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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Early genome activation in Drosophila is extensive with an initial tendency for aborted transcripts and retained introns

Control of metazoan embryogenesis shifts from maternal to zygotic gene products as the zygotic genome becomes transcriptionally activated. In Drosophila, zygotic genome activation (ZGA) begins with a minor wave, but technical challenges have hampered the identification of early transcripts or obscured the onset of their transcription. Here, we develop an approach to isolate transcribed mRNAs and apply it over the course of the minor wave and the start of the major wave of Drosophila ZGA. Our results increase known genes of the minor wave by 10 fold and show that this wave is continuous and gradual. Transposable-element mRNAs are also produced, but discontinuously. Genes in the early and middle part of the minor wave are short with few if any introns, and their transcripts are frequently aborted and tend to have retained introns, suggesting that inefficient splicing as well as rapid cell divisions constrain the lengths of early transcripts.

developmental biology↗

bioRxiv2018

Multiscale analysis of 3D nuclear morphology reveals new insights into growth plate organization in mice

The shape of the nucleus is tightly associated with cell morphology, the mechanical environment, and differentiation and transcriptional states. Yet, imaging of nuclei in three dimensions while preserving the spatial context of the tissue has been highly challenging. Here, using the embryonic tibial growth plate as a model for cell differentiation, we study nuclear morphology by imaging cleared samples by light-sheet fluorescence microscopy. Next, we quickly segmented tens of thousands of nuclei using several open-source tools including machine learning. Finally, segmented nuclei underwent morphometric analysis and 3D spatial reconstruction using newly designed algorithms. Our method revealed differences in nuclear morphology between chondrocytes at different differentiation stages. Additionally, we identified different morphological patterns in opposing growth plates, such as gradients of volume and surface area, as well as features characteristic of specific growth plate zones, such as sphericity and orientation. Altogether, this work supports a link between nuclear morphology and cell differentiation. Moreover, it demonstrates the suitability of our approach for studying the relationships between nuclear morphology and organ development.\n\nAuthor summaryThere has been a growing interest in the relationship between nuclear morphology and its regulation of gene expression. However, to study global patterns of nuclear morphology within a tissue we must address the problem of acquiring and analyzing multiscale data, ranging from the tissue level through to subcellular resolution. We have established a new pipeline that enables acquisition and segmentation of hundreds of thousands of nuclei at a resolution that allows quantitative analysis. Moreover we have developed new algorithms that allow superimposing morphological aspects of hundreds of thousands of nuclei onto a visual representation of the entire tissue, allowing us to study nuclear morphology at an organ level. Using mouse growth plates as a model for the relationship between nuclear morphology and tissue differentiation, we show that nuclei change different aspects of their morphology during chondrocyte differentiation. Growth plates are usually described generically in the literature, suggesting they lack unique characteristics. We challenge this dogma by showing that morphological features such as volume distribute differently in opposing growth plates. Altogether, this work highlights the possible role of nuclear shape in the regulation of cell differentiation and demonstrates that our approach enables the study of nuclear morphology patterns within a tissue.

developmental biology↗

bioRxiv2018

millepattes micropeptides are an ancient developmental switch required for embryonic patterning

Small open reading frames (smORFs) that code for \"micropeptides\" (10-100 amino acids) exhibit remarkable evolutionary complexity. Conserved micropeptides encoded by the millepattes (mlpt) gene are essential in Tribolium for embryogenesis but in Drosophila, function only in leg and cuticle differentiation. We find that a module identified in Drosophila trichome patterning, comprising Mlpt, UBR3, and Shaven-baby (Svb), coordinates early embryo patterning in several insect orders. Intriguingly, Mlpt segmentation function can be re-awakened in the Drosophila blastoderm, demonstrating the potency of an ancestral developmental switch retained despite evolving embryonic patterning modes. smORFs like millepattes thus illustrate plasticity of micropeptide functions despite constraints of essential genetic networks.\n\nOne sentence summaryA module comprising the small ORFs mlpt/pri/tal, the transcription factor Svb, and the ubiquitin ligase UBR3, possesses an ancestral function in insect embryo patterning which is lost in flies but reactivated when Svb expression is restored.

developmental biology↗

bioRxiv2018

Compensatory mechanisms render Tcf7l1a dispensable for eye formation despite its requirement in eye field specification

