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in category developmental biology
1,976 results found. For more information, click each entry to expand.
1,411 downloads developmental biology
In plants the dorsoventral boundary of leaves defines an axis of symmetry through the centre of the organ separating the top (dorsal) and bottom (ventral) tissues. Although the positioning of this boundary is critical for leaf morphogenesis, how the boundary is established and how it influences development remains unclear. Using live-imaging and perturbation experiments we show that leaf orientation, morphology and position are pre-patterned by HD-ZIPIII and KAN gene expression in the shoot, leading to a model in which dorsoventral genes coordinate to regulate plant development by localizing auxin response between their expression domains. However we also find that auxin levels feedback on dorsoventral patterning by spatially organizing HD-ZIPIII and KAN expression in the shoot periphery. By demonstrating that the regulation of these genes by auxin also governs their response to wounds, our results also provide a parsimonious explanation for the influence of wounds on leaf dorsoventrality.
1,410 downloads developmental biology
Is the human early embryo unique in lacking an inner cell mass (ICM) and having parallel rather than step-wise development? Here we reanalyse single-cell transcriptomic data and stain human embryos in situ to reveal both classical step-wise development and the missing ICM, a transcriptomic homolog of macaque ICM, that differentiates to epiblast and primitive endoderm. This apparent classicism obscures numerous features that render our blastocyst phylogenetically distinct: unlike mice, human epiblast has hallmarks of self-renewal and we have abundant, previously unrecognized, blastocyst non-committed cells (NCCs), part of an apoptosis-mediated quality control/purging process. Comparative transcriptomics further reveals the transcriptomes of the pluripotent cells to be especially fast evolving, rendering all primate embryos unique. Rapid transcriptome turnover is in large part owing to endogenous retrovirus H (ERVH) activity, ERVH being associated with recent major gene expression gain and loss events of pluripotency-associated genes. Each species is characterised by the ERVHs that are active and the neighbour genes whose expression are in turn modulated. The current portfolio of naive cultures, putative in vitro mimics of pluripotent cells, are both developmentally and phylogenetically "confused" in part owing to a lack of HERVH expression.
1,401 downloads developmental biology
Kidney organoids generated from human pluripotent stem cells have the potential to revolutionize how kidney development and injury are studied. Current protocols are technically complex and suffer from poor reproducibility and high reagent costs restricting scalability. To overcome these issues, we have established a simple, inexpensive and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day (d) 8 and show optimal tissue morphology at d14. A comparison with fetal human kidney suggests that d14 organoid renal structures most closely resemble capillary loop stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant human renal development.
1,398 downloads developmental biology
Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to the significant reduction of signal intensities. To overcome this limitation, we enhanced the translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.
1,398 downloads developmental biology
During embryonic development, cells undertake a series of cell fate decisions to form a complete organism, epitomising a branching process. In some instances, these decisions are reversible, particularly during the onset of disease. Single cell transcriptomics provides a rich resource to explore the temporal progression of bifurcations in gene activity and are therefore useful for elucidating the mechanisms of cell fate decisions, provided that the cells can be suitably ordered over a developmental axis. Most methods for inferring this ordering have been developed for heterogeneous populations of cells collected at single time points, with few approaches specifically designed with structured data in mind, such as single cell time-series data. Recent advances based on Gaussian process latent variable models (GPLVMs) address this by allowing the incorporation of prior information, such as capture time, to yield more accurate ordering of cells. However, such approaches do not allow the ordering of cells over developmental processes with more than one branch. Here we develop a pseudotime approach that allows the ordering of cells over developmental trajectories with arbitrary numbers of branches, in the form of branch-recombinant Gaussian process latent variable models (B-RGPLVM). We use first demonstrate the advantage of our approach compared to existing pseudotime algorithms. Subsequently, we use B-RGPLVMs to infer cell ordering that occurs during early human development as primordial germ cells (PGCs), the precursors of sperm and egg, and somatic cells diverge in the developing embryo. Using our approach, we identify known master regulators of human PGC development, and predict roles for a variety of signalling pathways, as well as transcription factors and epigenetic modifiers. By concentrating on the earliest branched signalling events, we identified an antagonistic role for FGF receptor (FGFR) signalling pathway in the acquisition of competence for human PGC fate. We experimentally validate our predictions using pharmacological blocking of FGFR or its downstream effectors (MEK, PI3K and JAK), and demonstrate enhanced competency for PGC fate in vitro. Thus, B-RGPLVMs represent a powerful and flexible data-driven approach for dissecting the temporal dynamics of cell fate decisions, providing unique insights into the mechanisms of early embryogenesis.
