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Results for “Developmental Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 289 records · Page 16Linked to original sources

bioRxiv2017

Gastruloids develop the three body axes in the absence of extraembryonic tissues and spatially localised signalling

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/{beta}-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/{beta}-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.\n\nOne sentence summaryCulture of aggregates of defined number of Embryonic Stem cells leads to self-organised embryo-like structures which, in the absence of localised signalling from extra embryonic tissues and under the autonomous influence of Wnt and Nodal signalling, develop the three main axes of the body.

developmental biology↗

bioRxiv2017

Morphogen signalling patterns calcium waves in the Drosophila wing disc

Organ development is driven by a set of patterned inductive signals. However, how these signals are integrated to coordinate tissue patterning is still poorly understood. Calcium ions (Ca2+) are critical signaling components involved in signal integration and are regulated by a core Ca2+ signaling toolkit. Ca2+ signaling encodes a significant fraction of information in cells through both amplitude and frequency-dependent regulation of transcription factors and key regulatory enzymes. A range of intercellular Ca2+ transients, including coordinated oscillations, recently have been reported in Drosophila wing discs. In an accompanying paper, we show that impaired Ca2+ signaling impacts the final size and shape of the wing. Here, we discover specific spatiotemporal signatures of Ca2+ transients during wing disc development. To do so, we developed a new neural-network-based approach for registration of oscillatory signals in organs that frequently move during imaging, and a pipeline for spatiotemporal analysis of intercellular Ca2+ oscillations. As a specific test case, we further demonstrated that the morphogen pathway, Hedgehog, controls frequencies of Ca2+ oscillations uniformly in the tissue and is required for spatial patterning of oscillation amplitudes. Thus, the time-averaged dynamics of spontaneous intercellular Ca2+ transients reflect the morphogenetic signaling state of the tissue during development. This suggests a general mechanism of physiological signaling that provides a memory of morphogenetic patterns. Additionally, our study provides a powerful approach for registering and quantifying oscillatory dynamics in developing organs.

developmental biology↗

bioRxiv2017

BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells (MSCs)

Signaling pathways are used reiteratively in different developmental processes yet produce distinct cell fates through activating specific downstream transcription factors. In this study, we used tooth root development as a model to investigate how the BMP signaling pathway regulates specific downstream transcriptional complexes to direct the fate determination of multipotent mesenchymal stem cells (MSCs). We first identified the MSC population supporting mouse molar root growth as Gli1+ cells. Using a Gli1-mediated transgenic animal model, our results provide the first in vivo evidence that BMP signaling activity is required for the odontogenic differentiation of MSCs. Specifically, we identified transcription factors that are downstream of BMP signaling and are expressed in a spatially restricted pattern consistent with their potential involvement in determining distinct cellular identities within the dental mesenchyme. Finally, we found that overactivation of one key transcription factor, Klf4, associated with the odontogenic region, promotes odontogenic differentiation of MSCs. Collectively, our results demonstrate the functional significance of BMP signaling in regulating the fate of MSCs during root development and shed light on how BMP signaling can achieve functional specificity in regulating diverse organ development.\n\nSummary StatementBMP signaling activity is required for the lineage commitment of MSCs and transcription factors downstream of BMP signaling may determine distinct cellular identities within the dental mesenchyme.

developmental biology↗

bioRxiv2017

The depletion of MARVELD1 leads to placenta accreta via integrin β4-dependent trophoblast cell invasion

AstractThe mammalian placenta is a remarkable organ. It serves as the interface between the mother and the fetus. Proper invasion of trophoblast cells into the maternal decidua is required for a successful pregnancy. Previous studies have found that the adhesion molecule integrin {beta}4 plays important roles during trophoblast cell invasion. Here, we found that the overall birth rate of the MARVELD1 knockout mouse is much lower than that of the wild-type mouse (P<0.001). In E18.5 MARVELD1 knockout mice, we observed an over-invasion of trophoblast cells, and indeed, the pregnant mice had a partial placenta accreta phenotype. The HTR8/SVneo cell line was used as an in vitro model to elucidate the underlying mechanisms of MARVELD1-mediated trophoblast invasion. We detected a diminished expression of integrin {beta}4 upon the downregulation of MARVELD1 and enhanced migration and invasive abilities of trophoblast cells both in vivo and in vitro. The integrin {beta}4 rescue assay also supported the results. In conclusion, this study found that MARVELD1 mediated the invasion of trophoblast cells via regulating the expression of integrin {beta}4.

