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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Glia mediated ionic balance in the stem cell niche is required for the proper proliferation of neurogenic tissues and wiring of neural circuits.

The cellular niche that surrounds stem cells modulates their self-renewal and proliferative properties. In the nervous system glial cells form part of the neural stem cell niche and express a wide variety of ion channels. While the physiological roles of ion channels in glia have been mainly studied in the mature nervous system, the contribution of glial ion channel function in the niche to nervous system development is poorly understood. To gain insight we explored the function of the Drosophila ClC-a chloride channel since its human ortholog CLCN2 is expressed in glial cells and impairment of its function results in neurodevelopmental disorders. We found ClC-a expressed in the niche in cortex glia. These glial cells are in close association with neurogenic tissues and also wrap neuronal cell bodies as they mature away from the niche. Characterization of loss of function ClC-a mutants showed these animals had smaller brains and widespread axon guidance defects. Here we show that ClC-a is required in cortex glia for the proliferation of neuroepithelial cells and neuroblasts, as well as for neuronal survival. We could also relate the axon guidance defects observed in photoreceptors to impaired proliferation in a neuroblast lineage that generates guidepost glial cells essential for this process. Taken together, our results show that ion channels expressed in glia forming part of the neural stem cell niche can have a significant impact on nervous system development. We propose that ion channels can non-autonomously modulate the number of neural progenitors and the progeny of neuroblast lineages, consequently affecting the correct assembly of neural circuits.

developmental biology↗

bioRxiv2018

A gene expression atlas of embryonic neurogenesis in Drosophila reveals complex spatiotemporal regulation of lncRNAs.

Cell type specification during early nervous system development in Drosophila melanogaster requires precise regulation of gene expression in time and space. Resolving the programs driving neurogenesis has been a major challenge owing to the complexity and rapidity with which distinct cell populations arise. To resolve the cell type-specific gene expression dynamics in early nervous system development, we have sequenced the transcriptomes of purified neurogenic cell types across consecutive time points covering critical events in neurogenesis. The resulting gene expression atlas comprises a detailed resource of global transcriptome dynamics that permits systematic analysis of how cells in the nervous system acquire distinct fates. We resolve known gene expression dynamics and uncover novel expression signatures for hundreds of genes among diverse neurogenic cell types, most of which remain unstudied. We also identified a set of conserved and processed long-noncoding RNAs (lncRNAs) that exhibit spatiotemporal expression during neurogenesis with exquisite specificity. LncRNA expression is highly dynamic and demarcates specific subpopulations within neurogenic cell types. Our spatiotemporal transcriptome atlas provides a comprehensive resource to investigate the function of coding genes and noncoding RNAs during critical stages of early neurogenesis.\n\nSummary statementWe present a spatiotemporal transcriptome during early Drosophila embryonic nervous system development, revealing a complex cell type-specific network of mRNAs and IncRNAs.

developmental biology↗

bioRxiv2018

Trynity controls epidermal barrier function and respiratory tube maturation in Drosophila by modulating apical extracellular matrix nano-patterning

The outer surface of insects is covered by the cuticle, which is derived from the apical extracellular matrix (aECM). The aECM is secreted by epidermal cells during embryogenesis. The aECM exhibits large variations in structure, function, and constituent molecules, reflecting the enormous diversity in insect appearances. To investigate the molecular principles of aECM organization and function, here we studied the role of a conserved aECM protein, the ZP domain protein Trynity, in Drosophila melanogaster. We first identified trynity as an essential gene for epidermal barrier function. trynity mutation caused disintegration of the outermost envelope layer of the cuticle, resulting in small- molecule leakage and in growth and molting defects. In addition, the tracheal tubules of trynity mutants showed defects in pore-like structures of the cuticle, and the mutant tracheal cells failed to absorb luminal proteins and liquid. Our findings indicated that trynity plays essential roles in organizing nano-level structures in the envelope layer of the cuticle that both restrict molecular trafficking through the epidermis and promote the massive absorption pulse in the trachea.\n\nSummary StatementThe zona pellucida domain protein Trynity controls the structural organization and function of the apical extracellular matrix in the epidermis and trachea of Drosophila.

developmental biology↗

bioRxiv2018

Exploring differentially expressed key genes related to development of follicle by RNA-seq in Peking ducks (Anas Platyrhynchos)

