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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Maintenance of spatial gene expression by Polycomb-mediated repression after formation of a vertebrate body plan

Polycomb group proteins are transcriptional repressors that are important regulators of cell fate during embryonic development. Among them, Ezh2 is responsible for catalyzing the epigenetic repressive mark H3K27me3 and is essential for animal development. The ability of zebrafish embryos lacking both maternal and zygotic ezh2 to form a normal body plan provides a unique model to comprehensively study Ezh2 function during early development in vertebrates. By using a multi-omics approach, we found that Ezh2 is required for the recruitment of H3K27me3 and Polycomb group protein Rnf2. However, in absence of Ezh2, only minor changes in global H3K4me3 levels and gene and protein expression occurred. These changes were mainly due to local deregulation of transcription factors outside their normal expression boundaries. Altogether, our results in zebrafish show that Polycomb-mediated gene repression is important only after the body plan is formed to maintain spatially restricted transcriptional profiles of Polycomb-targeted transcription factors.

developmental biology↗

bioRxiv2018

Body composition and growth in full-term small for gestational age and large for gestational age Swedish infants assessed with air displacement plethysmography at birth and at 3-4 months of age.

BackgroundBeing born small for gestational age (SGA) or large for gestational age (LGA) has short and long term metabolic consequences. There is a growing interest in the extent to which body composition, both in the short and the long term, differs in infants born at the extremes of these birth weights.\n\nMethodsBody composition in 25 SGA and 25 LGA infants were assessed during the first days of life and at 3-4 months of age using air displacement plethysmography.\n\nResultsSGA infants had significantly lower body fat (%) at birth compared to LGA infants. SGA infants increased their body weight and length at a significantly higher rate between birth and 3-4 months than LGA infants. Fat mass (g) in SGA infants increased 23 times between birth and 3-4 months of age compared to 2.8 times for LGA infants. At 3-4 months of age LGA infants reached a threshold in body fat (%) while SGA infants were still gaining body fat (%).\n\nConclusionSeveral significant differences have been identified between SGA and LGA infants, indicating that the effects of intrauterine life continues to play an important role in body composition and growth during the first 3-4 months of life.

developmental biology↗

bioRxiv2018

Culling less fit neurons protects against amyloid-β induced brain damage and cognitive and motor decline

Alzheimers disease (AD) is the most common form of dementia, impairing cognitive and motor functions. One of the pathological hallmarks of AD is neuronal loss, which is not reflected in mouse models of AD. Therefore, the role of neuronal death is still uncertain. Here, we used a Drosophila AD model expressing a secreted form of human amyloid-{beta}42 peptide and show that it recapitulates key aspects of AD pathology, including neuronal death and impaired long-term memory. We found that neuronal apoptosis is mediated by cell fitness-driven neuronal culling, which selectively eliminates impaired neurons from brain circuits. We show that removal of less fit neurons delays amyloid-{beta}42-induced brain damage and protects against cognitive and motor decline, suggesting that - contrary to common knowledge - neuronal death may have a beneficial effect in AD.

developmental biology↗

bioRxiv2018

ABC-transporter activity and eicosanoid-signaling are required for germ cell migration a basal chordate

In the colonial ascidian Botryllus schlosseri, long-lived germline stem cells (GSCs) migrate to new germline niches as they develop during repetitive cycles of asexual reproduction. ABC-transporters are involved in the export of lipid-signaling molecules, but their roles in germ cell migration are poorly understood. Here, we show that in Botryllus, abcc1 and abcb1 are highly expressed in germ cells, and inhibition of ABC-transporter activity leads to failure of germ cell migration. Phospholipase A2 (PLA2) produces arachidonic acid, which is further metabolized to eicosanoid signaling molecules. In humans, 12-lipoxygenase (LOX) metabolizes arachidonic acid to12-Hydroxyeicosatetraenoic acid (12-S-HETE), which stimulates migration of mammalian cancer cells and smooth muscle cells. We show that PLA2 and LOX activity are required for germ cell migration. A potential homolog to the human receptor for 12-S-HETE, BSgpr31, is expressed in germ cells. 12-S-HETE rescues migration towards S1P in the presence of inhibitors of ABCC1, ABCB1, PLA2 or LOX, and a gradient of 12-S-HETE enhances chemotaxis towards S1P and stimulates motility. We conclude that 12-S-HETE is a secondary chemoattractant exported by ACB-transporters that is required for migration of germ cells towards S1P. We also find that in the presence of S1P, detection of an 12-S-HETE gradient initiates an autologous positive feedback loop that may sustain migration. This is the first report of an eicosanoid-signaling molecule regulating germ cell migration.

