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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2019

Robo2 regulates synaptic oxytocin content by affecting actin state

The regulation of neuropeptide level at the site of release is essential for proper neurophysiological functions. We focused on a prominent neuropeptide, oxytocin (OXT), and used the zebrafish as an in vivo model to visualize and quantify OXT content at the resolution of a single synapse. We found that OXT-loaded synapses were surrounded by polymerized actin. Perturbation of actin filaments by cytochalasin-D resulted in decreased synaptic OXT levels. Live imaging of the actin probe, Lifeact-EGFP, showed reduced mobility in OXT synapses in robo2 mutants, which displayed decreased synaptic OXT content. Using a novel transgenic reporter line allowing real-time monitoring of OXT-loaded vesicles, we showed that robo2 mutants display slower rate of vesicles accumulation. OXT-specific expression of dominant-negative Cdc42, which is a key regulator of actin dynamics and a downstream effector of Robo2, led to a dose-dependent increase in OXT content in WT, and a dampened effect in robo2 mutants. Our results link Robo2-Cdc42 signalling, which controls local actin dynamics, with the maintenance of synaptic neuropeptide levels.

developmental biology↗

bioRxiv2019

Auto-inhibition of myoblast fusion by cyclic receptor signalling.

Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGF{beta} (SMAD2/3-dependent) signalling acts as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGF{beta} signalling in the embryonic muscle relies on a unique receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion is a self-regulated process that relies on cyclic TGF{beta} signalling to regulate its pace.

developmental biology↗

bioRxiv2019

Tbx1 interacts genetically with Vegfr3 to regulate cardiac lymphangiogenesis in mice

The transcription factor TBX1 is the major gene implicated in 22q11.2 deletion syndrome. The complex clinical phenotype includes vascular anomalies and a recent report presented new cases of primary lymphedema in 22q11.2DS patients. We have previously shown that Tbx1 activates Vegfr3 gene expression in lymphatic endothelial cells and that this activation is critical for lymphatic vessel development in prenatal mice and for their survival post-natally. Using loss-of-function genetics and transgenesis, we show a strong genetic interaction between Tbx1 and Vegfr3 in cardiac lymphangiogenesis that causes cardiac lymphatic vessel anomalies in compound heterozygotes. Intriguingly, different aspects of the cardiac lymphatic phenotype were regulated independently by the two genes. Tbx1Cre-activated Vegfr3 transgene expression was able to rescue the morphological abnormalities in the cardiac lymphatic vessels of compound heterozygotes, but it did not rescue the severe cardiac lymphatic vessel hypoplasia observed in Tbx1 homozygotes. Moreover, our study revealed a differential sensitivity between the ventral and dorsal cardiac lymphatic networks to the effects of altered Tbx1 and Vegfr3 gene dosage. Overall, our study demonstrates that a fine dosage balance between Tbx1 and Vegfr3 is required to regulate the number and morphology of cardiac lymphatic vessels.

developmental biology↗

bioRxiv2019

Micro-indentation and optical coherence tomography for the mechanical characterization of embryos: Experimental setup and measurements on fixed chicken embryos.

Summary statementWe introduce an experimental technique that combines micro-indentation and optical coherence tomography to map the viscoelastic properties of embryonic tissue and investigate correlations between local mechanical features and tissue morphology. AbstractThe investigation of the mechanical properties of embryos is expected to provide valuable information on the phenomenology of morphogenesis. It is thus believed that, by mapping the viscoelastic features of an embryo at different stages of growth, it may be possible to shed light on the role of mechanics in embryonic development. To contribute to this field, we present a new instrument that can determine spatiotemporal distributions of mechanical properties of embryos over a wide area and with unprecedented accuracy. The method relies on combining ferrule-top micro-indentation, which provides local measurements of viscoelasticity, with Optical Coherence Tomography, which can reveal changes in tissue morphology and help the user to localize the indentation locations. To prove the working principle, we have collected viscoelasticity maps of fixed HH11-HH12 chicken embryos. Our study highlights the nonlinear behavior of the tissue and qualitatively shows the correlation between local mechanical properties and tissue morphology for different regions of interest.

