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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

Spatio-temporal elasticity patterns in extracellular matrix during Hydra morphogenesis

Albeit ample evidence has suggested the remodeling of extracellular matrix (ECM) in animals plays crucial roles in development and diseases, little is understood how ECM mechanics correlates with tissue morphogenesis. In this study, we quantitatively determined how spatio-temporal elasticity patterns in ECM change during the asexual reproduction of freshwater polyp Hydra. We first determined the mesoscopic protein arrangement in Hydra ECM (mesoglea) by grazing-incidence small-angle X-ray scattering with nano-beam (nano-GISAXS). Our data unraveled fibrillar type I collagen in Hydra mesoglea (Hcol-I) takes an anisotropic, more strongly distorted hexagonal lattice compared to those in vertebrates that could be attributed to the lower proline content and lack of lysin-crosslinks in Hcol-1 fibers. Then, we "mapped" the spatio-temporal changes in ECM stiffness ex vivo with aid of nano-indentation. We identified three representative elasticity patterns during tissue growth along the oral-aboral body axis of the animals. Our complementary proteome analysis demonstrated that the elasticity patterns of the ECM correlate with a gradient like distribution of proteases. Perturbations of the oral Wnt/{beta}-catenin signaling center further indicated that ECM elasticity patterns are governed by Wnt/{beta}-catenin signaling. The ex vivo biomechanical phenotyping of Hydra mesoglea established in this study will help us gain comprehensive insights into the spatio-temporal coordination of biochemical and biomechanical cues in tissue morphogenesis in vivo.

developmental biology↗

bioRxiv2017

Minimum requirements for reprogramming and maintaining cell fate in the Arabidopsis root

Changes in gene regulation during differentiation are governed by networks of transcription factors. To identify the minimal network for endodermal differentiation in the Arabidopsis root, we asked what transcription factors are sufficient to program a non-native cell-type into endodermis. Our results show the transcription factors SHORTROOT and MYB36 have limited ability to reprogram a non-native cell-type (the epidermis) and that this reprogramming is reversible in the absence of additional cues. The stele-derived signaling peptide CIF2 stabilizes SHORTROOT-induced reprogramming. The outcome is a partially impermeable barrier deposited in the sub-epidermal cell layer that has a transcriptional signature similar to endodermis. The trans-differentiation mechanism depends on the expression of genes downstream in the gene regulatory network but is independent of SHORTROOT movement. These results highlight a non cell-autonomous induction mechanism for endodermis that resembles differentiation in many animal systems.\n\nOne Sentence SummarySHORTROOT and CIF2 combined can induce and stabilize an endodermis in sub-epidermal cells and do so in a non cell-autonomous manner.

developmental biology↗

bioRxiv2017

PI(4,5)P2 forms dynamic cortical structures and directs actin distribution and cell polarity in C. elegans embryos

Asymmetric division is crucial for embryonic development and stem cell lineages. In the one-cell C. elegans embryo, a contractile cortical actomyosin network contributes to anterior-posterior (A-P) polarity and asymmetric division by segregating PAR proteins to discrete cortical domains. Here, we discovered that the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms dynamic structures in C. elegans zygotes, distributing in a polarized and PAR-dependent manner along the A-P axis. PIP2 cortical structures overlap with F-actin and coincide with the actin regulators RHO-1, CDC-42 and ECT-2. Particle image velocimetry analysis revealed that PIP2 and F-actin cortical movements are coupled, with PIP2 structures moving slightly ahead. Importantly, we established that PIP2 cortical structures form in an actin-dependent manner and, conversely, that decreasing or increasing the level of PIP2 results in severe F-actin disorganization, revealing the interdependence between these components. Furthermore, we uncovered that PIP2 regulates the sizing of PAR cortical domains. Overall, our work establishes for the first time that a lipid membrane component, PIP2, is a critical modulator of actin organization and cell polarity in C. elegans embryos.\n\nSummary statementPI(4,5)P2 is distributed in dynamic cortical structures and regulates asymmetric division by controlling actin organization and cell polarity in the one-cell C. elegans embryo.

developmental biology↗

bioRxiv2017

Gene expression in cord blood links genetic risk for neurodevelopmental disorders with maternal psychological distress and adverse childhood outcomes

