, we examined the function of five leg patterning genes in the crustacean Parhyale hawaiensis and compared this to previous functional data from insects. By comparing gene knockout phenotypes of leg patterning genes in a crustacean with those of insects, we show that two ancestral crustacean leg segments were incorporated into the insect body, moving the leg's epipod dorsally, up onto the back to form insect wings. Thus, our data shows that much of the body wall of insects, including the entire wing, is derived from these two ancestral proximal leg segments. This model explains all observations in favor of either the body wall origin or proximal leg origin of insect wings. Thus, our results show that insect wings are not novel structures, but instead evolved from existing, ancestral structures.

23: A general theory of individuated multicellularity
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Posted to bioRxiv 24 Mar 2015

A general theory of individuated multicellularity
2,873 downloads developmental biology

Felipe A. Veloso

Changes in gene expression are thought to regulate the differentiation process intrinsically through complex epigenetic mechanisms. In fundamental terms, however, this assumed regulation refers only to the intricate propagation of changes in gene expression or else leads to logical inconsistencies. The evolution and self-regulatory dynamics of individuated multicellularity also lack a unified and falsifiable description. To fill this gap, I computationally analyzed publicly available high-throughput data of histone H3 post-translational modifications and mRNA abundance for different Homo sapiens, Mus musculus, and Drosophila melanogaster cell-type/developmental-periods samples. My analysis of genomic regions adjacent to transcription start sites generated a profile from pairwise partial correlations between histone modifications controlling for the respective mRNA levels for each cell-type/developmental-period dataset. I found that these profiles, while explicitly uncorrelated to transcript abundance by construction, associate strongly with cell differentiation states. This association is not expected if cell differentiation is, in effect, regulated by epigenetic mechanisms. Based on these results, I propose a falsifiable theory of individuated multicellularity, which relies on the synergistic coupling across the extracellular space of two stochastically independent "self-organizing" systems constraining histone modification states at the same sites. This theory describes how the multicellular individual—understood as an intrinsic, higher-order constraint—emerges from proliferating undifferentiated cells, and may explain the intrinsic regulation of gene transcriptional changes for cell differentiation and the evolution of individuated multicellular organisms.

24: Hair-bearing human skin generated entirely from pluripotent stem cells
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Posted to bioRxiv 08 Jul 2019

Hair-bearing human skin generated entirely from pluripotent stem cells
2,851 downloads developmental biology

Jiyoon Lee, Cyrus Rabbani, Hongyu Gao, Matthew Steinhart, Benjamin M. Woodruff, Zachary Pflum, Alexander Kim, Stefan Heller, Yunlong Liu, Taha Z Shipchandler, Karl R Koehler

The skin is important for regulating bodily fluid retention and temperature, guarding against external stresses, and mediating touch and pain sensation. The skin is also susceptible to damage from burns, diseases, or genetic defects, which affect nearly one billion people worldwide. For the advancement of skin regenerative therapies, it remains challenging to construct new skin with hair follicles and nerves in tissue cultures and in bioengineered skin grafts. Here, we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use step-wise modulation of the TGF and FGF signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During 4-5 months incubation, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis, and pigmented hair follicles equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking human touch circuitry. Single-cell RNA sequencing and direct comparison to foetal specimens suggest that skin organoids are equivalent to human facial skin in the second-trimester of development. Moreover, we show that skin organoids produce planar hair-bearing skin when grafted on nude mice. Together, our results demonstrate the self-assembly of nearly complete skin tissue in vitro that can be used to reconstitute skin in vivo. We anticipate that our skin organoid model will be foundational to future studies of human skin development, disease modelling, or reconstructive surgery.

25: A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in Drosophila
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Posted to bioRxiv 11 Aug 2014

A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in Drosophila
2,847 downloads developmental biology

Xu Zhang, Wouter Koolhaas, Frank Schnorrer

The development of CRISPR/Cas9 technologies promises a quantum leap in genome-engineering of model organisms. However, CRISPR-mediated gene targeting reports in Drosophila are still restricted to a few genes, use variable experimental conditions and vary in efficiency, questioning the universal applicability of the method. Here, we developed an efficient, two-step strategy to flexibly engineer the fly genome by combining CRISPR with recombinase-mediated cassette exchange (RMCE). In the first step, two sgRNAs, whose activity had been tested in cell culture, were co-injected together with a donor plasmid into transgenic Act5C-Cas9, Ligase4 mutant embryos and the homologous integration events were identified by eye fluorescence. In the second step, the eye marker was replaced with DNA sequences of choice using RMCE enabling flexible gene modification. We applied this strategy to engineer four different loci, including a gene on the fourth chromosome, at comparably high efficiencies, suggesting that any fly lab can engineer their favourite gene for a broad range of applications within about three months.