Duck follicle enter different reproductive phases throughout life, and follicle gene expression patterns differ according to these phases. In particular, differentially expressed genes and related to development of follicle (mRNAs) play an important role to explore the key genes in this process; however, the expression profiles of these genes remain unclear. In this study, transcriptome sequencing was used to investigate the expression levels of duck ovarian genes, and comparative transcriptional analysis was carried out to identify differential genes, cluster them into groups and function identification. The results showed differential expression of 593 coding genes between young and laying ducks, and of 518 coding genes between laying and old ducks. In further GO analysis, 35 genes from the comparison bewtween old ducks and laying ducks have significant been changed involved in hormones related to follicle development. They include up-regulated genes StAR, CYP17, EPOX, 3{beta}-HSD, CYP1B1 CYP19A1 and down-regulated genes SR-B1 in laying ducks hormone synthesis than old ducks. Among which EPOX is a key gene for time special highly expression during egg laying stage, and other key regulatory genes highly expression showed in young and laying stage and lower expression showing with follicular development stopping. Therefore, EPOX is key regulator for duck follicle development in laying period, when its expression level decrease 98% the follicular development will stopping in duck life cycle.

developmental biology↗

bioRxiv2018

Hierarchical stem cell topography splits growth and homeostatic functions in the fish gill

While lower vertebrates contain adult stem cells (aSCs) that maintain homeostasis and drive unexhaustive organismal growth, mammalian aSCs display mainly the homeostatic function. Understanding aSC-driven growth is of paramount importance to promote organ regeneration and prevent tumor formation in mammals. Here we present a clonal approach to address common or dedicated populations of aSCs for homeostasis and growth. Our functional assays on medaka gills demonstrate the existence of separate homeostatic and growth aSCs, which are clonal but differ in their topology. While homeostatic aSCs are fixed, embedded in the tissue, growth aSCs locate at the expanding peripheral zone. Modifications in tissue architecture can convert the homeostatic zone into a growth zone, indicating a leading role for the physical niche defining stem cell output. We hypothesize that physical niches are main players to restrict aSCs to a homeostatic function in animals with a fixed adult size.

developmental biology↗

bioRxiv2018

Longitudinal dissection in brain organoids at single cell resolution uncovers the developmental role of GSK3 in human corticogenesis

The regulation of proliferation and polarity of neural progenitors is crucial for the development of the brain cortex, with modes and timings of cell division intimately related to the stereotypical acquisition of layer-specific neuronal identities. Animal studies have implicated glycogen synthase kinase 3 (GSK3) as a pivotal regulator of both proliferation and polarity, yet the functional relevance of its signaling for the unique features of human corticogenesis remain to be elucidated. Here we harness human cortical brain organoids to probe, at single cell resolution, the longitudinal impact of GSK3 inhibition through multiple developmental stages. Our results indicate that chronic GSK3 inhibition increases the proliferation of neural progenitors and causes massive derangement of cortical tissue architecture. Surprisingly, single cell transcriptome profiling revealed only a discrete impact on early neurogenesis and uncovered the outer radial glia and the astrogenic lineage as the main GSK3-dependent developmental domains. Through this first single cell-level dissection of the GSK3 regulatory network in human corticogenesis, our work uncovers a remarkably specific conduit between the architecture of progenitor niches and lineage specification.

developmental biology↗

bioRxiv2018

Speeding up anterior-posterior patterning of insects by differential initialization of the gap gene cascade

Recently, it was shown that anterior-posterior patterning genes in the red flour beetle Tribolium castaneum are expressed sequentially in waves. However, in the fruit fly Drosophila melanogaster, an insect with a derived mode of embryogenesis compared to Tribolium, anterior-posterior patterning genes quickly and simultaneously arise as mature gene expression domains that, afterwards, undergo slight posterior-to-anterior shifts. This raises the question of how a fast and simultaneous mode of patterning, like that of Drosophila, could have evolved from a rather slow sequential mode of patterning, like that of Tribolium. In this paper, we elucidate a mechanism for this evolutionary transition based on a switch from a uniform to a gradient-mediated initialization of the gap gene cascade by maternal Hb. The model is supported by computational analyses and experiments.

developmental biology↗

bioRxiv2018

Temporal identity establishes columnar neuron morphology, connectivity, and function in a Drosophila navigation circuit