Prenatal exposure to maternal stress and depression has been identified as a risk factor for adverse behavioral and neurodevelopmental outcomes in early childhood. However, the molecular mechanisms through which maternal psychopathology shapes offspring development remain poorly understood. We applied transcriptome-wide screens to 149 umbilical cord blood samples from neonates born to mothers with posttraumatic stress disorder (PTSD; n=20), depression (n=31) and PTSD with comorbid depression (n=13), compared to carefully matched trauma exposed controls (n=23) and healthy mothers (n=62). Analyses by maternal diagnoses revealed a clear pattern of gene expression signatures distinguishing neonates born to mothers with a history of psychopathology from those without. Co-expression network analysis identified distinct gene expression perturbations across maternal diagnoses, including two depression-related modules implicated in axon-guidance and mRNA stability, as well as two PTSD-related modules implicated in TNF signaling and cellular response to stress. Notably, these disease-related modules were enriched with brain-expressed genes and genetic risk loci for autism spectrum disorder and schizophrenia, which may imply a causal role for impaired developmental outcomes. These molecular alterations preceded changes in clinical measures at twenty-four months, including reductions in cognitive and socio-emotional outcomes in affected infants. Collectively, these findings indicate that prenatal exposure to maternal psychological distress induces neuronal, immunological and behavioral abnormalities in affected offspring and support the search for early biomarkers of exposures to adverse in utero environments and the classification of children at risk for impaired development.

developmental biology↗

bioRxiv2017

Generation of Mouse-Zebrafish Hematopoietic Tissue Chimeric Embryos for Hematopoiesis and Host-Pathogen Interaction Studies

Xenografts of the hematopoietic system are extremely useful as disease models and for translational research. Zebrafish xenografts have been widely used to monitor blood cancer cell dissemination and homing due to the optical clarity of embryos and larvae, which allow unrestricted in vivo visualization of migratory events. To broaden the scope of xenotransplantation studies in zebrafish, we have developed a technique that transiently generates hematopoietic tissue chimeras by transplanting murine bone marrow cells into zebrafish blastulae. This procedure leads to mammalian cell integration into the fish developmental hematopoietic program. Monitoring zebrafish chimeras at different time points post fertilization using in vivo time-lapse and confocal imaging showed murine cell co-localization with developing primitive and definitive hematopoietic tissues, intravasation into fish circulation, and dynamic hematopoietic cell-vascular endothelial and hematopoietic cell-niche interactions. Immunohistochemistry assays performed in chimeric animals showed that, after engraftment, murine cells expressed antigens related to i) hematopoietic stem and progenitor cells, ii) active cell proliferation, and iii) myeloid cell lineages. Lastly, xenografted zebrafish larvae infected with Klebsiella pneumoniae showed murine immune cells trafficking to bacterial foci and interacting with bacterial cells. Overall, these results show that mammalian bone marrow cells xenografted in zebrafish integrate into the host hematopoietic system revealing highly conserved molecular mechanisms of hematopoiesis between zebrafish and mammals. In addition, this procedure introduces a useful and simple method that improves and broadens the scope of hematopoietic tissue xenotransplantation studies in zebrafish.

developmental biology↗

bioRxiv2017

EpiGraph: an open-source platform to quantify epithelial organization

During development, cells must coordinate their differentiation with their growth and organization to form complex multicellular structures such as tissues and organs. Healthy tissues must maintain these structures during homeostasis. Epithelia are packed ensembles of cells from which the different tissues of the organism will originate during embryogenesis. A large barrier to the analysis of the morphogenetic changes in epithelia is the lack of simple tools that enable the quantification of cell arrangements. Here we present EpiGraph, an image analysis tool that quantifies epithelial organization. Our method combines computational geometry and graph theory to measure the degree of order of any packed tissue. EpiGraph goes beyond the traditional polygon distribution analysis, capturing other organizational traits that improve the characterization of epithelia. EpiGraph can objectively compare the rearrangements of epithelial cells during development and homeostasis to quantify how the global ensemble is affected. Importantly, it has been implemented in the open-access platform FIJI. This makes EpiGraph very user friendly, with no programming skills required.

developmental biology↗

bioRxiv2017

Diversification of heart progenitor cells by EGF signaling and differential modulation of ETS protein activity