developmental biology↗

bioRxiv2019

Effects of the secondhand smoking exposure in the early stages of the bone development

ObjectiveThe objective of this study was to evaluate the effects of the secondhand smoking in the trabecular bone micro-architecture of the mandible of rats, offsprings of passive smoking matrices. Materials and MethodsFifty-five rats, Rattus norvegicus albinus, offsprings of passive smoking and non-passive smoking matrices, were divided into three groups: continuous smoking offsprings (CSO), interrupted smoking offsprings (ISO) and non-smoking offsprings (NSO/control). After the 21st, 42nd, 63rd and 128th days, the mandibles were analyzed by micro-computer tomography(micro-CT). Images of inter-radicular alveolar bone of the mandibular first molars underwent three-dimensional reconstruction and were analyzed. The bone volume fraction (BV/TV, bone volume/total volume), the trabecular thickness (Tb.Th), the trabecular spacing (Tb.Sp), the trabecular number (Tb.N) and the structure model index (SMI) were analyzed. ResultsThe BV/TV analysis revealed increase of the average values in the CSO group, at 21st and 42nd days (p=0,0124), tending to decrease related to the mean from the 42nd day. The animals of ISO group did not show significant difference in BV/TV, about the control group (p=0,9751). The results of Tb.Th were different and significant during all the experimental period among the three groups: CSO and control (p<0,0001), ISO and control (p=0,0030) and CSO / ISO (p=0,0020). About Tb.Sp, the differences were not significant among the three groups. About Tb.N, the difference was significant into each group, with increasing values (p<0.0001). The SMI showed significant difference between the CSOs and control, CSO and ISO, both with (p<0,0001). The difference between control and ISO group was not significant (p=0,1253). ConclusionThe passive inhalation of cigarette smoke by the offsprings of smoking matrices had a harmful effect in the micro-archicteture of the trabecular bone of the rats mandible in developing. About the ISO groups, the recovery of the micro-archicteture occurred partially.

developmental biology↗

bioRxiv2019

Control of Hematopoietic Stem Cell Function Through Epigenetic Regulation of Energy Metabolism and Genome Integrity

It remains largely unclear how stem cells regulate bioenergetics and genome integrity to ensure tissue homeostasis. Here, our integrative gene analyses suggest metabolic and genotoxic stresses may underlie the common functional defects of both fetal and adult hematopoietic stem and progenitor cells (HSPCs) upon loss of DPY30, an epigenetic modulator that facilitates H3K4 methylation. DPY30 directly regulates expression of several key glycolytic genes, and its loss in HSPCs critically impaired energy metabolism, including both glycolytic and mitochondrial pathways. We also found significant increase in DNA breaks as a result of impaired DNA repair upon DPY30 loss, and inhibition of DNA damage response partially rescued clonogenicity of the DPY30-deficient HSPCs. Moreover, CDK inhibitor p21 was upregulated in DPY30-deficient HSPCs, and p21 deletion alleviated their functional defect. These results demonstrate that epigenetic mechanisms by H3K4 methylation play a crucial role in HSPC function through control of energy metabolism and protecting genome integrity.

developmental biology↗

bioRxiv2019

A chromatin modulator sustains self-renewal and enables differentiation of postnatal neural stem and progenitor cells