The insect central complex (CX) is a conserved brain region containing 60+ neuronal subtypes, several of which contribute to navigation. It is not known how CX neuronal diversity is generated or how developmental origin of subtypes relates to function. We mapped the developmental origin of four key CX subtypes and found that neurons with similar origin have matching axon/dendrite targeting. Moreover, we found that the temporal transcription factor (TTF) Eyeless/Pax6 regulates the development of two recurrently-connected CX subtypes: Eyeless loss simultaneously produces ectopic P-EN neurons with normal axon/dendrite projections, and reduces the number of E-PG neurons. Furthermore, the transient loss of Eyeless during development impairs adult flies capacity to perform celestial navigation. We conclude that neurons with similar developmental origin have similar connectivity, that Eyeless maintains equal E-PG and P-EN neuron number, and that Eyeless is required for the development of circuits that control adult navigation.

developmental biology↗

bioRxiv2018

Neuroblast-specific chromatin landscapes allow integration of spatial and temporal cues to generate neuronal diversity in Drosophila

During early neurogenesis in flies and mice, spatial and temporal cues interact to specify neuronal diversity, yet in no organism is it known how spatial and temporal cues are integrated. We used Targeted DamID (TaDa) to identify the genomic binding sites of the temporal transcription factor Hunchback in two adjacent Drosophila neuroblasts (NB5-6 and NB7-4). Hunchback targets were different in each neuroblast. Profiling chromatin accessibility showed that each neuroblast had a distinct chromatin landscape: Hunchback-bound loci in NB5-6 were in open chromatin, but the same loci in NB7-4 were in closed chromatin. Moreover, binding of the spatial factor Gsb/Pax3, essential for NB5-6 specification, was correlated with open chromatin and Hunchback-enriched loci in NB5-6, but not NB7-4. We propose early-acting spatial factors establish a unique chromatin landscape in each neuroblast, thereby restricting temporal factor binding to different loci in each neuroblast, resulting in different neurons in each neuroblast lineage.\n\nImpact statementIntegration of spatial and temporal identity during Drosophila neurogenesis is due to spatial factors generating neuroblast-specific chromatin thereby biasing subsequent temporal transcription factor binding and producing neuroblast-specific neurons.

developmental biology↗

bioRxiv2018

Volumetric reconstruction of main Caenorhabditis elegans neuropil at two different time points

Detailed knowledge of both synaptic connectivity and the spatial proximity of neurons is crucial for understanding wiring specificity in the nervous system. Here, we volumetrically reconstructed the C. elegans nerve ring from legacy serial-sectioned electron micrographs at two distinct time points: the L4 and young adult. The new volumetric reconstructions provide detailed spatial and morphological information of neural processes in the nerve ring. Our analysis suggests that the nerve ring exhibits three levels of wiring specificity: spatial, synaptic and subcellular. Neuron classes innervate well defined neighborhoods and aggregate functionally similar synapses to support distinct computational pathways. Connectivity fractions vary based on neuron class and synapse type. We find that the variability in process placement accounts for less than 20% of the variability in synaptic connectivity and models based only on spatial information cannot account for the reproducibility of synaptic connections among homologous neurons. This suggests that additional, non-spatial factors also contribute to synaptic and subcellular specificity. With this in mind, we conjecture that a spatially constrained, genetic model could provide sufficient synaptic specificity. Using a model of cell-specific combinatorial genetic expression, we show that additional specificity, such as sub-cellular domains or alternative splicing, would be required to reproduce the wiring specificity in the nerve ring.

developmental biology↗

bioRxiv2018

Lgr5+ stem/progenitor cells reside at the apex of the embryonic hepatoblast pool

AbstractDuring mouse embryogenesis, progenitors within the liver known as hepatoblasts give rise to adult hepatocyte and cholangiocyte cells. Hepatoblasts, which are specified at E8.5-E9.0, have been regarded as a homogeneous population of progenitors, which initiate differentiation into hepatocytes and cholangiocytes from E13.5 onwards. Recently, sub-populations of transcriptionally different hepatoblasts have been identified as already present at E11.5 by single cell RNAseq (scRNAseq) analysis. However, whether these transcriptional differences result from functionally heterogeneous hepatoblast populations is unknown. Here we show that the hepatoblast pool is not only transcriptionally but also functionally heterogeneous and that a sub-population of E9.5-E10.0 hepatoblasts exhibits a previously unidentified early commitment to cholangiocyte fate. Importantly, we also identify a sub-population of bona-fide E9.5 hepatoblasts which express the adult stem cell marker Lgr5 and contribute to liver development by generating both hepatocyte and cholangiocyte progeny that persist for the life-span of the mouse. Using a combination of lineage tracing and scRNAseq, we show that Lgr5 marks E9.5-E10.0 bi-potent liver progenitors residing at the apex of a hierarchy of the hepatoblast population. Notably, isolated Lgr5+ hepatoblasts can be clonally expanded in vitro into embryonic liver organoids, which can commit to hepatocyte or cholangiocyte fates dependent upon the culture conditions. Our study represents the first functional demonstration of heterogeneity within E9.5 hepatoblasts and identifies Lgr5 as a marker for a sub-population of truly bi-potent liver progenitors.\n\nSummary StatementLgr5 positive bi-potential hepatoblasts contribute to liver development and reside at the apex of an embryonic liver progenitor pool.

developmental biology↗

bioRxiv2018

A refutation to ‘A new A-P compartment boundary and organizer in holometabolous insect wings.’