26: Comparative analysis of kidney organoid and adult human kidney single cell and single nucleus transcriptomes
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Posted to bioRxiv 11 Dec 2017

Comparative analysis of kidney organoid and adult human kidney single cell and single nucleus transcriptomes
2,818 downloads developmental biology

Haojia Wu, Kohei Uchimura, Erinn Donnelly, Yuhei Kirita, Samantha A. Morris, Benjamin D. Humphreys

Kidney organoids differentiated from human pluripotent stem cells hold great promise for understanding organogenesis, modeling disease and ultimately as a source of replacement tissue. Realizing the full potential of this technology will require better differentiation strategies based upon knowledge of the cellular diversity and differentiation state of all cells within these organoids. Here we analyze single cell gene expression in 45,227 cells isolated from 23 organoids differentiated using two different protocols. Both generate kidney organoids that contain a diverse range of kidney cells at differing ratios as well as non-renal cell types. We quantified the differentiation state of major organoid kidney cell types by comparing them against a 4,259 single nucleus RNA-seq dataset generated from adult human kidney, revealing immaturity of all kidney organoid cell types. We reconstructed lineage relationships during organoid differentiation through pseudotemporal ordering, and identified transcription factor networks associated with fate decisions. These results define impressive kidney organoid cell diversity, identify incomplete differentiation as a major roadblock for current directed differentiation protocols and provide a human adult kidney snRNA-seq dataset against which to benchmark future progress.

27: Species-specific developmental timing is associated with global differences in protein stability in mouse and human
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Posted to bioRxiv 31 Dec 2019

Species-specific developmental timing is associated with global differences in protein stability in mouse and human
2,718 downloads developmental biology

Teresa Rayon, Despina Stamataki, Ruben Perez-Carrasco, Lorena Garcia-Perez, Christopher Barrington, Manuela Melchionda, Katherine Exelby, Victor Tybulewicz, Elizabeth M. C. Fisher, James Briscoe

What determines the pace of embryonic development? Although many molecular mechanisms controlling developmental processes are evolutionarily conserved, the speed at which these operate can vary substantially between species. For example, the same genetic programme, comprising sequential changes in transcriptional states, governs the differentiation of motor neurons in mouse and human, but the tempo at which it operates differs between species. Using in vitro directed differentiation of embryonic stem cells to motor neurons, we show that the programme runs twice as fast in mouse as in human. We provide evidence that this is neither due to differences in signalling, nor the genomic sequence of genes or their regulatory elements. Instead, we find an approximately two-fold increase in protein stability and cell cycle duration in human cells compared to mouse. This can account for the slower pace of human development, indicating that global differences in key kinetic parameters play a major role in interspecies differences in developmental tempo.

28: CRISPR-Cas13d induces efficient mRNA knock-down in animal embryos
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Posted to bioRxiv 14 Jan 2020

CRISPR-Cas13d induces efficient mRNA knock-down in animal embryos
2,696 downloads developmental biology

Gopal Kushawah, Joaquin Abugattas-Nuñez del Prado, Juan R. Martinez-Morales, Michelle DeVore, Javier R. Guelfo, Emry O. Brannan, Wei Wang, Timothy J. Corbin, Andrea M. Moran, Alejandro Sánchez Alvarado, Edward Málaga-Trillo, Carter M Takacs, Ariel A. Bazzini, Miguel A. Moreno-Mateos

Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to induce RNA degradation in yeast, plants and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-Cas13d is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that both zygotically-expressed and maternally-provided transcripts are efficiently targeted, resulting in an 80% average decrease in transcript level and the recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish and mouse embryos. Altogether our results demonstrate that CRISPR-Cas13d is an efficient knock-down platform to interrogate gene function in animal embryos.