1,393 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.
1,389 downloads developmental biology
Understanding male fertility requires an in-depth characterisation of spermatogenesis, the developmental process by which male gametes are generated. Spermatogenesis occurs continuously throughout a male's reproductive window and involves a complex sequence of developmental steps, both of which make this process difficult to decipher at the molecular level. To overcome this, we transcriptionally profiled single cells from multiple distinct stages during the first wave of spermatogenesis, where the most mature germ cell type is known. This naturally enriches for spermatogonia and somatic cell types present at very low frequencies in adult testes. Our atlas, available as a shiny app (https://marionilab.cruk.cam.ac.uk/SpermatoShiny), allowed us to reconstruct the three main processes of spermatogenesis: spermatogonial differentiation, meiosis, and spermiogenesis. Additionally, we profiled the chromatin changes associated with meiotic silencing of the X chromosome, revealing a set of genes specifically and strongly repressed by H3K9me3 in the spermatocyte stage, but which escape post-meiotic silencing in spermatids.
1,381 downloads developmental biology
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.
1,375 downloads developmental biology
The asymmetric distribution of cytoplasmic components by mRNA localization is critical for eukaryotic cells and affects large numbers of transcripts. How such global subcellular localization of mRNAs is regulated is still unknown. We combined transcriptomics and systematic imaging to determine tissue-specific expression and subcellular localizations of 5862 mRNAs during Drosophila oogenesis. While the transcriptome is stable and alternative splicing and polyadenylation is rare, cytoplasmic localization of mRNAs is widespread. Localized mRNAs have distinct gene features and diverge in expression level, 3'UTR length and sequence conservation. We show that intracellular localization of mRNAs depends on an intact microtubule cytoskeleton and that specifically the posterior enrichment requires the localization of oskar mRNA to the posterior cortex. Using cross-tissue comparison we revealed that the localization landscape differs substantially between epithelial, germline and embryonic cells and the localization status of mRNAs also changes considerably within the oocyte over the course of oogenesis.
1,344 downloads developmental biology
The early mammalian conceptus (blastocyst) contains two supporting extraembryonic tissues - the trophectoderm and the primitive endoderm (PrE) - that encase and guide the epiblast (Epi) to eventually form the all body. Modifications of the conceptus exposed key genes regulating these tissues co-development. However, the combinations of signalling pathways underlying the interplay of PrE and Epi remains elusive. Stem cell-based models including embryoid bodies and blastoids can be generated in large numbers and subjected to high-content screens. Here, we use combinatorial screens of proteins, GPCR ligands and small molecules to rapidly (72 hours) and efficiently (80%) guide embryoid bodies to form a three-dimensional PrE-/Epiblast-like niche in chemically-defined conditions (gel-free, serum-free). This bipotent niche spontaneously progresses, without growth factors, to form a pro-amniotic cavity surrounded by a polarized Epi covered with parietal and visceral endoderm-like cells. In blastoids, these molecules enhance the ratio and number of Gata6+/Nanog+ cells and promote the survival, expansion and morphogenesis of a post-implantation-like Epi in vitro . Altogether, modelling early development in chemically-defined conditions delineates the pathways sufficient to form a functional PrE/Epiblast niche that fuels post-implantation development.
1,342 downloads developmental biology
Tissues undergoing morphogenesis impose mechanical effects on one another. How developmental programs adapt to or take advantage of these effects remains poorly explored. Here, using a combination of live imaging, modeling, and microsurgical perturbations, we show that the axial and paraxial tissues in the forming avian embryonic body coordinate their rates of elongation through mechanical interactions. First, a cell motility gradient drives paraxial presomitic mesoderm (PSM) expansion, resulting in compression of the axial neural tube and notochord; second, elongation of axial tissues driven by PSM compression and polarized cell intercalation pushes the caudal progenitor domain posteriorly; finally, the axial push drives progenitors to emigrate into the PSM to maintain tissue growth and cell motility. These interactions form an engine-like positive feedback loop, which ensures the tissue-coupling and self-sustaining characteristics of body elongation. Our results suggest a general role of inter-tissue forces in the coordination of complex morphogenesis involving distinct tissues.
