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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

MTA proteins act redundantly within the NuRD complex to ensure fidelity of ES cell lineage commitment

Multiprotein chromatin remodelling complexes show remarkable conservation of function amongst metazoans, even though components present in invertebrates are often present as multiple paralogous proteins in vertebrate complexes. In some cases these paralogues specify distinct biochemical and/or functional activities in vertebrate cells. Here we set out to define the biochemical and functional diversity encoded by one such group of proteins within the mammalian Nucleosome Remodelling and Deacetylation (NuRD) complex: Mta1, Mta2 and Mta3. We find that, in contrast to what has been described in somatic cells, MTA proteins are not mutually exclusive within ES cell NuRD and, despite subtle differences in chromatin binding and biochemical interactions, serve largely redundant functions. Nevertheless, ES cells lacking all three MTA proteins represent a complete NuRD null and are viable, allowing us to identify a previously undetected function for NuRD in maintaining differentiation trajectory during early stages of lineage commitment.

developmental biology↗

bioRxiv2018

Notch signalling patterns retinal composition by regulating atoh7 during post-embryonic growth

Patterning of a continuously growing naive field in the context of a life-long growing organ, the teleost eye is of highest functional relevance. Intrinsic and extrinsic signals were proposed to regulate lineage specification in progenitors that exit the stem cell niche in the ciliary marginal zone (CMZ). The proper cell type composition arising from those progenitors is prerequisite for retinal function. Our findings in the teleost medaka (Oryzias latipes) uncover that the Notch-Atoh7 axis continuously patterns the CMZ. The complement of cell-types originating from the two juxtaposed progenitors marked by Notch or Atoh7 activity contains all constituents of a retinal column. Modulation of Notch signalling specifically in Atoh7-expressing cells demonstrates the crucial role of this axis in generating the correct cell type proportions. After transiently blocking Notch signalling, retinal patterning and differentiation is reinitiated de novo. Taken together we show that Notch activity in the CMZ continuously structures the growing retina by juxtaposing Notch and Atoh7 progenitors giving rise to distinct, complementary lineages, revealing a coupling of de novo patterning and cell-type specification in the respective lineages.

developmental biology↗

bioRxiv2018

Transposon silencing in the Drosophila female germline ensures genome stability in progeny embryos

The piRNA pathway functions in transposon control in the germ line of metazoans. The conserved RNA helicase Vasa is an essential piRNA pathway component, but has additional important developmental functions. Here we address the importance of Vasa-dependent transposon control in the Drosophila female germline and early embryos. We find that transient loss of vasa expression during early oogenesis leads to transposon up-regulation in supporting nurse cells of the fly egg-chamber. We show that elevated transposon levels have dramatic consequences, as de-repressed transposons accumulate in the oocyte where they cause DNA damage. We find that suppression of Chk2-mediated DNA damage signaling in vasa mutant females restores oogenesis and egg production. Damaged DNA and up-regulated transposons are transmitted from the mother to the embryos, which sustain severe nuclear defects and arrest development. Our findings reveal that the Vasa-dependent protection against selfish genetic elements in the nuage of nurse cell is essential to prevent DNA damage-induced arrest of embryonic development.

developmental biology↗

bioRxiv2018

Evolution of Endothelin signaling and diversification of adult pigment pattern in Danio fishes