developmental biology↗

bioRxiv2018

A Scube2-Shh feedback loop links morphogen release to morphogen signaling to enable scale invariant patterning of the ventral neural tube

To enable robust patterning, morphogen systems should be resistant to variations in gene expression and tissue size. Here we explore how the Sonic Hedgehog (Shh) morphogen gradient in the ventral neural tube enables scaled patterning in embryos of varying sizes. Using zebrafish eggs that have been surgically reduced in size, we find that ventral neural tube patterning remains proportional in smaller embryos. Intriguingly, a secreted protein implicated in Shh release, Scube2, is expressed in the dorsal-intermediate neural tube far from Shh producing cells. Overexpression of scube2 expands the Shh gradient whereas loss of scube2 causes gradient contraction. Conversely, upregulation of Shh represses scube2 expression while Shh downregulation increases scube2 expression thus establishing a negative feedback loop. This regulatory feedback is necessary for scaling, as demonstrated by its loss in scube2 overexpressing embryos. Using mathematical modeling, we show that feedback control on diffusion and release rates of Shh allows the morphogen gradient to be robust to differences in field length and Shh gene dosage. We conclude that Scube2 promotes release and diffusion of Shh to allow gradient scaling in an extension to the expander-repressor model. Summary StatementThe Shh morphogen gradient can scale to different size tissues by feedback between Scube2 mediated release and diffusion of Shh and Shh based inhibition of Scube2 expression

developmental biology↗

bioRxiv2018

Single-cell RNA-seq of the Developing Cardiac Outflow Tract Reveals Convergent Development of the Vascular Smooth Muscle Cells at the Base of the Great Arteries

RationaleCardiac outflow tract (OFT) is a major hotspot for congenital heart diseases (CHDs). A thorough understanding of the cellular diversity, transitions and regulatory networks of normal OFT development is essential to decipher the etiology of OFT malformations.\n\nObjectiveWe sought to explore the cellular diversity and transitions between cell lineages during OFT development.\n\nMethods and ResultsWe performed single-cell transcriptomic sequencing of 55,611 mouse OFT cells from three developmental stages that generally correspond to the early, middle and late stages of OFT remodeling and septation. We identified 17 cell clusters that could be assigned to six cell lineages. Among these lineages, the macrophage and VSMC lineages of the developing OFT have seldom been previously described. Known cellular transitions, such as endothelial to mesenchymal transition, have been recapitulated. In particular, we identified convergent development of the VSMC lineage, where intermediate cell subpopulations were found to be involved in either myocardial to VSMC trans-differentiation or mesenchymal to VSMC transition. Through single-molecule in situ hybridization, we observed that cells expressing the myocardial marker Myh7 co-expressed the VSMC marker gene Cxcl12 in OFT walls, thus confirming the existence of myocardial to VSMC trans-differentiation. Moreover, we found that the Penk+ cluster c8, a relatively small mesenchymal subpopulation that was undergoing mesenchymal to VSMC transition, was associated with the fusion of OFT cushions. We also uncovered the expression dynamics and critical transcriptional regulators potentially governing cell state transitions. Finally, we developed web-based interactive interfaces to facilitate further data exploration.\n\nConclusionsWe provide a single-cell reference map of cell states for normal OFT development, which will be a valuable resource for the CHD community. Our data support the existence of myocardial to VSMC trans-differentiation and convergent development of the VSMC lineage at the base of the great arteries.