It remains unknown whether H3K4 methylation, an epigenetic modification associated with gene activation, regulates fate determination of the postnatal neural stem and progenitor cells (NSCs and NPCs, or NSPCs). Here we show that the Dpy30 subunit of the major H3K4 methyltransferase complexes is preferentially expressed at a high level in NSCs and NPCs. By genetically inactivating Dpy30 in specific regions of mouse brain, we demonstrate a crucial role of efficient H3K4 methylation in maintaining both the self-renewal and differentiation capacity of postnatal NSPCs. Dpy30 inactivation results in deficiency in global H3K4 methylation, and disrupts development of hippocampus and especially the dentate gyrus and subventricular zone, the major regions for postnatal NSC activities. By in vitro assays on neurospheres from mouse brains as well as human and mouse NPCs, we show that Dpy30 is indispensable for sustaining the self-renewal and proliferation of NSPCs in a cell-intrinsic manner, and also enables the differentiation of mouse and human NPCs to neuronal and glial lineages. Dpy30 directly regulates H3K4 methylation and the induction of several genes critical in neurogenesis. These findings link a prominent epigenetic mechanism of gene expression to the fundamental properties of NSPCs, and may have implications in neurodevelopmental disorders. SIGNIFICANCE STATEMENTAs a highly prominent epigenetic mark that is associated with gene activation and a number of neurodevelopmental disorders in human, the role of histone H3K4 methylation in the fate determination of neural stem cells is unclear. Result of this study uncover a profound role of this epigenetic modification in the fundamental properties of postnatal neural stem cells, including self-renewal and differentiation, and may have implications for a better understanding and treatment of a broad spectrum of neurodevelopmental disorders associated with H3K4 methylation modulators.

developmental biology↗

bioRxiv2019

On the front line of Klebsiella pneumoniae surface structures understanding: establishment of Fourier Transform Infrared (FT-IR) spectroscopy as a capsule typing method

Genomics-based population analysis of multidrug resistant (MDR) Klebsiella pneumoniae (Kp) motivated a renewed interest on capsule (K) types given their importance as evolutionary and virulence markers of clinically relevant strains. However, there is a gap between genotypic based predictions and information on capsular polysaccharide structure and composition. We used molecular genotypic, comparative genomics, biochemical and phenotypic data on the cps locus to support the usefulness of Fourier-Transform Infrared (FT-IR) spectroscopy as a phenotypic approach for K-type characterization and identification. The approach was validated with a collection of representative MDR Kp isolates from main lineages/Clonal Groups (CGs) involved in local or nationwide epidemics in 6 European and South American countries. FT-IR-based K-type assignments were compared with those obtained by genotypic methods and WGS (cps operon), and further complemented with data on the polysaccharide composition and structure of known K-types. We demonstrate that our FT-IR-based spectroscopy approach can discriminate all 21 K-types identified with a resolution comparable (or even higher) to that provided by WGS, considered gold-standard methodology. Besides contributing to enlighten K-type diversity among a significant MDR Kp collection, the specific associations between certain K-types and Kp lineages identified in different geographic regions over time support the usefulness of our FT-IR-based approach for strain typing. Additionally, we demonstrate that FT-IR discriminatory ability is correlated with variation on the structure/composition of known K-types and, supported on WGS data, we were able to predict the sugar composition and chemical structure of new KL-types. Our data revealed an unprecedent resolution at a quick and low-cost rate of Kp K-types at the phenotypic level. Our FT-IR spectroscopy-based approach might be extremely useful not only as a cost-effective Kp typing tool, but also to improve our understanding on sugar-based coating structures of high relevance for strain evolution and host adaptation.

developmental biology↗

bioRxiv2019

BioCell2XML: A novel tool for converting cell lineage data from SIMI BioCell to MaMuT (Fiji)

Computer-assisted 4D manual cell tracking has been a valuable method for understanding spatial-temporal dynamics of embryogenesis (e.g., Stach & Anselmi, 2015; Vellutini et al., 2017; Wolff et al., 2018) since the method was introduced in the late 1990s. Since two decades SIMI(R) BioCell (Schnabel et al., 1997), a software which initially was developed for analyzing data coming from the, at that time new technique of 4D microscopy, is in use. Many laboratories around the world use SIMI BioCell for the manual tracing of cells in embryonic development of various species to reconstruct cell genealogies with high precision. However, the software has several disadvantages: Limits in handling very large data sets, the virtually no maintenance over the last ten years (bound to older Windows versions), the difficulty to access the created cell lineage data for analyses outside SIMI BioCell, and the high cost of the program. Recently, bioinformatics, in close collaboration with biologists, developed new lineaging tools that are freely available through the open source image processing platform Fiji. Here we introduce a software tool that allows conversion of SIMI BioCell lineage data to a format that is compatible with the Fiji plugin MaMuT (Wolff et al., 2018). Hereby we intend to maintain the usability of SIMI BioCell created cell lineage data for the future and, for investigators who wish to do so, facilitate the transition from this software to a more convenient program.