We respond to a recent report by Abbasi and Marcus who present two main findings: first they argue that there is an organiser and a compartment boundary within the posterior compartment of the butterfly wing. Second, they present evidence for a previously undiscovered lineage boundary near wing vein 5 in Drosophila, a boundary that delineates a \"far posterior\" compartment. Clones of cells were marked with the yellow mutation and they reported that these clones always fail to cross a line close to vein 5 on the Drosophila wing. In our hands yellow proved an unusable marker for clones in the wing blade and therefore we reexamined the matter. We marked clones of cells with multiple wing hairs or forked and found a substantial proportion of these clones cross the proposed lineage boundary near vein 5, in conflict with their findings and conclusion. As internal controls we showed that these same clones respect the other two well established compartment boundaries: the anteroposterior compartment boundary is always respected. The dorsoventral boundary is mostly respected, and is crossed only by clones that are induced early in development, consistent with many reports. We question the validity of Abbasi and Marcus conclusions regarding the butterfly wing but present no new data.\n\nArising from: R. Abbasi and J. M. Marcus Sci. Rep. 7, 16337 (2017); https://doi.org/10.1038/s41598-017-16553-5

developmental biology↗

bioRxiv2018

Arkadia degrades SNON to activate level-specific NODAL responses

How cells assess levels of signaling and select to transcribe different target genes depending on the levels of activated effectors remains elusive. High NODAL-signalling levels specify anterior/head, lower specify posterior, and complete loss abolishes anterior-posterior patterning in the mammalian embryo. Here we show that cells assess NODAL-activated SMAD2 and SMAD3 (SMAD2/3) effector-levels by complex formation and pairing each effector with the co-repressor SNON, which is present in the cell before signaling. These complexes enable the E3-ubiquitin ligase Arkadia (RNF111) to degrade SNON. High SMAD2/3 levels can saturate and remove SNON, leading to derepression and activation of a subset of targets (high targets) that are highly susceptible to SNON repression. However, low SMAD2/3 levels can only reduce SNON preventing derepression/activation of high targets. Arkadia degrades SNON transiently only upon signaling exposure, leading to dynamic signaling-responses, which most likely initiate level-specific cell-fate decisions. Arkadia-null mouse embryos and Embryonic Stem Cells (ESC) cannot develop anterior tissues and head. However, SnoN/Arkadia, double-null embryos and ESCs are rescued confirming that Arkadia removes SNON, to achieve level-dependent cell-fates One Sentence SummarySignaling intensity induces equivalent degradation of a transcriptional repressor leading to level-dependent responses.

developmental biology↗

bioRxiv2018

The Spatio-Temporal Control of Zygotic Genome Activation

One of the earliest and most significant events in embryonic development is zygotic genome activation (ZGA). In several species, bulk transcription begins at the mid-blastula transition (MBT) when, after a certain number of cleavages, the embryo attains a particular nuclear-to-cytoplasmic (N/C) ratio, maternal repressors become sufficiently diluted, and the cell cycle slows down. Here we resolve the frog ZGA in time and space by profiling RNA polymerase II (RNAPII) engagement and its transcriptional readout. We detect a gradual increase in both the quantity and the length of RNAPII elongation before the MBT, revealing that >1,000 zygotic genes disregard the N/C timer for their activation, and that the sizes of newly transcribed genes are not necessarily constrained by cell cycle duration. We also find that Wnt, Nodal and BMP signaling together generate most of the spatio-temporal dynamics of regional ZGA, directing the formation of orthogonal body axes and proportionate germ layers.

developmental biology↗

bioRxiv2018

Cadherin switch marks germ layer formation in the diploblastic sea anemone Nematostella vectensis