developmental biology↗

bioRxiv2018

A dual function of FGF signaling in Xenopus left-right axis formation

Organ left-right (LR) asymmetry is a conserved vertebrate feature, which is regulated by left-sided activation of Nodal signaling. Nodal asymmetry is established by a leftward fluid-flow generated at the ciliated LR organizer (LRO). While the role of fibroblast growth factor (FGF) signaling pathways during mesoderm development are conserved, diverging results from different model organisms suggested a non-conserved function in LR asymmetry. Here, we demonstrate that FGF is required during gastrulation in a dual function at consecutive stages of Xenopus embryonic development. In the early gastrula, FGF is necessary for LRO precursor induction, acting in parallel to FGF-mediated mesoderm induction. During late gastrulation, the FGF/Ca2+-branch is required for specification of the flow sensing lateral LRO cells, a function related to FGF-mediated mesoderm morphogenesis. This second function in addition requires input from the calcium channel Polycystin-2. Thus, analogous to mesoderm development, FGF activity is required in a dual role for laterality specification, namely for generating and sensing of leftward flow. Moreover, our data show that FGF functions in LR asymmetric development are conserved across vertebrate species, from fish to mammals.

developmental biology↗

bioRxiv2018

The Toll pathway inhibits tissue growth and regulates cell fitness in an infection-dependent manner

The Toll pathway regulates the cellular response to infection via the transcriptional upregulation of antimicrobial peptides. Apart from its role in innate immunity, this pathway has also been reported to be important for the elimination of loser cells in a process referred to as cell competition, which can be locally triggered by secreted factors released from winner cells. In this work we provide evidence that the inhibition of Toll signaling not only increases the fitness of loser cells, but also bestows a clonal growth advantage on wild-type cells. We further demonstrate that this growth advantage depends on basal infection levels since it is no longer present under axenic conditions but exacerbated upon intense pathogen exposure. Thus, the Toll pathway functions as a fine-tuned pro-apoptotic and anti-proliferative regulator, underlining the existence of a trade-off between innate immunity and growth during development.

developmental biology↗

bioRxiv2018

Charting the emergent organotypic landscape of the mammalian gut endoderm at single-cell resolution

To comprehensively delineate the ontogeny of an organ system, we generated 112,217 singlecell transcriptomes representing all endoderm populations within the mouse embryo until midgestation. We employed graph-based approaches to model differentiating cells for spatio-temporal characterization of developmental trajectories. Our analysis reveals the detailed architecture of the emergence of the first (primitive or extra-embryonic) endodermal population and pluripotent epiblast. We uncover an unappreciated relationship between descendants of these lineages, before the onset of gastrulation, suggesting that mixing of extra-embryonic and embryonic endoderm cells occurs more than once during mammalian development. We map the trajectories of endoderm cells as they acquire embryonic versus extra-embryonic fates, and their spatial convergence within the gut endoderm; revealing them to be globally similar but retaining aspects of their lineage history. We observe the regionalized localization of cells along the forming gut tube, reflecting their extra-embryonic or embryonic origin, and their coordinate patterning into organ-specific territories along the anterior-posterior axis.

developmental biology↗

bioRxiv2018

Wolf-Hirschhorn Syndrome-associated genes are enriched in motile neural crest and affect craniofacial development in Xenopus laevis