developmental biology↗

bioRxiv2019

Immunohistochemical and ultrastructural analysis of the maturing larval zebrafish enteric nervous system reveals the formation of a neuropil pattern

The gastrointestinal tract is constructed with an intrinsic series of interconnected ganglia that span its entire length, called the enteric nervous system (ENS). The ENS exerts critical local reflex control over many essential gut functions; including peristalsis, water balance, hormone secretions and intestinal barrier homeostasis. ENS ganglia exist as a collection of neurons and glia that are arranged in a series of plexuses throughout the gut: the myenteric plexus and submucosal plexus. While it is known that enteric ganglia are derived from a stem cell population called the neural crest, mechanisms that dictate final neuropil plexus organization remain obscure. Recently, the vertebrate animal, zebrafish, has emerged as a useful model to understand ENS development, however knowledge of its developing myenteric plexus architecture was unknown. Here, we examine myenteric plexus of the maturing zebrafish larval fish histologically over time and find that it consists of a series of tight axon layers and long glial cell processes that wrap the circumference of the gut tube to completely encapsulate it, along all levels of the gut. By late larval stages, complexity of the myenteric plexus increases such that a layer of axons is juxtaposed to concentric layers of glial cells. Ultrastructurally, glial cells contain glial filaments and make intimate contacts with one another in long, thread-like projections. Conserved indicators of vesicular axon profiles are readily abundant throughout the larval plexus neuropil. Together, these data extend our understanding of myenteric plexus architecture in maturing zebrafish, thereby enabling functional studies of its formation in the future.

developmental biology↗

bioRxiv2019

MicroRNA-mediated control of developmental lymphangiogenesis

The post-transcriptional mechanisms contributing to molecular regulation of developmental lymphangiogenesis and lymphatic network assembly are not well understood. Here, we use high throughput small RNA sequencing to identify miR-204, a highly conserved miRNA dramatically enriched in lymphatic vs. blood endothelial cells, and we demonstrate that this miRNA plays a critical role during lymphatic development. Suppressing miR-204 leads to loss of lymphatic vessel formation, while overproducing miR-204 in lymphatic vessels accelerates lymphatic vessel formation, suggesting a positive role during developmental lymphangiogenesis. We also identify the NFATC1 transcription factor as a key conserved target for post-transcriptional regulation by miR-204 during lymphangiogenesis. While miR-204 suppression leads to loss of lymphatics, knocking down its target NFATC1 leads to lymphatic hyperplasia, and the loss of lymphatics in miR-204-deficient animals can be rescued by NFATC1 knockdown. Together, our results highlight a miR-204/NFATC1 molecular regulatory axis required for proper lymphatic development.

developmental biology↗

bioRxiv2019

Dynamics of Sox2 expression during rat germ cell development and its relationship with emergence of spermatogonia

1.The gonocytes represent a specific phase of male gem cell development that precedes spermatogonial stem cell differentiation. Here, we describe the expression of Sox2, an OCT4 partner, during rat germ cell development. Our hypothesis is that SOX2 has a cytoplasmic role during gonocyte-to-spermatogonia transition. Male rat embryos and testes were submitted to the analysis of Sox2 expression. Sox2 was detected in germ cells from 14 days post-conception (dpc) to 8dpp. SOX2 was present in 14dpc and 15dpc embryos and absent at 17 and 19dpc; however, it did not show direct correlation with mRNA. SOX2 labelling was detected after birth and its expression increased from 1dpp to 5dpp. SOX2 was localized in the cytoplasm and showed a granulated pattern similar to P-bodies. Indeed, GW182/SOX2 and LIN28/SOX2 double-labelling showed that SOX2 partially co-localized with the P-bodies components GW182 and LIN28. At 8dpp SOX2 was detected in the nucleus and/or in the cytoplasm of spermatogonia, whereas at 25dpp it was detected in the nucleus of rare spermatogonia. This suggests that SOX2 localization changes during gonocytes to spermatogonia transition.