developmental biology↗

bioRxiv2017

A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila

Investigating the role of protein localization is crucial to understand protein function in cells or tissues. However, in many cases the role of different subcellular fractions of given proteins along the apical-basal axis of polarized cells has not been investigated in vivo, partially due to lack of suitable tools. Here, we present the GrabFP system, a nanobody-based toolbox to modify the localization and the dispersal of GFP-tagged proteins along the apical-basal axis of polarized cells. We show that the GrabFP system is an effective and easy-to-implement tool to mislocalize cytosolic and transmembrane GFP-tagged proteins and thereby functionally investigate protein localization along the apical-basal axis. We use the GrabFP system as a tool to study the extracellular dispersal of the Decapentaplegic (Dpp) protein and show that the Dpp gradient forming in the lateral plane of the Drosophila wing disc epithelium is essential for patterning of the wing imaginal disc.

developmental biology↗

bioRxiv2017

Viscoelastic dissipation stabilizes cell shape changes during tissue morphogenesis

Tissue morphogenesis relies on the production of active cellular forces. Understanding how such forces are mechanically converted into cell shape changes is essential to our understanding of morphogenesis. Here we use Myosin II pulsatile activity during Drosophila embryogenesis to study how transient forces generate irreversible cell shape changes. Analyzing the dynamics of junction shortening and elongation resulting from Myosin II pulses, we find that long pulses yield less reversible deformations, typically a signature of dissipative mechanics. This is consistent with a simple viscoelastic description, which we use to model individual shortening and elongation events. The model predicts that dissipation typically occurs on the minute timescale, a timescale commensurate with that of force generation by Myosin II pulses. We test this estimate by applying time-controlled forces on junctions with optical tweezers. Our results argue that active junctional deformation is stabilized by dissipation. Hence, tissue morphogenesis requires coordination between force generation and dissipation.

developmental biology↗

bioRxiv2017

A high-resolution mRNA expression time course of embryonic development in zebrafish

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1-cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3' end transcript counting method we characterise the temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5,024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. The data reveal complex and widespread differential use of exons and previously unidentified 3' ends across development, new primary microRNA transcripts and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data are accessible to browse and download at Expression Atlas and Ensembl.

developmental biology↗

bioRxiv2017

SUPERMAN prevents stamen formation and promotes stem cell termination in the fourth whorl of the Arabidopsis flower

The molecular and genetic networks underlying the determination of floral organ identity are well studied, but much less is known about how the flower is partitioned into four developmentally distinct whorls. The SUPERMAN gene is required for proper specification of the boundary between stamens in whorl 3 and carpels in whorl 4, as superman mutants exhibit supernumerary stamens but usually lack carpels. However, it has remained unclear whether extra stamens in superman mutants originate from an organ identity change in whorl 4 or the overproliferation of whorl 3. Using live confocal imaging, we show that the extra stamens in superman mutants arise from cells in whorl 4, which change their fate from female to male, while floral stem cells proliferate longer, allowing for the production of additional stamens.

developmental biology↗

bioRxiv2017

Bursting on a two states stochastic model for gene transcription in Drosophila embryos

Recent experimental data on the transcription dynamics of eve gene stripe two formation of Drosophila melanogaster embryos occurs in bursts of multiple sizes and durations. That has motivated the proposition of a transcription model having multiple ON states for the promoter of the eve gene each of them characterized by different synthesis rate. To understand the role of multiple ON states on gene transcription we approach the exact solutions for a two state stochastic model for gene transcription in D. melanogaster embryos and derive its bursting limit. Simulations based on the Gillespie algorithm at the bursting limit show the occurrence of bursts of multiple sizes and durations. Based on our theoretical approach, we interpret the aforementioned experimental data as a demonstration of the intrinsic stochasticity of the transcriptional processes in fruit fly embryos. Then, we conceive the experimental arrangement to determine when gene transcription has multiple ON promoter state in a noisy environment.

developmental biology↗

bioRxiv2017

Novel methods of isolation and amplification of progenitor cells applied to avian primordial germ cells

BackgroundThe progenitor cells in adult tissues are scarce and have a great regenerative potential. In this study novel methods were used to improve the isolation and culture of the chicken primordial germ cells (PGCs) from stage X and HH 8-9 embryos. The cellular size and external glycoprotein envelope were the two criteria studied and used.\n\nResultsPGCs were segregated with high efficiency and purity, from stage X and HH 8-9 gross cell suspensions through cell strainers with 10 m of pore size. In embryos in toto, WGA Alexa 594 (affinity for N-acetylglucosamine) and Con A Alexa 488 (binding D-mannosyl) were used to characterize external polysaccharides of the PGCs. The PGCs in stage X embryos (zone pellucida), have predominately Nacetylglucosamine and later on, in HH 8-9 embryos (cephalic zone), -D mannosyl residues, in a specific manner. In coated plates with the appropriate lectin and in alkaline conditions, isolated cells from stage X and HH 8-9 embryos formed numerous clumped PGC-LCs with spherical shape \"germspheres\". In all isolates from single embryo, immunohistochemistry confirmed that they were PGCs and revealed that the \"germspheres\" were formed by hundreds of positive cells to VASA and SSEA-1. N-acethyl D+glucosamine supplementation to the culture media greatly enhances the amplification of isolated PGC-LCs.\n\nConclusionsThese gentle and quick strategies with high yields of PGCs can be potentially useful for many progenitor cells in Regenerative Medicine.