Wolf-Hirschhorn Syndrome (WHS) is a human developmental disorder arising from a hemizygous perturbation, typically a microdeletion, on the short arm of chromosome four. In addition to pronounced intellectual disability, seizures, and delayed growth, WHS presents with a characteristic facial dysmorphism and varying prevalence of microcephaly, micrognathia, cartilage malformation in the ear and nose, and facial asymmetries. These affected craniofacial tissues all derive from a shared embryonic precursor, the cranial neural crest, inviting the hypothesis that one or more WHS-affected genes may be critical regulators of neural crest development or migration. To explore this, we characterized expression of multiple genes within or immediately proximal to defined WHS critical regions, across the span of craniofacial development in the vertebrate model system Xenopus laevis. This subset of genes, WHSC1, WHSC2, LETM1, and TACC3, are diverse in their currently-elucidated cellular functions; yet we find that their expression demonstrates shared tissue-specific enrichment within the anterior neural tube, pharyngeal arches, and later craniofacial structures. We examine the ramifications of this by characterizing craniofacial development and neural crest migration following individual gene depletion. We observe that several WHS-associated genes significantly impact facial patterning, cartilage formation, pharyngeal arch migration, and neural crest motility, and can separately contribute to forebrain scaling. Thus, we have determined that numerous genes within and surrounding the defined WHS critical regions potently impact craniofacial patterning, suggesting their role in WHS presentation may stem from essential functions during neural crest-derived tissue formation.\n\nAuthor SummaryWolf-Hirschhorn Syndrome (WHS), a developmental disorder caused by small deletions on chromosome four, manifests with pronounced and characteristic facial malformation. While genetic profiling and case studies provide insights into how broader regions of the genome affect the syndromes severity, we lack a key component of understanding its pathology; a basic knowledge of how individual WHS-affected genes function during development. Importantly, many tissues affected by WHS derive from shared embryonic origin, the cranial neural crest. This led us to hypothesize that genes deleted in WHS may hold especially critical roles in this tissue. To this end, we investigated the roles of four WHS-associated genes during neural crest cell migration and facial patterning. We show that during normal development, expression of these genes is enriched in migratory neural crest and craniofacial structures. Subsequently, we examine their functional roles during facial patterning, cartilage formation, and forebrain development, and find that their depletion recapitulates features of WHS craniofacial malformation. Additionally, two of these genes directly affect neural crest cell migration rate. We report that depletion of WHS-associated genes is a potent effector of neural crest-derived tissues, and suggest that this explains why WHS clinical presentation shares so many characteristics with classic neurochristopathies.

developmental biology↗

bioRxiv2018

DAZL is a master translational regulator of murine spermatogenesis

Expression of DAZ-like (DAZL) is a hallmark of vertebrate germ cells and essential for embryonic germ cell development and differentiation, yet gametogenic function of DAZL has not been fully characterized with most of its in vivo direct targets unknown. We showed that postnatal stage-specific deletion of Dazl in mouse germ cells did not affect female fertility, but caused complete male sterility with gradual loss of spermatogonial stem cells (SSCs), meiotic arrest and spermatid arrest respectively. Using the genome-wide HITS-CLIP and mass spectrometry approach, we found that DAZL bound to a large number of testicular mRNA transcripts (at least 3008) at 3' UnTranslated Region (3' UTR) and interacted with translation proteins including PABP. In the absence of DAZL, polysome-associated target transcripts, but not their total transcripts were significantly decreased, resulting in drastic reduction of an array of spermatogenic proteins and thus developmental arrest. Thus, DAZL is a master translational regulator essential for spermatogenesis.

developmental biology↗

bioRxiv2018

Single cell transcriptomics reveals spatial and temporal dynamics of gene expression in the developing mouse spinal cord

The coordinated spatial and temporal regulation of gene expression in the vertebrate neural tube determines the identity of neural progenitors and the function and physiology of the neurons they generate. Progress has been made deciphering the gene regulatory programmes responsible for this process, however, the complexity of the tissue has hampered the systematic analysis of the network and the underlying mechanisms. To address this, we used single cell mRNA sequencing to profile cervical and thoracic regions of the developing mouse neural tube between embryonic days (e)9.5-e13.5. We confirmed the data accurately recapitulates neural tube development, allowing us to identify new markers for specific progenitor and neuronal populations. In addition, the analysis highlighted a previously underappreciated temporal component to the mechanisms generating neuronal diversity and revealed common features in the sequence of transcriptional events that lead to the differentiation of specific neuronal subtypes. Together the data provide a compendium of gene expression for classifying spinal cord cell types that will support future studies of neural tube development, function, and disease.

developmental biology↗

bioRxiv2018

Fetal and trophoblast PI3Kp110α have distinct roles in regulating resource supply to the growing fetus