Fishes of the genus Danio exhibit diverse pigment patterns that serve as useful models for understanding the genes and cell behaviors underlying the evolution of adult form. Among these species, zebrafish D. rerio exhibit several dark stripes of melanophores with sparse iridophores that alternate with light interstripes of dense iridophores and xanthophores. By contrast, the closely related species D. nigrofasciatus has an attenuated pattern with fewer melanophores, stripes and interstripes. Here we demonstrate species differences in iridophore development that presage the fully formed patterns. Using genetic and transgenic approaches we identify the secreted peptide Endothelin-3 (Edn3)--a known melanogenic factor of tetrapods--as contributing to reduced iridophore proliferation and fewer stripes and interstripes in D. nigrofasciatus. We further show the locus encoding this factor is expressed at lower levels in D. nigrofasciatus owing to cis-regulatory differences between species. Finally, we show that functions of two paralogous loci encoding Edn3 have been partitioned between skin and non-skin iridophores. Our findings reveal genetic and cellular mechanisms contributing to pattern differences between these species and suggest a model for evolutionary changes in Edn3 requirements across vertebrates.\n\nAuthor SummaryNeural crest derived pigment cells generate the spectacular variation in skin pigment patterns among vertebrates. Mammals and birds have just a single skin pigment cell, the melanocyte, whereas ectothermic vertebrates have several pigment cells including melanophores, iridophores and xanthophores, that together organize into a diverse array of patterns. In the teleost zebrafish, Danio rerio, an adult pattern of stripes depends on interactions between pigment cell classes and between pigment cells and their tissue environment. The close relative, D. nigrofasciatus has fewer stripes and prior analyses suggested a difference between these species that lies extrinsic to the pigment cells themselves. A candidate for mediating this difference is Endothelin-3 (Edn3), essential for melanocyte development in warm-blooded animals, and required by all three classes of pigment cells in an amphibian. We show that Edn3 specifically promotes iridophore development in Danio, and that differences in Edn3 expression contribute to differences in iridophore complements, and striping, between D. rerio and D. nigrofasciatus. Our study reveals a novel function for Edn3 and provides new insights into how changes in gene expression yield morphogenetic outcomes to effect diversification of adult form.

developmental biology↗

bioRxiv2018

Disparate expression specificities coded by a shared Hox-C enhancer

Can a single regulatory sequence be shared by two genes undergoing functional divergence? Here we describe a single promiscuous enhancer within the Drosophila Antennapedia Complex, EO053, that directs aspects of the expression of two adjacent genes, pb (a Hox2 ortholog) and zen2 (a divergent Hox3 paralog), with disparate spatial and temporal expression patterns. We were unable to separate the pb-like and zen2-like specificities within EO053, and we identify sequences affecting both expression patterns. Importantly, deletion of EO053 affects expression of both genes as well, despite each gene having additional enhancers. We examine sequence conservation of EO053 within the Schizophora, and show that patterns of synteny between the Hox2 and Hox3 orthologs in Arthropods are consistent with a shared regulatory relationship extending prior to the Hox3/zen divergence. Thus, EO053 represents an example of two genes having evolved disparate outputs while utilizing this shared regulatory region.

developmental biology↗

bioRxiv2018

Heterogeneity of transposon expression and activation of the repressive network in human fetal germ cells

Epigenetic resetting in germ cells during development leads to the de-repression of transposable elements (TEs). piRNAs protect fetal germ cells from potentially harmful TEs by targeted destruction of mRNA and deposition of repressive epigenetic marks. Here we provide the first evidence for an active piRNA pathway and TE repression in germ cells of human fetal testis. We identify pre-pachytene piRNAs with features of secondary amplification that map most abundantly to L1 family TEs. We find that L1-ORF1p expression is heterogeneous in fetal germ cells, peaks at mid-gestation and declines concomitantly with increasing levels of piRNAs and H3K9me3, as well as nuclear localization of HIWI2. Surprisingly, following this decline, the same cells with accumulation of L1-ORF1p display highest levels of HIWI2 and H3K9me3, whereas L1-ORF1p low cells are also low in HIWI2 and H3K9me3. Conversely, earlier in development, the germ cells lacking L1-ORF1p express high levels of the chaperone HSP90a. We propose that a subset of HSP90a-armed germ cells resists L1 expression, whereas only those vulnerable L1-expressing germ cells activate the PIWI-piRNA repression pathway which leads to epigenetic silencing of L1 via H3K9me3.

developmental biology↗

bioRxiv2018

Insulin signalling activates multiple feedback loops to elicit hunger-induced feeding in Drosophila