Morphogenesis is a shape-building process during development of multicellular organisms. During this process the establishment and modulation of cell-cell contacts play an important role. Cadherins, the major cell adhesion molecules, form adherens junctions connecting ephithelial cells. Numerous studies in Bilateria have shown that cadherins are associated with the regulation of cell differentiation, cell shape changes, cell migration and tissue morphogenesis. To date, the role of Cadherins in non-bilaterians is unknown. Here, we study the expression and the function of two paralogous classical cadherins, cadherin1 and cadherin3, in the diploblastic animal, the sea anemone Nematostella vectensis. We show that a cadherin switch is accompanying the formation of germ layers. Using specific antibodies, we show that both cadherins are localized to adherens junctions at apical and basal positions in ectoderm and endoderm. During gastrulation, partial EMT of endodermal cells is marked by a step-wise downregulation of cadherin3 and upregulation of cadherin1. Knockdown experiments show that both cadherins are required for maintenance of tissue integrity and tissue morphogenesis. This demonstrates that cnidarians convergently use cadherins to differentially control morphogenetic events during development.

developmental biology↗

bioRxiv2018

The coordination of terminal differentiation and cell cycle exit is mediated through the regulation of chromatin accessibility

During terminal differentiation most cells will exit the cell cycle and enter into a prolonged or permanent G0. Cell cycle exit is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called DREAM. However when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. Here we provide evidence that developmentally programmed, temporal changes in chromatin accessibility at a subset of critical cell cycle genes acts to enforce cell cycle exit during terminal differentiation in the Drosophila melanogaster wing. We show that during terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators cycE, e2f1 and stg. This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle re-activation. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains largely unaffected and the closing of enhancers at cycE, e2f1 and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. Our results uncover a mechanism that acts as a cell cycle-independent timer to limit aberrant cycling in terminally differentiating tissues. In addition, we provide a new molecular description of the cross-talk between cell cycle exit and terminal differentiation during metamorphosis.

developmental biology↗

bioRxiv2018

High temperature limits on developmental canalization in the ascidian Ciona intestinalis

The normal embryogenesis of marine animals is typically confined to a species-specific range of temperatures. Within that temperature range development results in a consistent, or canalized, phenotype, whereas above and below the range abnormal phenotypes are produced. This study reveals an abrupt high temperature limit, occurring over a 1-2{degrees}C range, for normal embryonic development in C. intestinalis. Above that threshold morphological abnormalities in the notochord and other organs are observed, beginning with cleavage and gastrula stages, and becoming more pronounced as embryogenesis proceeds. However, even in highly morphologically abnormal temperature disrupted (TD) embryos, cell type specification, including muscle, endoderm, notochord, and sensory pigment cells is accomplished. An explanation for this finding is that in C. intestinalis cell type specification occurs relatively early in embryogenesis, due to cleavage stage segregation of maternal cytoplasmic determinants and short-range cell interactions, which are largely intact in TD embryos. On the other hand, morphogenesis of the notochord and other structures is dependent on precise cell movement and shape changes after the gastrula stage, which appear to be disrupted above the high temperature threshold. These findings have implications for the relationship between ecology and reproduction in C. intestinalis. More broadly they point to mechanisms behind canalization in animals, such as ascidians, characterized by early, largely autonomous, cell type specification.

developmental biology↗

bioRxiv2018

Wnt/β-catenin signaling is required for the development of multiple nephron segments

The nephron is composed of distinct segments that perform unique physiological functions to generate urine. Little is known about how multipotent nephron progenitor cells differentiate into different nephron segments. It is well known that Wnt/{beta}-catenin signaling regulates the maintenance and commitment of mesenchymal nephron progenitors during kidney development. However, it is not fully understood how it regulates nephron patterning after nephron progenitors undergo mesenchymal-to-epithelial transition. To address this, we performed {beta}-catenin loss-of-function and gain-of-function studies in epithelial nephron progenitors in the mouse kidney. Consistent with a previous report, the formation of the renal corpuscle was defective in the absence of {beta}-catenin. Interestingly, we found that epithelial nephron progenitors lacking {beta}-catenin were able to form presumptive proximal tubules but that they failed to further develop into differentiated proximal tubules, suggesting that Wnt/{beta}-catenin signaling plays a critical role in proximal tubule development. We also found that epithelial nephron progenitors lacking {beta}-catenin failed to form the distal tubules. Constitutive activation of Wnt/{beta}-catenin signaling blocked the proper formation of all nephron segments, suggesting tight regulation of Wnt/{beta}-catenin signaling during nephron patterning. This work shows that Wnt/{beta}-catenin signaling regulates the patterning of multiple nephron segments along the proximo-distal axis of the mammalian nephron.

developmental biology↗