1,338 downloads developmental biology
Intracellular transcriptional regulators and extracellular signaling pathways together regulate the allocation of cell fates during development, but how their molecular activities are integrated to establish the correct proportions of cells with particular fates is not known. Here we study this question in the context of the decision between the epiblast (Epi) and the primitive endoderm (PrE) fate that occurs in the mammalian preimplantation embryo. Using an embryonic stem (ES) cell model, we discover two successive functions of FGF/MAPK signaling in this decision. First, the pathway needs to be inhibited to make the PrE-like gene expression program accessible for activation by GATA transcription factors in ES cells. In a second step, MAPK signaling levels determine the threshold concentration of GATA factors required for PrE-like differentiation, and thereby control the proportion of cells differentiating along this lineage. Our findings can be explained by a simple mutual repression circuit modulated by FGF/MAPK signaling. This may be a general network architecture to integrate the activity of signal transduction pathways and transcriptional regulators, and serve to balance proportions of cell fates in several contexts.
1,334 downloads developmental biology
The Caudal Lateral Epiblast of mammalian embryos harbours bipotent progenitors that contribute to the spinal cord and the paraxial mesoderm in concert with the elongation of the body axis. These progenitors, called Neural Mesodermal Progenitors (NMPs) are identified as cells coexpressing Sox2 and T/Brachyury, a criterion used to derive NMP-like cells from Embryonic Stem Cells in vitro. However, these progenitors do not self renew, as embryonic NMPs do. Here we find that protocols that yield NMP-like cells in vitro first produce a multipotent population that, additional to NMPs, generates progenitors for the lateral plate and intermediate mesoderm. We show that Epiblast Stem Cells (EpiSCs) are an effective source for these multipotent progenitors that are further differentiated by a balance between BMP and Nodal signalling. Importantly, we show that NMP-like cells derived from EpiSCs self renew in vitro and exhibit a gene expression signature similar to that of their embryo counterparts.
1,330 downloads developmental biology
How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here, we mutated an invertebrate orphan relaxin receptor, the Drosophila Lgr3, and found body asymmetries similar to those found in insulin/relaxin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNAi against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By fluorescently-tagging the endogenous Lgr3 protein and performing CNS-specific RNAi, we find that Lgr3 is expressed and required in a novel subset of CNS neurons to transmit the peripheral tissue stress signal, Dilp8, to the neuroendocrine centers controlling developmental timing. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.
1,315 downloads developmental biology
Early mammalian development entails a series of cell fate transitions that includes transit through naive pluripotency to post-implantation epiblast. This subsequently gives rise to primordial germ cells (PGC), the founding population of the germline lineage. To investigate the gene regulatory networks that control these critical cell fate decisions, we developed a compound-reporter system to track cellular identity in a model of PGC specification (PGC-like cells; PGCLC), and coupled it with unbiased genome-wide CRISPR screening. This enabled identification of key genes both for exit from pluripotency and for acquisition of PGC fate, with further characterisation revealing a central role for the transcription factors Nr5a2 and Zfp296 in germline ontogeny. Abrogation of these genes results in significantly impaired PGCLC development due to widespread activation (Nr5a2-/-) or inhibition (Zfp296-/-) of WNT pathway components. This leads to aberrant upregulation of the somatic programme or failure to appropriately activate germline genes in PGCLC, respectively, and consequently loss of germ cell identity. Overall our study places Zfp296 and Nr5a2 as key components of an expanded PGC gene regulatory network, and outlines a transferable strategy for identifying critical regulators of complex cell fate transitions.
1,313 downloads developmental biology
Tissue morphogenesis emerges from coordinated cell shape changes driven by actomyosin contractions. Patterns of gene expression regionalize and polarize cell behaviours by controlling actomyosin contractility. Yet how mechanical feedbacks affect tissue morphogenesis is unclear. We report two modes of control over Rho1 and MyosinII activation in the Drosophila endoderm. First, Rho1/MyoII are induced in a primordium via localized transcription of the GPCR ligand Fog. Second, a tissue-scale wave of Rho1/MyoII activation and cell invagination progresses anteriorly. The wave does not require sustained gene transcription, and is not governed by regulated Fog delivery. Instead, MyoII inhibition blocked acute Rho1 activation and propagation, revealing a mechanical feedback driven by MyoII. Last, we identify a cycle of 3D cell deformations whereby MyoII activation and invagination in each row of cells drives adhesion to the vitelline membrane, apical spreading, MyoII activation and invagination in the next row. Thus endoderm morphogenesis emerges from local transcriptional initiation and a mechanically driven wave of cell deformation.