Previous studies suggest that the placental supply of nutrients to the fetus adapts according to fetal demand. However, the signaling events underlying placental adaptations remain largely unknown. Earlier work in mice has revealed that loss of the phosphoinositide 3-kinase p110 impairs feto-placental growth but placental nutrient supply is adaptively increased. Here we explore the role of p110 in the epiblast-derived (fetal) and trophoblast lineages of the conceptus in relation to feto-placental growth and placental development and transfer function. Using conditional gene manipulations to knock-down p110 either by [~]50% or [~]100% in the fetal lineages and/or trophoblast, this study shows that p110 in the fetus is essential for prenatal development and a major regulator of placental phenotype in mice. Complete loss of fetal p110 caused embryonic death, whilst heterozygous loss resulted in fetal growth restriction and impaired placental formation and nutrient transport. Loss of trophoblast p110 also resulted in abnormal placental development, although fetuses were viable. However, in response to complete loss of trophoblast p110, the placenta failed to transport sufficient amino acid to match fetal demands for growth. Using RNA-seq, we identified several genes downstream of p110 in the trophoblast that are important in adapting placental phenotype to support fetal growth. Further work using CRISPR/Cas9 genome targeting showed that loss of p110 differentially affects the expression of genes in trophoblast and embryonic stem cells. Our findings thus reveal important, but distinct roles for p110 signaling in the different compartments of the conceptus, which control fetal resource acquisition and ultimately affect healthy growth.\n\nOne Sentence SummaryFetal and trophoblast p110 modify resource allocation

developmental biology↗

bioRxiv2018

A kinesin Klp10A mediates cell cycle-dependent shuttling of Piwi between nucleus and nuage

The piRNA pathway protects germline genomes through transcript cleavage of selfish genetic elements, such as transposons, in the cytoplasm and their transcriptional silencing in the nucleus. Here, we describe a mechanism by which the nuclear and cytoplasmic arms of the silencing mechanism are linked. During mitosis of Drosophila spermatogonia, nuclear Piwi interacts with nuage, the compartment that mediates the cytoplasmic arm of piRNA-mediated silencing. At the end of mitosis, Piwi leaves nuage to return to the nucleus. We found that dissociation of Piwi from nuage occurs at the depolymerizing microtubules of the central spindle, mediated by a microtubule-depolymerizing kinesin Klp10A. Depletion of klp10A delays Piwis return to the nucleus and affects piRNA production, suggesting the importance of nuclear-cytoplasmic communication in piRNA biogenesis. We propose that cell cycle-dependent communication between the nuclear and cytoplasmic arms of the piRNA pathway plays important roles in coordinated piRNA production.

developmental biology↗

bioRxiv2018

Osteocyte death and bone overgrowth in mice lacking Fibroblast Growth Factor Receptors 1 and 2 in mature osteoblasts and osteocytes

Fibroblast Growth Factor (FGF) signaling pathways have well established roles in skeletal development, with essential functions in both chondrogenesis and osteogenesis. In mice, previous conditional knockout studies suggested distinct roles for FGF receptor 1 (FGFR1) signaling at different stages of osteogenesis and a role for FGFR2 in osteoblast maturation. However, the potential for redundancy among FGFRs and the mechanisms and consequences of stage-specific osteoblast lineage regulation were not addressed. Here, we conditionally inactivate Fgfr1 and Fgfr2 in mature osteoblasts with an Osteocalcin-Cre or Dentin matrix protein 1-CreER driver. We find that young mice lacking both receptors or only FGFR1 are phenotypically normal. However, after 6 weeks of age these Fgfr1/Fgfr2 double- and Fgfr1 single-conditional knockout mice develop a high bone mass phenotype with increased periosteal apposition, increased endocortical woven bone with increased porosity, and biomechanical properties that reflect increased bone mass but impaired material properties. Histopathological and gene expression analyses show that this phenotype is preceded by a striking loss of osteocytes, and gradual activation of the Wnt/{beta}Catenin signaling pathway. These data identify a role for FGFR1 signaling in mature osteoblasts/osteocytes that is required for osteocyte survival during postnatal bone growth.