The vertebrate eye originates from the eyefield, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7la is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much reduced eyefield in tcf7l1a mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through compensation by paralogous genes; instead, the smaller optic vesicle of tcf7l1a mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in hesx1, cct5 and gdf6a, which give synthetically enhanced eye specification or growth phenotypes when in combination with the tcf7l1a mutation.

developmental biology↗

bioRxiv2018

The USTC complex co-opts an ancient machinery to drive piRNA transcription in C. elegans

Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and non-self nucleic acids, maintain genome integrity, and are essential for fertility in a variety of organisms. In C. elegans most piRNA precursors are transcribed from two genomic clusters that contain thousands of individual piRNA transcription units. While a few genes have been shown to be required for piRNA biogenesis the mechanism of piRNA transcription remains elusive. Here we used functional proteomics approaches to identify an upstream sequence transcription complex (USTC) that is essential for piRNA biogenesis. The USTC complex contains PRDE-1, TOFU-4, TOFU-5 and SNPC-4. The USTC complex form a unique piRNA foci in germline nuclei and coat the piRNA cluster genomic loci. USTC factors associate with the Ruby motif just upstream of type I piRNA genes. USTC factors are also mutually dependent for binding to the piRNA clusters and to form the piRNA foci. Interestingly, USTC components bind differentially to piRNAs in the clusters and other non-coding RNA genes. These results reveal USTC as a striking example of the repurposing of a general transcription factor complex to aid in genome defence against transposons.

developmental biology↗

bioRxiv2018

TUBA1A mutations identified in lissencephaly patients dominantly disrupt neuronal migration and impair dynein activity

Tubulinopathies are severe human brain malformations associated with mutations in tubulin genes. Despite the identification of many tubulin mutations in patients, we do not understand how these mutations impact the microtubule cytoskeleton, how the changes to microtubule function lead to brain malformations, or how different tubulin isotypes regulate microtubules to support normal neurodevelopment. TUBA1A -tubulin is the most commonly affected tubulin isotype in tubulinopathy patients. Heterozygous mutations in TUBA1A have been identified in patients with diverse cortical malformations including microlissencephaly, lissencephaly, pachygyria, and polymicrogyria. Here we focus on mutations affecting the conserved arginine at position 402 (R402), which account for 30% of all reported TUBA1A mutations in patients. We demonstrate that exogenous expression of TUBA1A-R402C and TUBA1A-R402H patient alleles is sufficient to dominantly disrupt cortical neuron migration in the developing mouse brain, recapitulating the human lissencephaly phenotype. Intriguingly, ectopic expression of TUBA1A-R402C/H alleles does not alter morphology, axonal trafficking, or microtubule polymerization rates in cultured neurons, but does lead to subtle changes in axonal microtubule orientation. Further, we find that budding yeast -tubulin with analogous R402C and R402H mutations assembles into microtubules but disrupts the activity of the microtubule motor dynein. The level of dynein impairment scales with abundance of R402 mutant -tubulin in the cell. Together, our results support a model in which tubulinopathy mutations at R402 poison the microtubule network in young neurons by creating defective binding sites for dynein at the microtubule surface.

developmental biology↗

bioRxiv2018

Sequential Specification of Oligodendrocyte and NG2 Cell Fates by Distinct Levels of Hedgehog Signaling

During development of the central nervous system oligodendrocyte precursor cells (OPCs) give rise to both myelinating oligodendrocytes and NG2 glia, which are the most proliferative cells in the adult mammalian brain. NG2 glia retain characteristics of OPCs, and some NG2 glia produce oligodendrocytes, but many others persist throughout adulthood. Why some OPCs differentiate as oligodendrocytes during development whereas others persist as OPCs and acquire characteristics of NG2 glia is not known. Using zebrafish spinal cord as a model, we found that OPCs that differentiate rapidly as oligodendrocytes and others that remain as OPCs arise in sequential waves from distinct neural progenitors. Additionally, oligodendrocyte and persistent OPC fates are specified during a defined critical period by small differences in Shh signaling and Notch activity, which modulates Shh signaling response. Thus, our data indicate that OPCs fated to produce oligodendrocytes or remain as OPCs during development are specified as distinct cell types, raising the possibility that the myelinating potential of OPCs is set by graded Shh signaling activity.

developmental biology↗

bioRxiv2018

AKT, NOTCH and GSK3β interact to trigger early myogenesis in vertebrate embryos.