developmental biology↗

bioRxiv2019

Evolutionary conserved canonical BMP activity controls cortical neurogenesis

The growth and evolutionary expansion of the cerebral cortex are defined by the spatial-temporal production of neurons, which itself depends on the decision of radial glial cells (RGCs) to self-amplify or to switch to neurogenic divisions. The mechanisms regulating these RGC fate decisions are still incompletely understood. Here we describe a novel and evolutionarily conserved role of the canonical BMP transcription factors SMAD1/5 in controlling neurogenesis and growth during corticogenesis. Reducing the expression of both SMAD1 and SMAD5 in neural progenitors at early mouse cortical development caused microcephaly and an increased production of early-born cortical neurons at the expense of late-born ones, which correlated with the premature differentiation and depletion of the pool of cortical progenitors. Gain- and loss-of-function experiments performed during early cortical neurogenesis in the chick revealed that SMAD1/5 activity supports self-amplifying RGC divisions and restrain the neurogenic ones. Furthermore, we demonstrate that SMAD1/5 stimulate RGC self-amplification through the positive post-transcriptional regulation of the Hippo signaling effector YAP. We anticipate this SMAD1/5-YAP signaling module to be fundamental in controlling growth and evolution of the amniote cerebral cortex.

developmental biology↗

bioRxiv2019

Cell-type specific mechanical response and actomyosin dynamics in the developing Drosophila retina

During organogenesis, different cell types need to work together to induce functional multicellular structures. To study this process, we made use of the genetically tractable fly retina, with a focus on the mechanisms that coordinate morphogenesis between the different epithelial cell types that make up the optical lens. Our work shows that these epithelial cells present contractile apical-medial MyosinII meshworks, which control the apical area and junctional geometry of these cells during lens development. Our study also suggests that MyosinII meshworks drive cell shape changes in response to external forces, and thus they mediate part of the biomechanical coupling that takes place between these cells. Importantly, our work, including mathematical modelling of forces and material stiffness during lens development, raises the possibility that increased cell stiffness acts as a mechanism for limiting this mechanical coupling. We propose this might be required in complex tissues, where different cell types undergo concurrent morphogenesis and where averaging out of forces across cells could compromise individual cell apical geometry and thereby organ function.

developmental biology↗

bioRxiv2019

YAP is involved in replenishment of granule cell progenitors following injury to the neonatal cerebellum

The cerebellum (CB) undergoes major rapid growth during the third trimester and early neonatal stage in humans, making it vulnerable to injuries in pre-term babies. Experiments in mice have revealed a remarkable ability of the neonatal CB to recover from injuries around birth. In particular, recovery following irradiation-induced ablation of granule cell precursors (GCPs) involves adaptive reprogramming of Nestin-expressing glial progenitors (NEPs). Sonic hedgehog signaling is required for the initial step in NEP reprogramming; however, the full spectrum of developmental signaling pathways that promote NEP-driven regeneration is not known. Since the growth regulatory Hippo pathway has been implicated in the repair of several tissue types, we tested whether Hippo signaling is involved in regeneration of the CB. Using mouse models, we found that the Hippo pathway transcriptional co-activator YAP (Yes-associated protein) but not TAZ (transcriptional coactivator with PDZ binding motif) is required in NEPs for full recovery of the CB following irradiation one day after birth. The size of the adult CB, and in particular the internal granule cell layer produced by GCPs, is significantly reduced in mutants, and the organization of Purkinje cells and Bergmann glial fibers is disrupted. Surprisingly, the initial proliferative response of Yap mutant NEPs to irradiation is normal and the cells migrate to the GCP niche, but then undergo increased cell death. Loss of Yap in NEPs or GCPs during normal development leads to only mild defects in differentiation. Moreover, loss of Taz does not abrogate regeneration of GCPs by Yap mutant NEPs or alter development of the cerebellum. Our study provides new insights into the molecular signaling underlying postnatal cerebellar development and regeneration.