developmental biology↗

bioRxiv2017

Synthetic DNA templates for the production of in situ hybridization probes

Generating RNA riboprobes for in situ hybridization generally requires the use of plasmids, which must be grown in bacteria, isolated, purified, and linearized prior to in vitro transcription. Here we report a simple method for generating DNA templates for the in vitro transcription of RNA probes from synthetic DNA (IDT gBlocks). Each synthetic DNA template contains sequences corresponding to the target mRNA flanked by bacteriophage promoters. Amplification of the template by a single round of PCR and subsequent in vitro transcription results in production of high quality RNA probes for in situ hybridization.

developmental biology↗

bioRxiv2017

Glycerophosphodiesterase GDE2 affects pancreas differentiation in zebrafish

Notch signaling plays an essential role in the proliferation, differentiation and cell fate determination of various tissues, including the developing pancreas. One regulator of the Notch pathway is GDE2 (or GDPD5), a transmembrane ecto-phosphodiesterase that cleaves GPI-anchored proteins at the plasma membrane, including a Notch ligand regulator. Here we report that Gde2 knockdown in zebrafish embryos leads to developmental defects, particularly, impaired motility and reduced pancreas differentiation, as shown by decreased expression of insulin and other pancreatic markers. Exogenous expression of human GDE2, but not catalytically dead GDE2, similarly leads to developmental defects. These data reveal functional conservation between zebrafish and human GDE2, and suggest that strict regulation of GDE2 expression and catalytic activity is critical for correct embryonic patterning. In particular, our data uncover a role for GDE2 in regulating pancreas differentiation.

developmental biology↗

bioRxiv2017

Integrated growth factor signaling promotes lung epithelial progenitor cell expansion and maintenance in mice and humans

The bud tip epithelium of the branching mouse and human lung contains multipotent progenitors that are able to self-renew and give rise to all mature lung epithelial cell types. The current study aimed to understand the developmental signaling cues that regulate bud tip progenitor cells in the human fetal lung, which are present during branching morphogenesis, and to use this information to induce a bud tip progenitor-like population from human pluripotent stem cells (hPSCs) in vitro. We identified that FGF7, CHIR-99021 and RA maintained isolated human fetal lung epithelial bud tip progenitor cells in an undifferentiated state in vitro, and led to the induction of a 3-dimensional lung-like epithelium from hPSCs. 3-dimensional hPSC-derived lung tissue was initially patterned, with airway-like interior domains and bud tip-like progenitor domains at the periphery. Epithelial bud tip-like domains could be isolated, expanded and maintained as a nearly homogeneous population by serial passaging. Comparisons between human fetal lung epithelial bud tip cells and hPSC-derived bud tip-like cells were carried out using immunostaining, in situ hybridization and transcriptome-wide analysis, and revealed that in vitro derived tissue was highly similar to native lung. hPSC-derived epithelial bud tip-like structures survived in vitro for over 16 weeks, could be easily frozen and thawed and maintained multi-lineage potential. Furthermore, hPSC-derived epithelial bud tip progenitors successfully engrafted in the proximal airways of injured immunocompromised NSG mouse lungs, where they persisted for up to 6 weeks and gave rise to several lung epithelial lineages.

developmental biology↗

bioRxiv2017

Assessment of asymmetric cell divisions in the early development of Caenorhabditis elegans

Asymmetric cell divisions are of fundamental importance for developmental processes, e.g. for the generation of founder cells. Prime examples are asymmetric cell divisions in the P lineage during early embryogenesis of the model organism Caenorhabditis elegans. However, due to a lack of quantitative data it has remained unclear how frequent unequal daughter cell sizes emerge in the nematodes early embryogenesis, and whether these originate from sterical or biochemical cues. Using quantitative light-sheet microscopy, we have found that about 40% of all cell divisions in C. elegans until gastrulation generate daughter cells with significantly different volumes. Removing the embryos rigid eggshell revealed asymmetric divisions in somatic cells to be primarily induced by steric effects. Division asymmetries in the germline remained unaltered and were correctly reproduced by a model based on a cell-size independent, eccentric displacement of the metaphase plate. Our data suggest asymmetric cell divisions to be essential for establishing important cell-cell interactions that eventually fuel a successful embryogenesis.\n\nSummary statementAbout 40% of all cell divisions in early C. elegans embryogenesis are found to be asymmetric. A cell-size independent displacement of the mitotic spindle explains division asymmetries in the germline whereas the confining eggshell induces asymmetries of somatic cells.

developmental biology↗

bioRxiv2017

Can the location of a trophectoderm biopsy contribute to human blastocyst development ?