During early embryonic development, migrating neural crest cells expressing the NOTCH ligand Delta1 (DLL1) trigger the activation of NOTCH1 signaling in selected epithelial cells within newly formed somites. A key event in this process is a dramatic inhibition of GSK3{beta} activity, initiated by the activation of NOTCH1 and that takes place independent of its transcriptional function. Here, we investigated the mechanism whereby NOTCH1 exerts its non-canonical function in somites. Using the activation of myogenesis as a read-out of the ability of NOTCH receptors to trigger transcription-independent responses in somites, we found that all NOTCH receptors (1-4) activate MYF5 expression and we showed that the RAM (RBPJ-Associated Molecule) domain of the NOTCH Intracellular Domain (NICD) is necessary and sufficient in this process. We then demonstrated that the NOTCH1 Intracellular Domain (NICD1) physically interacts in the cytosol with GSK3{beta} and with the serine threonine protein kinase AKT. Activating AKT triggers myogenesis, likely via the inhibition of GSK3{beta}. We found that AKT, in a dose-dependent manner, decreases the transcriptional activity of NOTCH, suggesting a role in the balance between the canonical and non-canonical functions of NOTCH. Altogether these data strongly support the hypothesis that transcription-independent function of NICD is a central mechanism driving myogenesis in early somites and suggests that, in this tissue, AKT, NOTCH and GSK3{beta} interact in the cytoplasm to trigger a signaling cascade that leads to the formation of the early myotome in vertebrates.

developmental biology↗

bioRxiv2018

Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos

The regulation of transcription requires the coordination of numerous activities on DNA, yet it remains poorly understood how transcription factors facilitate these multiple functions. Here we use lattice light-sheet microscopy to integrate single-molecule and high-speed 4D imaging in developing Drosophila embryos to study the nuclear organization and interactions of the key patterning factors Zelda and Bicoid. In contrast to previous studies suggesting stable, cooperative binding, we show that both factors interact with DNA with surprisingly high off-rates. We find that both factors form dynamic subnuclear hubs, and that Bicoid binding is enriched within Zelda hubs. Remarkably, these hubs are both short lived and interact only transiently with sites of active Bicoid dependent transcription. Based on our observations we hypothesize that, beyond simply forming bridges between DNA and the transcription machinery, transcription factors can organize other proteins into hubs that transiently drive multiple activities at their gene targets.

developmental biology↗

bioRxiv2018

Tbx1 and Foxi3 genetically interact in the third pharyngeal pouch endoderm required for thymus and parathyroid development

The mechanisms required for segmentation of the pharyngeal apparatus to individual arches are not precisely delineated in mammalian species. Here, using conditional mutagenesis, we found that two transcription factor genes, Tbx1, the gene for 22q11.2 deletion syndrome and Foxi3, genetically interact in the third pharyngeal pouch endoderm for thymus and parathyroid gland development. We found that Tbx1 is autonomously required for the endoderm to form a temporary multilayered epithelium while invaginating. E-cadherin for adherens junctions remains expressed and cells in the apical boundary express ZO-1. Foxi3 is required autonomously to modulate proliferation and promote later restoration of the endoderm to a monolayer once the epithelia meet after invagination. Completion of this process cooccurs with expression of Alcam needed to stabilize adherens junctions and extracellular, Fibronectin. These processes are required in the third pharyngeal pouch to form the thymus and parathyroid glands, disrupted in 22q11.2 deletion syndrome patients.

developmental biology↗

bioRxiv2018

Multiple Mechanisms Inactivate the LIN-41 RNA-Binding Protein to Ensure A Robust Oocyte-to-Embryo Transition in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the conserved LIN-41 RNA-binding protein is a translational repressor that coordinately controls oocyte growth and meiotic maturation. LIN-41 exerts these effects, at least in part, by preventing the premature activation of the cyclin-dependent kinase CDK-1. Here we investigate the mechanism by which LIN-41 is rapidly eliminated upon the onset of meiotic maturation. Elimination of LIN-41 requires the activities of CDK-1 and multiple SCF-type ubiquitin ligase subunits, including the conserved substrate adaptor protein SEL-10/Fbw7/Cdc4, suggesting that LIN-41 is a target of ubiquitin-mediated protein degradation. Within the LIN-41 protein, two non-overlapping regions, Deg-A and Deg-B, are individually necessary for LIN-41 degradation; both contain several potential phosphodegron sequences, and at least one of these sites is required for LIN-41 degradation. Finally, Deg-A and Deg-B are sufficient, in combination, to mediate SEL-10-dependent degradation when transplanted into a different oocyte protein. Although LIN-41 is a potent inhibitor of protein translation and M-phase entry, the failure to eliminate LIN-41 from early embryos does not result in the continued translational repression of LIN-41 oocyte mRNA targets. Based on these observations, we propose a molecular model for the elimination of LIN-41 by SCFSEL-10 and suggest that LIN-41 is inactivated before it is degraded. Furthermore, we provide evidence that another RNA-binding protein, the GLD-1 tumor suppressor, is regulated similarly. Redundant mechanisms to extinguish translational repression by RNA-binding proteins may both control and provide robustness to irreversible developmental transitions, including meiotic maturation and the oocyte-to-embryo transition.