For coordinated circulation, vertebrate and invertebrate hearts require stereotyped arrangements of diverse cell populations. This study explores the process of cardiac cell diversification in the Drosophila heart, focusing on the two major cardioblast subpopulations: generic working myocardial cells and inflow valve-forming ostial cardioblasts. By screening a large collection of randomly induced mutants we identified several genes involved in cardiac patterning. Further analysis revealed an unexpected, specific requirement of EGF signaling for the specification of generic cardioblasts and a subset of pericardial cells. We demonstrate that the Tbx20 ortholog Midline acts as a direct target of the EGFR effector Pointed to repress ostial fates. Furthermore, we identified Edl/Mae, an antagonist of the ETS factor Pointed, as a novel cardiac regulator crucial for ostial cardioblast specification. Combining these findings we propose a regulatory model in which the balance between activation of Pointed and its inhibition by Edl controls cardioblast subtype-specific gene expression.

developmental biology↗

bioRxiv2017

High fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins

Accurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions of mammalian development. Lineage tracing using global labeling techniques is complicated by increasing cell density and rapid embryo rotation, impeding automatic alignment and rendering accurate cell tracking of obtained four-dimensional imaging data sets highly challenging. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of H2B-pr-mEosFP labeled nuclei combined with light-sheet imaging greatly facilitates segmentation, classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using green and red labels as fiducial markers, we computationally correct for rotational and translational drift and accomplish high fidelity lineage tracing combined with a reduced data size - addressing majors concerns in the field of volumetric embryo imaging.

developmental biology↗

bioRxiv2017

Cell-nonautonomous local and systemic responses to cell arrest enable long-bone catch-up growth

Catch-up growth after insults to growing organs is paramount to achieving robust body proportions. In fly larvae, local injury is followed by local and systemic compensatory mechanisms that allow damaged tissues to regain proportions with other tissues. In vertebrates, local catch-up growth has been described after transient reduction of bone growth, but the underlying cellular responses are controversial. We developed an approach to study catch-up growth in foetal mice by inducing mosaic expression of the cell cycle suppressor p21 in the cartilage cells (chondrocytes) that drive long bone elongation. By specifically targeting the left hindlimb, the right limb served as an internal control. Strikingly, left-right limb symmetry was not altered, revealing deployment of compensatory mechanisms. Above a certain threshold of insult, an orchestrated response was triggered involving local enhancement of bone growth and systemic growth reduction that ensured body proportions were maintained. The local response entailed hyper-proliferation of spared left-limb chondrocytes that was associated with reduced chondrocyte density. The systemic effect involved impaired placental IGF signalling and function, revealing bone-placenta communication. Thus, vertebrates, much like invertebrates, can mount coordinated local and systemic responses to developmental insults to ensure normal body proportions are maintained.

developmental biology↗

bioRxiv2017

Maternal proteins that are phospho-regulated upon egg activation include crucial factors for oogenesis, egg activation and embryogenesis in Drosophila melanogaster

Egg activation is essential for the successful transition from a mature oocyte to a developmentally competent egg. It consists of a series of events including the resumption and completion of meiosis, initiation of translation of some maternal mRNAs and destruction of others, and changes to the vitelline envelope. This drastic change of cell state is accompanied by large scale alteration of the phospho-proteome of the cell. Despite the importance of this transition in cell and developmental state, it has been difficult to find many of its regulators. We hypothesize that phosphorylation state changes between oocyte and early embryo regulate the activities of proteins that are necessary during or after this transition, and thus that the set of phospho-regulated proteins would be an enriched source for finding critical players in the egg-to-embryo transition. To test this, we used germline-specific RNAi to examine the function of 189 maternal proteins that are phospho-regulated during egg activation in Drosophila melanogaster. We identified 53 genes whose knockdown reduced or abolished egg production, as well as 50 genes for which maternal knockdown led to significant impairment or abolishment of the eggs ability to hatch (hatchability). We observed different stages of developmental arrest in the embryos with impaired hatchability and several distinct categories of abnormalities in the maternal knockdown embryos that arrest early in development, suggesting potential roles of the candidates in egg activation and early embryogenesis. Our results, validated by our detection of multiple genes with previously-documented maternal-effect phenotypes among the proteins we tested, revealed 15 genes with new roles in egg activation and early embryogenesis. Given that protein phospho-regulation also occurs during this transition in other organisms, we suggest that the phospho-regulated proteins may provide an enriched dataset for identifying important players in the egg-to-embryo transition.

developmental biology↗

bioRxiv2017

Hox genes pattern the primary body axis of an anthozoan cnidarian prior to gastrulation