29: Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development
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Posted to bioRxiv 27 Jun 2019

Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development
2,664 downloads developmental biology

Sabina Kanton, Michael James Boyle, Zhisong He, Malgorzata Santel, Anne Weigert, Fatima Sanchis Calleja, Leila Sidow, Jonas Fleck, Patricia Guijarro, Dingding Han, Zhengzong Qian, Michael Heide, Wieland Huttner, Philipp Khaitovich, Svante Pääbo, Barbara Treutlein, J. Gray Camp

The human brain has changed dramatically since humans diverged from our closest living relatives, chimpanzees and the other great apes. However, the genetic and developmental programs underlying this divergence are not fully understood. Here, we have analyzed stem cell-derived cerebral organoids using single-cell transcriptomics (scRNA-seq) and accessible chromatin profiling (scATAC-seq) to explore gene regulatory changes that are specific to humans. We first analyze cell composition and reconstruct differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. We find that brain region composition varies in organoids from different iPSC lines, yet regional gene expression patterns are largely reproducible across individuals. We then analyze chimpanzee and macaque cerebral organoids and find that human neuronal development proceeds at a delayed pace relative to the other two primates. Through pseudotemporal alignment of differentiation paths, we identify human-specific gene expression resolved to distinct cell states along progenitor to neuron lineages in the cortex. We find that chromatin accessibility is dynamic during cortex development, and identify instances of accessibility divergence between human and chimpanzee that correlate with human-specific gene expression and genetic change. Finally, we map human-specific expression in adult prefrontal cortex using single-nucleus RNA-seq and find developmental differences that persist into adulthood, as well as cell state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene regulatory features that are unique to humans.

30: 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
2,657 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.

31: Broad applicability of a streamlined Ethyl Cinnamate-based clearing procedure
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Posted to bioRxiv 13 Jun 2018

Broad applicability of a streamlined Ethyl Cinnamate-based clearing procedure
2,629 downloads developmental biology

Wouter Masselink, Daniel Reumann, Prayag Murawala, Pawel Pasierbek, Yuka Taniguchi, Jürgen A. Knoblich, Elly M. Tanaka

Turbidity and opaqueness are inherent properties of tissues which limit the capacity to acquire microscopic images through large tissues. Creating a uniform refractive index, known as tissue clearing, overcomes most of these issues. These methods have enabled researchers to image large and complex 3D structures with unprecedented depth and resolution. However, tissue clearing has been adopted to a limited extent due to a combination of cost, time, complexity of existing methods and potential negative impact on fluorescence signal. Here we describe 2Eci (2nd generation Ethyl cinnamate based clearing method) which can be used to clear a wide range of tissues, including cerebral organoids, Drosophila melanogaster, zebrafish, axolotl, and Xenopus laevis in as little as 1-5 days while preserving a broad range of fluorescence proteins including GFP, mCherry, Brainbow, and alexa-fluorophores. Ethyl cinnamate is non-toxic and can easily be used in multi-user microscope facilities. This method will open up clearing to a much broader group of researchers, due to its broad applicability, ease of use, and non-toxic nature of Ethyl cinnamate.

32: Guided self-organization recapitulates tissue architecture in a bioengineered brain organoid model
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Posted to bioRxiv 19 Apr 2016

Guided self-organization recapitulates tissue architecture in a bioengineered brain organoid model
2,588 downloads developmental biology

Madeline A. Lancaster, Nina S Corsini, Thomas R Burkard, Jürgen A. Knoblich

Recently emerging methodology for generating human tissues in vitro has the potential to revolutionize drug discovery and disease research. Currently, three-dimensional cell culture models either rely on the pronounced ability of mammalian cells to self organize in vitro1-6, or use bioengineered constructs to arrange cells in an organ-like configuration7,8. While self-organizing organoids can recapitulate developmental events at a remarkable level of detail, bioengineered constructs excel at reproducibly generating tissue of a desired architecture. Here, we combine these two approaches to reproducibly generate micropatterned human forebrain tissue while maintaining its self-organizing capacity. We utilize poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a scaffold to generate elongated embryoid bodies and demonstrate that this influences tissue identity. Micropatterned engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, we reconstitute the basement membrane at later stages leading to characteristic cortical tissue architecture including formation of a polarized cortical plate and radial units. enCORs provide the first in vitro system for modelling the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. We demonstrate their utility by modelling teratogenic effects of ethanol and show that defects in leading process formation may be responsible for the neuronal migration deficits in fetal alcohol syndrome. Our data demonstrate that combining 3D cell culture with bioengineering can significantly enhance tissue identity and architecture, and establish organoid models for teratogenic compounds.