1,311 downloads developmental biology
In Drosophila larvae, growth and developmental timing are regulated by nutrition in a tightly coordinated fashion. The networks that couple these processes are far from understood. Here, we show that the intestine responds to nutrient availability by regulating production of a circulating lipoprotein-associated form of the signaling protein Hedgehog (Hh). Levels of circulating Hh tune the rates of growth and developmental timing in a coordinated fashion. Circulating Hh signals to the fat body to control larval growth. It regulates developmental timing by controlling ecdysteroid production in the prothoracic gland. Circulating Hh is especially important during starvation, when it is also required for mobilization of fat body triacylglycerol (TAG) stores. Thus, we demonstrate that Hh, previously known only for its local morphogenetic functions, also acts as a lipoprotein-associated endocrine hormone, coordinating the response of multiple tissues to nutrient availability.
1,308 downloads developmental biology
Gene expression studies have typically focused on finding differentially expressed genes or pathways between two or more conditions. More recently, single-cell RNA-seq has been established as a reliable and accessible technique enabling new types of analyses, such as the study of gene expression variation within cell types from cell lines or from relatively similar cells in tissues, organs or tumors. However, although single-cell RNA-seq provides quantitative and comprehensive expression data in a developing embryo, it is not yet clear whether this can replace conventional in situ screens for finding developmentally important genes; moreover, current single-cell data analysis approaches typically cluster cells into types based on a common set of genes or identify more variable or differentially expressed genes using predefined groups of cells, limiting their use for finding genes with novel expression patterns. Here we present a method that comprehensively finds cell-specific patterns of developmentally important regulators directly from single-cell gene expression data of the Ciona embryo, a marine chordate. We recover many of the known expression patterns directly from our single-cell RNA-seq data and despite extensive previous screens, we succeed in finding new cell-specific patterns and genes, which we validate by in situ and single-cell qPCR.
1,300 downloads developmental biology
Planarian flatworms have an indefinite capacity to regenerate missing or damaged body parts owing to a population of pluripotent adult stems cells called neoblasts (NBs). Currently, little is known about the importance of the epigenetic status of NBs and how histone modifications regulate homeostasis and cellular differentiation. We have developed an improved and optimized ChIP-seq protocol for NBs in Schmidtea mediterranea and have generated genome-wide profiles for the active marks H3K4me3 and H3K36me3, and suppressive marks H3K4me1 and H3K27me3. The genome-wide profiles of these marks were found to correlate well with NB gene expression profiles. We found that genes with little transcriptional activity in the NB compartment but which switch on in post-mitotic progeny during differentiation are bivalent, being marked by both H3K4me3 and H3K27me3 at promoter regions. In further support of this hypothesis bivalent genes also have a high level of paused RNA Polymerase II at the promoter-proximal region. Overall, this study confirms that epigenetic control is important for the maintenance of a NB transcriptional program and makes a case for bivalent promoters as a conserved feature of animal stem cells and not a vertebrate specific innovation. By establishing a robust ChIP-seq protocol and analysis methodology, we further promote planarians as a promising model system to investigate histone modification mediated regulation of stem cell function and differentiation.
1,292 downloads developmental biology
During gastrulation, the pluripotent epiblast is patterned into the three germ layers, which form the embryo proper. This patterning requires a signaling cascade involving the BMP, WNT and NODAL pathways; however, how these pathways regulate one another in space and time to generate cell-fate patterns remains unknown. Using a human gastruloid model, we show that BMP signaling initiates a wave of WNT signaling, which, in turn, initiates a wave of NODAL signaling. While WNT propagation depends on continuous BMP activity, NODAL propagates independently of upstream signals. We further show that the duration of BMP signaling determines the position of mesodermal differentiation while WNT and NODAL synergize to achieve maximal differentiation. The waves of both WNT and NODAL signaling activity extend farther into the colony than mesodermal differentiation. Combining dynamic measurements of signaling activity with mathematical modeling revealed that the formation of signaling waves is inconsistent with WNT and NODAL forming a stable spatial pattern in signaling activities, and the final signaling state is spatially homogeneous. Thus, dynamic eventsin the BMP, WNT, and NODAL signaling cascade, in the absence of a signaling gradient, have the potential to mediate epiblast patterning.
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