developmental biology↗

bioRxiv2018

Comprehensive analysis of retinal development at single cell resolution identifies NFI factors as essential for mitotic exit and specification of late-born cells.

Precise temporal control of gene expression in neuronal progenitors is necessary for correct regulation of neurogenesis and cell fate specification. However, the extensive cellular heterogeneity of the developing CNS has posed a major obstacle to identifying the gene regulatory networks that control these processes. To address this, we used single cell RNA-sequencing to profile ten developmental stages encompassing the full course of retinal neurogenesis. This allowed us to comprehensively characterize changes in gene expression that occur during initiation of neurogenesis, changes in developmental competence, and specification and differentiation of each of the major retinal cell types. These data identify transitions in gene expression between early and late-stage retinal progenitors, as well as a classification of neurogenic progenitors. We identify here the NFI family of transcription factors (Nfia, Nfib, and Nfix) as genes with enriched expression within late RPCs, and show they are regulators of bipolar interneuron and Muller glia specification and the control of proliferative quiescence.

developmental biology↗

bioRxiv2018

A test of the general occurrence and predictive utility of isochronal, equiproportional, ‘variable proportional’, and ‘mixed’ development among arthropods

In isochronal (ICD) and equiproportional development (EPD), the proportion of total immature (egg, larval, and/or juvenile) development spent in each stage (developmental proportion) does not vary among stages or temperatures, respectively. ICD and EPD have mainly been reported in copepods, and whether they occur in other arthropods is not known. If they did, then rearing studies could be simplified because the durations of later developmental stages could be predicted based on those of earlier ones. The goal of this study was to test whether different taxa have ICD, EPD, or an alternative development type in which stage-specific proportions depend on temperature, termed variable proportional development (VPD), and also how well each development type allowed later-stage durations to be predicted from earlier ones. Data for 71 arthropods (arachnids, copepod and decapod crustaceans, and insects) were tested, and most (85.9 %) species were concluded to have VPD, meaning that ICD and EPD do not occur generally. However, EPD predicted later-stage durations comparably well to VPD (within 19-23 %), and thus may still be useful. Interestingly, some species showed a mixed form of development, where some stages developmental proportions varied with temperature while those of others did not, which should be further investigated.\n\nHighlightsO_LIWhether arthropod development is generally isochronal or equiproportional was tested\nC_LIO_LIDevelopmental proportions of most species stages varied with temperature\nC_LIO_LIMany species had mixed development between variable and equiproportional types\nC_LIO_LIThe general occurrence of isochronal and equiproportional development was rejected\nC_LIO_LIEquiproportional development did make reasonable predictions of stage durations\nC_LI

developmental biology↗

bioRxiv2018

Evolution of limb development in cephalopod mollusks

Cephalopod mollusks evolved numerous anatomical innovations, including specialized arms and tentacles, but little is known about the developmental mechanisms underlying the evolution of cephalopod limbs. Here we show that all three axes of cuttlefish limbs are patterned by the same signaling networks that act in vertebrates and arthropods, although they evolved limbs independently. In cuttlefish limb buds, Hedgehog is expressed anteriorly. Posterior transplantation of Hedgehog-expressing cells induced mirror-image limb duplications. Bmp and Wnt signaling, which establishes dorsoventral polarity in vertebrate and arthropod limbs, is similarly polarized in cuttlefish. Inhibition of the dorsal Bmp signal caused ectopic expression of Notum, a ventral sucker field marker, and development of ectopic suckers. Cuttlefish limbs also show proximodistally regionalized expression of Htx, Exd, Dll, Dac, Sp8/9, and Wnt genes, which delineate arm and tentacle sucker fields. These results suggest that cephalopod limbs evolved by parallel activation of an ancient genetic program for appendage development that was present in the bilaterian common ancestor.