developmental biology↗

bioRxiv2018

Deriving Cardiomyocytes from Human Amniocytes

Many forms of congenital heart disease (CHD) have high morbidity-mortality rates and require challenging surgeries. Human amniocytes have important stem cell characteristics and could potentially provide patient-specific tissue for repairs of some types of CHDs. We report that amniocytes express features of poised cardiomyocytes. However, a variety of direct reprogramming approaches failed to convert their fetal and transcriptionally repressed state into bona fide cardiomyocytes. Induced-pluripotent stem cell (iPSC) reprogramming removes repression and converts amniocytes to a baseline pluripotent state. Based on molecular and electrophysiological signatures, iPSC reprogrammed amniocytes can be induced to differentiate into functionally immature, predominantly ventricular cardiomyocytes and a heterogeneous mixture of vascular and unspecified epithelial cells. Developmental time course analyses and pattern clustering of amniocyte-derived cardiomyocytes identifies numerous temporal co-regulators of cardiac induction and maturation as well as distinct sarcomeric and ion channel gene signatures. Normal fetal cardiomyocytes are derived by overcoming complex forms of transcriptional repression that suppress direct transdifferentiation of human amniocytes. These results suggest the possibility of using amniocytes as a source of patient-specific ventricular cardiomyocytes for cell therapies.\n\nSUMMARY STATEMENTAmniocytes are a possible source of patient-specific cardiomyocytes for newborns with congenital heart disease. Genome-wide DNA methylation patterns and transcriptional repressors preclude direct differentiation, but pluripotent reprogramming provides cardiomyocytes for dissecting genetic pathways contributing to this disease.

developmental biology↗

bioRxiv2018

Glutamate signaling at cytoneme synapses

We investigated the roles of neuronal synapse components for development of the Drosophila air sac primordium (ASP). The ASP, an epithelial tube, extends specialized signaling filopodia called cytonemes that take up signals such as Dpp from the wing imaginal disc. Dpp signaling in the ASP requires that disc cells express Dpp, Synaptobrevin, Synaptotagmin-1, the glutamate transporter, and a voltage-gated calcium channel, and that ASP cells express the Dpp receptor, Synaptotagmin-4 and the AMPA-type glutamate receptor GluRII. Calcium transients in ASP cytonemes correlate with signaling activity. Calcium transients in the ASP require GluRII, are activated by L-glutamate and by stimulation of an optogenetic ion channel expressed in the wing disc, and are inhibited by EGTA and NASPM. Activation of GluRII is essential but not sufficient for signaling. Cytoneme-mediated signaling is glutamatergic.\n\nSummaryParacrine signals transfer between Drosophila epithelial cells at glutamatergic synapses.

developmental biology↗

bioRxiv2018

Generation of Cynomolgus Monkeys (Macaca fascicularis) Embryos with ICSI Based on the MII-Stage Oocytes Acquired by Personalized Superovulation Protocol

The use of cynomolgus monkeys (Macaca fascicularis) has facilitated the establishment of gene-modified animal models, and the appropriate number of high-quality mature oocytes (MII-stage oocytes) are crucial in assisted reproductive technologies (ART) of cynomolgus macaques. In this study we performed 3 different superovulation protocols on 24 female cynomolgus monkeys aimed to improve the proportion of MII-stage oocyte retrieval. The results suggested some differences in the proportion of MII-stage oocytes between the personalized superovulation protocol and the superovulation protocols I and II. Afterwards, we performed Intracytoplasmic sperm injection (ICSI) on 22 MII-stage oocytes derived from one animal with personalized superovulation protocol, obtained 15 embryos of 2-4-cells, 2 cases of successful pregnancy after transferring to 4 surrogate female, and obtained 3 aborted fetuses. These results suggested that personalized superovulation protocol incorporated the menstrual cycle length of each cynomolgus monkey, and the timing of oocytes retrieval based on the equation: menstrual cycle length/2 {+/-} 1, which increased the rate of MII-stage oocytes acquired and generation of cynomolgus monkeys embryos with ICSI based on these oocytes, and finally could successfully develop into fetus under conditions that effectively guarantee the synchronization of the surrogate mothers.\n\nSUMMARY STATEMENTThe personalized superovulation protocol based on the menstrual cycle length of each cynomolgus monkey can increase the rate of MII-stage oocytes acquired, and finally could successfully develop into fetus under conditions that minimize damage to the surrogate mothers and effectively guarantee the synchronization of the surrogate mothers.

developmental biology↗