The influence of the location of a trophectoderm biopsy in human blastocysts on the development of those blastocysts has not yet been investigated. In our prospective study (n=92), our multivariate logistic regression analysis indicated that blastocoel development was influenced by the location of the trophectoderm biopsy (p=0.049) and by the type of human blastocyst used (fresh or thawed) (p=0.037), regardless of the patients age (p=0.507) and the number of days for the human blastocyst in the pretrophectoderm biopsy (p=0.239). Therefore, when a trophectoderm biopsy is close to the inner cell mass (ICM) in human blastocysts, it improves the progress of blastocoel development.\n\nClinical evidence suggests that the progress of blastocoel development is a predictor of clinical outcomes after single blastocyst transfer. Therefore, when the trophectoderm biopsy is done from near the ICM, improvement of clinical outcomes after single blastocyst transfer may be expected.

developmental biology↗

bioRxiv2017

Micromanagment of stem cell proliferation by the Drosophila testis stem cell niche

Niches have traditionally been characterized as signalling microenvironments that allow stem cells to maintain their fate. This definition implicitly assumes that the various niche signals are integrated towards a binary fate decision between stemness and differentiation. However, observations in multiple systems have demonstrated that stem cell properties such as proliferation and self renewal can be uncoupled at the level of niche signalling input, which is incompatible with this simplified view. We have studied the role of the transcriptional regulator Zfh1, a shared target of the Hedgehog and Jak/Stat niche signalling pathways, in the somatic stem cells of the Drosophila testis. We found that Zfh1 binds and downregulates salvador and kibra, two tumour suppressor genes of the Hippo/Wts/Yki pathway, thereby restricting Yki activation and proliferation to the Zfh1 positive stem cells. These observations provide an unbroken link from niche signal input to an individual aspect of stem cell behaviour that does not, at any step, involve a fate decision. We discuss the relevance of our observations and other reports in the literature for an overall concept of stemness and niche function.\n\nSummary statementWe demonstrate that the fly testis niche controls stem cell proliferation by repressing Hippo pathway genes independent of a binary cell fate decision between stemness and proliferation.

developmental biology↗

bioRxiv2017

The Pax9/Wnt pathway regulates secondary palate formation in mice

Clefts of the palate and/or lip arise in about 1/700 human live births and are caused by multiple genetic and environmental factors. Studies of mouse knockout models of cleft palate have improved our understanding of the molecular control of palatogenesis. While it is known that Pax9 regulates palatogenesis through Bmp, Fgf and Shh signaling, there is still much to learn about its precise relationship with other pathways. Here we show that alterations of Wnt expression and decreased Wnt activity in Pax9-/- palatal shelves are a result of Pax9s ability to directly bind and repress the promoters of Dkk1 and Dkk2, proteins that antagonize Wnt signaling. The delivery of small-molecule Dkk inhibitors (Wnt agonists) into the tail-veins of pregnant Pax9+/- mice from E10.5 to E14.5 restored Wnt signaling, promoted cell proliferation, bone formation and restored the fusion of palatal shelves in Pax9-/- embryos. In contrast, other organ defects in Pax9 mutants were not corrected. These data uncover a unique molecular relationship between Pax9 and Wnt genes in palatogenesis and offer a new approach for treating cleft palates in humans.\n\nSummary StatementThese studies demonstrate that the Pax9/Wnt genes regulate murine palatogenesis. This unique molecular relationship is proven by the correction of cleft defects in Pax9-deficient mice through Wnt agonist therapies.

developmental biology↗

bioRxiv2017

Decoding temporal interpretation of the morphogen Bicoid in the early Drosophila embryo

Morphogen gradients provide essential spatial information during development. Not only the local concentration but also duration of morphogen exposure is critical for correct cell fate decisions. Yet, how and when cells temporally integrate signals from a morphogen remains unclear. Here, we use optogenetic manipulation to switch off Bicoid-dependent transcription in the early Drosophila embryo with high temporal resolution, allowing time-specific and reversible manipulation of morphogen signalling. We find that Bicoid transcriptional activity is dispensable for embryonic viability in the first hour after fertilization, but persistently required throughout the rest of the blastoderm stage. Short interruptions of Bicoid activity alter the most anterior cell fate decisions, while prolonged inactivation expands patterning defects from anterior to posterior. Such anterior susceptibility correlates with high reliance of anterior gap gene expression on Bicoid. Therefore, cell fates exposed to higher Bicoid concentration require input for longer duration, demonstrating a previously unknown aspect of morphogen decoding.

developmental biology↗