developmental biology↗

bioRxiv2018

Mutation of sequences flanking and separating transcription factor binding sites in a Drosophila enhancer significantly alter its output

Here we explore how mutating different sequences in an enhancer that regulates patterned gene expression in Drosophila melanogaster embryos can affect its output. We used quantitative imaging to analyze the effects of a wide variety of mutations in the hunchback distal anterior enhancer. This enhancer has been shown to respond to the anterior morphogen Bicoid, but we found that mutations in only one of the five strong Bicoid sites in the enhancer has a significant effect on its binding. The pioneer factor Zelda, which binds to this enhancer and is the only other factor implicated in its activity besides Bicoid. However, we found that mutations of all its sites only has modest effect that is limited to reduction of its output in more posterior regions of the embryo, where Bicoid levels are low. In contrast to the modest effects of mutating known transcription factor binding sites, randomizing the sequences between Zelda and Bicoid sites significantly compromised enhancer activity. Finer mapping suggested that the sequences that determine activity are broadly distributed in the enhancer. Mutations in short sequences flanking Bicoid binding sites have stronger effects than mutations to Bicoid sites themselves, highlighting the complex and counterintuitive nature of the relationship between enhancer sequence and activity.

developmental biology↗

bioRxiv2018

Activin signaling informs the graded pattern of terminal mitosis and hair cell differentiation in the mammalian cochlea

The mammalian auditory sensory epithelium has one of the most stereotyped cellular patterns known in vertebrates. Mechano-sensory hair cells are arranged in precise rows, with one row of inner and three rows of outer hair cells spanning the length of the spiral-shaped sensory epithelium. Aiding such precise cellular patterning, differentiation of the auditory sensory epithelium is precisely timed and follows a steep longitudinal gradient. The molecular signals that promote auditory sensory differentiation and instruct its graded pattern are largely unknown. Here, we identify Activin A as an activator of hair cell differentiation and show, using mouse genetic approaches, that a local gradient of Activin A signaling within the auditory sensory epithelium times the longitudinal gradient of hair cell differentiation. Furthermore, we provide evidence that Activin-type signaling regulates a radial gradient of terminal mitosis within the auditory sensory epithelium, which constitutes a novel mechanism for limiting the number of inner hair cells being produced.

developmental biology↗

bioRxiv2018

Damage-induced reactive oxygen species enable regenerative signalling by the rapid repositioning of Hedgehog expressing cells.

Aquatic vertebrates have a remarkable ability to regenerate limbs and tails after amputation. Previous studies indicate that reactive oxygen species (ROS) signaling initiates regeneration, but the mechanism by which this takes place is poorly understood. Developmental signalling pathways have been shown to have pro-regenerative roles in many systems. However, whether these are playing roles that are specific to regeneration, or are simply recapitulating their developmental functions is unclear. We have analysed zebrafish larval tail regeneration and find evidence that ROS released upon wounding cause repositioning of notochord cells to the damage site. These cells secrete Hedgehog ligands which are required for regeneration. Hedgehog signalling is not required for normal tail development suggesting that it has a regeneration specific role. Our results provide a model for how ROS initiate tail regeneration, and indicate that developmental signaling pathways can play regenerative functions that are not directly related to their developmental roles.

developmental biology↗

bioRxiv2018

Choline Transporter in α/β core neurons of Drosophila mushroom body non-canonically regulates pupal eclosion and maintains neuromuscular junction integrity

Insect mushroom bodies (MB) have an ensemble of synaptic connections well-studied for their role in experience-dependent learning and several higher cognitive functions. MB requires neurotransmission for an efficient flow of information across synapses with the different flexibility to meet the demand of the dynamically changing environment of an insect. Neurotransmitter transporters coordinate appropriate changes for an efficient neurotransmission at the synapse. Till date, there is no transporter reported for any of the previously known neurotransmitters in the intrinsic neurons of MB. In this study, we report a highly enriched expression of Choline Transporter (ChT) in Drosophila MB. We demonstrate that knockdown of ChT in a sub-type of MB neurons called /{beta} core (/{beta}c) neurons leads to eclosion failure, peristaltic defect in larvae, and altered NMJ phenotype. These defects were neither observed on knockdown of proteins of the cholinergic locus in /{beta}c neurons nor by knockdown of ChT in cholinergic neurons. Thus, our study provides insights into non-canonical roles of ChT in MB.

developmental biology↗