33: Stem cell differentiation trajectories in Hydra resolved at single-cell resolution
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Posted to bioRxiv 03 Nov 2018

Stem cell differentiation trajectories in Hydra resolved at single-cell resolution
2,582 downloads developmental biology

Stefan Siebert, Jeffrey A. Farrell, Jack F. Cazet, Yashodara L. Abeykoon, Abby S. Primack, Christine E. Schnitzler, Celina E. Juliano

The adult Hydra polyp continuously renews all of its cells using three separate stem cell populations, but the genetic pathways enabling homeostatic tissue maintenance are not well understood. We used Drop-seq to sequence transcriptomes of 24,985 single Hydra cells and identified the molecular signatures of a broad spectrum of cell states, from stem cells to terminally differentiated cells. We constructed differentiation trajectories for each cell lineage and identified the transcription factors expressed along these trajectories, thus creating a comprehensive molecular map of all developmental lineages in the adult animal. We unexpectedly found that neuron and gland cell differentiation transits through a common progenitor state, suggesting a shared evolutionary history for these secretory cell types. Finally, we have built the first gene expression map of the Hydra nervous system. By producing a comprehensive molecular description of the adult Hydra polyp, we have generated a resource for addressing fundamental questions regarding the evolution of developmental processes and nervous system function.

34: High throughput single cell RNA-seq of developing mouse kidney and human kidney organoids reveals a roadmap for recreating the kidney
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Posted to bioRxiv 16 Dec 2017

High throughput single cell RNA-seq of developing mouse kidney and human kidney organoids reveals a roadmap for recreating the kidney
2,494 downloads developmental biology

Alexander N Combes, Belinda Phipson, Luke Zappia, Kynan T Lawlor, Pei Xuan Er, Alicia Oshlack, Melissa H. Little

Recent advances in our capacity to differentiate human pluripotent stem cells to human kidney tissue are moving the field closer to novel approaches for renal replacement. Such protocols have relied upon our current understanding of the molecular basis of mammalian kidney morphogenesis. To date this has depended upon population based-profiling of non-homogenous cellular compartments. In order to improve our resolution of individual cell transcriptional profiles during kidney morphogenesis, we have performed 10x Chromium single cell RNA-seq on over 6000 cells from the E18.5 developing mouse kidney, as well as more than 7000 cells from human iPSC-derived kidney organoids. We identified 16 clusters of cells representing all major cell lineages in the E18.5 mouse kidney. The differentially expressed genes from individual murine clusters were then used to guide the classification of 16 cell clusters within human kidney organoids, revealing the presence of distinguishable stromal, endothelial, nephron, podocyte and nephron progenitor populations. Despite the congruence between developing mouse and human organoid, our analysis suggested limited nephron maturation and the presence of off target populations in human kidney organoids, including unidentified stromal populations and evidence of neural clusters. This may reflect unique human kidney populations, mixed cultures or aberrant differentiation in vitro. Analysis of clusters within the mouse data revealed novel insights into progenitor maintenance and cellular maturation in the major renal lineages and will serve as a roadmap to refine directed differentiation approaches in human iPSC-derived kidney organoids.

35: Single-cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development
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Posted to bioRxiv 07 Aug 2018

Single-cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development
2,474 downloads developmental biology

Thorsten Boroviak, Giuliano G Stirparo, Sabine Dietmann, Irene Hernando-Herraez, Hisham Mohammed, Wolf Reik, Austin Smith, Erika Sasaki, Jennifer Nichols, Paul Bertone

The mouse embryo is the canonical model for mammalian preimplantation development. Recent advances in single-cell profiling allow detailed analysis of embryogenesis in other eutherian species, including human, to distinguish conserved from divergent regulatory programs and signalling pathways in the rodent paradigm. Here, we identify and compare transcriptional features of human, marmoset and mouse embryos by single-cell RNA-seq. Zygotic genome activation correlates with the presence of Polycomb Repressive Complexes in all three species, while ribosome biogenesis emerges as a predominant attribute in primate embryos, supporting prolonged translation of maternally deposited RNAs. We find that transposable element expression signatures are species-, stage- and lineage-specific. The pluripotency network in the primate epiblast lacks certain regulators operative in mouse, but encompasses WNT components and genes associated with trophoblast specification. Sequential activation of GATA6, SOX17 and GATA4 markers of primitive endoderm identity is conserved in primates. Unexpectedly, OTX2 is also associated with primitive endoderm specification in human and nonhuman primate blastocysts. Our cross-species analysis demarcates both conserved and primate-specific features of preimplantation development and underscores the molecular adaptability of early mammalian embryogenesis.