developmental biology↗

bioRxiv2019

Loss of function mutation of mouse Snap29 on a mixed genetic background phenocopy abnormalities found in CEDNIK and 22q11.2 Deletion Syndrome patients

Synaptosomal-associated protein 29 (SNAP29) is a member of the SNARE family of proteins involved in maintenance of various intracellular protein trafficking pathways. SNAP29 maps to the 22q11.2 region and is deleted in 90% of patients with 22q11.2 deletion syndrome (22q11.2DS). However, the contribution of hemizygosity of SNAP29 to developmental abnormalities in 22q11.2DS remains to be determined. Mutations in SNAP29 are responsible for the developmental syndrome called CEDNIK (cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma). On an inbred C57Bl/6J genetic background, only the ichthyotic skin defect associated with CEDNIK was reported. In this study, we show that loss of function mutation of Snap29 on a mixed genetic background not only models skin abnormalities found in CEDNIK, but also phenocopy ophthalmological, neurological, and motor defects found in these patients and a subset of 22q11.2DS patients. Thus, our findings indicate that mouse models of human syndromes should be analyzed on a mixed genetic background. Our work also reveals an unanticipated requirement for Snap29 in male fertility, and support contribution of hemizygosity for SNAP29 to the phenotypic spectrum of abnormalities found in 22q11.2DS patients.

developmental biology↗

bioRxiv2019

Basal protrusions mediate spatiotemporal patterns of spinal neuron differentiation

During early spinal cord development, neurons of particular subtypes differentiate with a sparse periodic pattern while later neurons differentiate in the intervening space to eventually produce continuous columns of similar neurons. The mechanisms that regulate this spatiotemporal pattern are unknown. In vivo imaging of zebrafish reveals differentiating spinal neurons transiently extend two long protrusions along the basal surface of the spinal cord prior to axon initiation. These protrusions express Delta protein consistent with the possibility they influence Notch signalling at a distance of several cell diameters. Experimental reduction of laminin expression leads to smaller protrusions and shorter distances between differentiating neurons. The experimental data and a theoretical model support the proposal that the pattern of neuronal differentiation is regulated by transient basal protrusions that deliver temporally controlled lateral inhibition mediated at a distance. This work uncovers novel, stereotyped protrusive activity of new-born neurons that organizes long distance spatiotemporal patterning of differentiation.

developmental biology↗

bioRxiv2019

Positive feedback loop of regulating ERK phosphorylation in mESCs mediated by Etv5-Tet2-Fgfr2 axis

Dynamic equilibrium of extracellular signal-regulated kinase (ERK) activity is regulated elaborately by multiple feedback loops to ensure the normal self-renewal of mouse embryonic stem cells (mESCs). Previous studies on mESCs have demonstrated that the negative feedback loops are engaged to prevent the overactivated ERK phosphorylation (pERK). It is not clear whether there is any positive feedback loop involved to maintain a minimum of pERK in mESCs. Here, we found that blocking fibroblast growth factor (FGF)-ERK pathway by chemical PD0325901 downregulated the transcription of E26 transformation-specific (ETS) family transcription factor Etv5 in mESCs. In turn, knockout (KO) of Etv5 by CRISPR/Cas9 decreased pERK. Moreover, Etv5 KO enhanced the DNA methylation at promoter of fibroblast growth factor receptor 2 (Fgfr2) by downregulating DNA hydroxylase Tet2, which further decreased the expression of Fgfr2 in mESCs. Collectively, a positive feedback loop of regulating pERK was revealed in mESCs, which was mediated by Etv5-Tet2-Fgfr2 axis. Our findings provide a new paradigm for pERK regulation in mESCs and will be useful to understand the cell fate determination during early embryo development.

developmental biology↗