Hox gene transcription factors are important regulators of positional identity along the anterior-posterior axis in bilaterian animals. Cnidarians (e.g. sea anemones, corals and hydroids) are the sister group to the Bilateria and possess genes related to both anterior and central/posterior class Hox genes. In the absence of a conserved set of Hox genes among other early branching animal clades, cnidarians provide the best opportunity to learn about the emergence of this gene family. We report a previously unrecognized domain of Hox expression in the starlet sea anemone, Nematostella vectensis, beginning at early blastula stages. Functional perturbation reveals that two Hox genes not only regulate their respective expression domains, but interact with one another to pattern the entire oral-aboral axis mediated by Wnt signaling. This suggests an ancient link between Hox/Wnt patterning of the oral-aboral axis and suggest that these domains are likely established during blastula formation in anthozoan cnidarians.

developmental biology↗

bioRxiv2017

Reorientation of the primary body axis by ectopic embryonic cWnt signaling.

Gastrulation is a crucial time during embryogenesis when cells make important decisions on what larval or adult tissues (i.e. ectodermal, mesodermal, or endodermal) are going to generate. The evolution of gastrulation was a pivotal event during metazoan evolution, as it paved the way for diversification of the metazoan clade from a hollow, ciliated, radially symmetrical ancestor (1, 2). The position of the site of gastrulation (that segregates internal endomesodermal precursors from outer ectodermal tissue) has played a role in our understanding patterns of body plan evolution (e.g. deuterostomes vs protostomes) and is tightly regulated during development. In bilaterians (a large clade of bilaterally symmetrical animals that represent over 99% of all extant species), the site of gastrulation is determined by a localized molecular asymmetry resulting from a differential distribution of maternal determinants (3) along the so-called an ...

developmental biology↗

bioRxiv2017

Hominin-specific NOTCH2 paralogs expand human cortical neurogenesis through regulation of Delta/Notch interactions.

The human cerebral cortex has undergone rapid expansion and increased complexity during recent evolution. Hominid-specific gene duplications represent a major driving force of evolution, but their impact on human brain evolution remains unclear. Using tailored RNA sequencing (RNAseq), we profiled the spatial and temporal expression of Hominid-specific duplicated (HS) genes in the human fetal cortex, leading to the identification of a repertoire of 36 HS genes displaying robust and dynamic patterns during cortical neurogenesis. Among these we focused on NOTCH2NL, previously uncharacterized HS paralogs of NOTCH2. NOTCH2NL promote the clonal expansion of human cortical progenitors by increasing self-renewal, ultimately leading to higher neuronal output. NOTCH2NL function by activating the Notch pathway, through inhibition of Delta/Notch interactions. Our study uncovers a large repertoire of recently evolved genes linking genomic evolution to human brain development, and reveals how hominin-specific NOTCH paralogs may have contributed to the expansion of the human cortex.

developmental biology↗

bioRxiv2017

Selective Filopodia Adhesion Ensures Robust Cell Matching in the Drosophila Heart

The ability to form specific cell-cell connections within complex cellular environments is critical for multicellular organisms. However, the underlying mechanisms of cell matching that instruct these connections remain elusive. Here, we explore the dynamic regulation of matching processes utilizing Drosophila cardiogenesis. During embryonic heart formation, cardioblasts (CBs) form precise contacts with their partners after long-range migration. We find that CB matching is highly robust at the boundaries between distinct CB subtypes. Filopodia in these CB subtypes have different binding affinities. We identify the adhesion molecules Fasciclin III (Fas3) and Ten-m as having complementary differential expression in CBs. Altering Fas3 expression influences the CB filopodia selective binding activities and CB matching. In contrast to single knockouts, loss of both Fas3 and Ten-m dramatically impairs CB alignment. We propose that differential expression of adhesion molecules mediates selective filopodia binding, and these molecules work in concert to instruct precise and robust cell matching.

developmental biology↗

bioRxiv2017

Ca2+-induced mitochondrial ROS regulate the early embryonic cell cycle

While it has long been appreciated that reactive oxygen species (ROS) can act as second messengers in both homeostastic and stress response signaling pathways, potential roles for ROS during early vertebrate development have remained largely unexplored. Here we show that fertilization in Xenopus embryos triggers a rapid increase in ROS levels, which oscillates with each cell division. Furthermore, we show that the fertilization induced Ca2+ wave is both necessary and sufficient to induce ROS production in activated or fertilized eggs. Using chemical inhibitors, we identified mitochondria as the major source of fertilization induced ROS production. Inhibition of mitochondrial ROS production in early embryos results in cell cycle arrest, in part, via ROS dependent regulation of Cdc25C activity. This study reveals for the first time, a role for oscillating ROS levels in the regulation of the early cell cycle in Xenopus embryos.\n\nHighlightsO_LIROS, including hydrogen peroxide, are produced after fertilization in Xenopus\nC_LIO_LICa2+ signaling after fertilization induces ROS production in mitochondria\nC_LIO_LIMitochondria are the major source of oscillating ROS levels\nC_LIO_LIROS regulate Cdc25C activity and the early cell cycle\nC_LI

developmental biology↗

bioRxiv2017

Elevated and sustained reactive oxygen species levels facilitate mesoderm formation during early Xenopus development