36: A single cell transcriptional roadmap for cardiopharyngeal fate diversification
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Posted to bioRxiv 15 Jun 2017

A single cell transcriptional roadmap for cardiopharyngeal fate diversification
2,469 downloads developmental biology

Wei Wang, Xiang Niu, Tim Stuart, Estelle Jullian, William Mauck, Robert G. Kelly, Rahul Satija, Lionel Christiaen

In vertebrates, multipotent progenitors located in the pharyngeal mesoderm form cardiomyocytes and branchiomeric head muscles, but the dynamic gene expression programs and mechanisms underlying cardiopharyngeal multipotency and heart vs. head muscle fate choices remain elusive. Here, we used single cell genomics in the simple chordate model Ciona, to reconstruct developmental trajectories forming first and second heart lineages, and pharyngeal muscle precursors, and characterize the molecular underpinnings of cardiopharyngeal fate choices. We show that FGF-MAPK signaling maintains multipotency and promotes the pharyngeal muscle fate, whereas signal termination permits the deployment of a pan-cardiac program, shared by the first and second lineages, to define heart identity. In the second heart lineage, a Tbx1/10-Dach pathway actively suppresses the first heart lineage program, conditioning later cell diversity in the beating heart. Finally, cross-species comparisons between Ciona and the mouse evoke the deep evolutionary origins of cardiopharyngeal networks in chordates.

37: 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
2,455 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.

38: A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation
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Posted to bioRxiv 21 Aug 2019

A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation
2,431 downloads developmental biology

Celia Alda-Catalinas, Danila Bredikhin, Irene Hernando-Herraez, Oana Kubinyecz, Fátima Santos, Mélanie A. Eckersley-Maslin, Oliver Stegle, Wolf Reik

Zygotic genome activation (ZGA) is a crucial developmental milestone that remains poorly understood. This first essential transcriptional event in embryonic development coincides with extensive epigenetic reprogramming processes and is orchestrated, in part, by the interplay of transcriptional and epigenetic regulators. Here, we developed a novel high-throughput screening method that combines pooled CRISPR-activation (CRISPRa) with single-cell transcriptomics to systematically probe candidate regulators of ZGA. We screened 230 epigenetic and transcriptional regulators by upregulating their expression with CRISPRa in mouse embryonic stem cells (ESCs). Through single-cell RNA-sequencing (scRNA-seq) of CRISPRa-perturbed cells, we generated approximately 200,000 single-cell transcriptomes, each transduced with a unique short-guide RNA (sgRNA) targeting a specific candidate gene promoter. Using integrative dimensionality reduction of the perturbation scRNA-seq profiles, we characterized molecular signatures of ZGA and uncovered 44 factors that promote a ZGA-like response in ESCs, both in the coding and non-coding transcriptome. Upon upregulation of these factors, including the DNA binding protein Dppa2, the chromatin remodeller Smarca5 and the transcription factor Patz1, ESCs adopt an early embryonic-like state. Supporting their roles as ZGA regulators, Dppa2 and Smarca5 knock-out ESCs lose expression of ZGA genes, however, overexpression of Dppa2 in Smarca5 knock-out ESCs, but not vice versa, rescues ZGA-like expression, suggesting that Smarca5 regulates ZGA upstream and via Dppa2. Together, our single-cell transcriptomic profiling of CRISPRa-perturbed cells provides comprehensive system-level insights into the molecular mechanisms that orchestrate ZGA.

39: Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos
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Posted to bioRxiv 05 Jun 2020

Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos
2,429 downloads developmental biology

Gregorio Alanis-Lobato, Jasmin Zohren, Afshan McCarthy, Norah M.E. Fogarty, Nada Kubikova, Emily Hardman, Maria Greco, Dagan Wells, James MA Turner, Kathy K. Niakan

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germline. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and Cas9-only control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss-of-heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in approximately 22% of the human embryo cells analysed and spanned 4 to 20kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology. ### Competing Interest Statement The authors have declared no competing interest.

40: 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
2,414 downloads developmental biology

Javier Frias-Aldeguer, Maarten Kip, Judith Vivié, 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.

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