Fertilisation triggers embryonic development culminating with the activation of a number of highly co-ordinated and evolutionarily conserved signalling pathways, which induce and pattern the mesoderm of the developing embryo. Previous studies in invertebrates have shown that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), can act as a signalling molecule for axis specification during early development. Using a HyPer transgenic Xenopus laevis line that expresses a H2O2-sensitive fluorescent protein sensor maternally, we recently found that fertilisation triggers a rapid increase in ROS production. Here we show that this increase in ROS levels is sustained throughout early embryogenesis, lasting until the tailbud stages. In addition we show that lowering ROS levels from the blastula stage through the gastrula stages via antioxidant treatments disrupts mesoderm formation. Furthermore, we show that attenuating ROS levels during the blastula / gastrula stages affects some, but not all, growth factor signalling pathways involved in mesoderm induction and patterning, including the PI3K/Akt, TGF-{beta}/Nodal, and Wnt/{beta}-catenin signalling pathways. These data suggest that sustained elevated ROS levels during the blastula and gastrula stages are essential for early vertebrate embryonic development, at least partly, through their roles in promoting growth factor signalling.

developmental biology↗

bioRxiv2017

Re-arrangements in the cytoplasmic distribution of small RNAs following the maternal-to-zygotic transition in Drosophila embryos

Small RNAs are known to regulate gene expression during early development. However, the dynamics of interaction between small RNAs and polysomes during this process is largely unknown. 0-1h and 7-8h Drosophila embryos were fractionated on sucrose density gradients into four fractions based on A254 reading (1) translationally inactive messengerribonucleoprotein (mRNP); (2) 60S; (3) monosome; and (4) polysome. Comparative analysis of deep-sequencing reads from fractionated and un-fractionated 0-1h and 8-h embryos revealed development-specific co-sedimentation pattern of small RNAs with the cellular translation machinery. Although most miRNAs did not have a specific preference for any state of the translational machinery, we detected fraction-specific enrichment of some miRNAs such as miR-1-3p, -184-39, 5-5p and 263-5p. More interestingly, we observed dysregulation of a subset of miRNAs in fractionated embryos despite no measurable difference in their amount in unfractionated embryos. Transposon-derived endosiRNAs are over-expressed in 7-8h embryos and are associated mainly with the mRNP fraction. However, transposon-derived piRNAs, which are more abundant in 0-1h embryos, co-sediment primarily with the polysome fractions. These results suggest that there appears to be a complex interplay among the small RNAs with respect to their polysome-cosedimention pattern during early development in Drosophila.

developmental biology↗

bioRxiv2017

Shared Genetic Requirements for Atf5 Translation in the Vomeronasal Organ and Main Olfactory Epithelium

BackgroundBoth olfactory sensory neurons (OSNs) and vomeronasal sensory neurons (VSNs) require the transcription factor Atf5 for maturation and survival. In OSNs, ATF5 translation is controlled by olfactory receptor (OR) expression-mediated activation of the PERK branch of the unfolded protein response. This study evaluated whether OSNs and VSNs share genetic requirements for ATF5 translation.\n\nMethodsATF5 immunoreactivity was assayed in whole vomeronasal organs from a series of genetic mutant animals identified in studies of OR gene choice, OR feedback, and regulation and OSN development.\n\nResultsATF5 expression in VSNs required the histone demethylase Lsd1, which has been previously reported to be required for OR expression. ATF5 expression also required PERK-mediated phosphorylation of the translation initiation factor eIF2. Finally, unlike previous observations in OSNs, ATF5 was found to be widespread in the mature VNO and co-expressed with mature VSN markers.\n\nConclusionsThese data suggest that the initiation of ATF5 translation in VSNs and OSNs is under similar regulation, and that persistent/prolonged ATF5 translation in VSNs may serve VSN-specific gene regulatory programs. This study firmly establishes the unfolded protein response as a major controller of sensory neuronal maturation and diversification.

developmental biology↗