Insulin, a highly conserved peptide hormone, links nutrient availability to metabolism and growth in animals. Besides this, in fed states insulin levels are high and insulin acts as a satiety hormone. In animals that are food deprived insulin levels remain low which facilitates hunger induced feeding. Contrary to expectations, we present evidence for persistent Drosophila insulin-like peptide gene expression and insulin signalling during initial phases of starvation. Maintenance of insulin signalling is crucial to sustain feeding responses during initial stages of starvation. Insulin signalling acts in a feedback loop involving the abdominal fatbody to maintain dilp gene expression in the early stages of food deprivation. Furthermore, another feedback regulatory loop between insulin-producing cells (IPCs) and neurons that produce the orectic hormone short-neuropeptide-F (sNPF), maintains sNPF levels and triggers feeding behavior. Thus, insulin acts through multiple feedback regulatory loops to elicit orexigenic responses and aid in efficient utilization of energy stores during early starvation.

developmental biology↗

bioRxiv2018

Non-adhesive alginate hydrogels support growth of pluripotent stem cell-derived intestinal organoids

Human intestinal organoids (HIOs) represent a powerful system to study human development and are promising candidates for clinical translation as drug-screening tools or engineered tissue. Experimental control and clinical use of HIOs is limited by growth in expensive and poorly defined tumor-cell-derived extracellular matrices, prompting investigation of synthetic ECM-mimetics for HIO culture. Since HIOs possess an inner epithelium and outer mesenchyme, we hypothesized that adhesive cues provided by the matrix may be dispensable for HIO culture. Here, we demonstrate that alginate, a minimally supportive hydrogel with no inherent cell adhesion properties, supports HIO growth in vitro and leads to HIO epithelial differentiation that is virtually indistinguishable from Matrigel-grown HIOs. Additionally, alginate-grown HIOs mature to a similar degree as Matrigel-grown HIOs when transplanted in vivo, both resembling human fetal intestine. This work demonstrates that purely mechanical support from a simple-to-use and inexpensive hydrogel is sufficient to promote HIO survival and development.

developmental biology↗

bioRxiv2018

YBX2 Dysregulation is a Potential Cause for Late Maturation Arrest in Men with Non-Obstructive Azoospermia

IntroductionYBX2 protein binds to Y-box promotors and to mRNAs in the cytoplasm of pachytene spermatocytes and round spermatids in rodents. Knock-out of YBX2 leads to maturation arrest in animal models. YBX2 binds PRM1 and 2 mRNA, which is transcribed early and sequestrated for translation during late spermatogenesis.\n\nObjectiveThis study aimed to determine if the loss of YBX2 is associated with MA arrest due to the loss of sequestration of protamines in the human testis. As a second aim, we examined the expression of YBX2, and its transcription factors in maturation arrest (MA)(early and late) and normal controls in men.\n\nMethodsRNAseq was performed using RNA extracted from human testis samples from 44 men with non-obstructive azoospermia and ten from healthy controls. Differential expression was performed using JMPgenomics, FDR<0.001. FANTOM5 was used to predict enhancers and inhibitors of YBX2 expression. Immunofluorescence (IF) was used to stain testis tissue sections with antibodies against YBX2, SYCP3, and PRM2 in normal and MA samples. Flow cytometry was utilized to characterize YBX2 positive cells.\n\nResultsExpression of YBX2 mRNA was significantly downregulated in early and late MA compared to controls. Surprisingly, PRM1&2 mRNAs were also depleted in men with MA. Multifactorial regression analysis demonstrated a decrease in YBX2 expression in MA is due to decrease in COMP levels (p<0.0001). IF localized YBX2 protein in spermatocytes and round spermatids among fertile men, with rare YBX2 positive spermatocytes stained in LMA. PRM1&2 proteins were absent or abnormally sequestrated within spermatocytes.\n\nConclusionsDecrease in YBX2 protein expression in men with LMA leads to loss of translational suppression and lack of PRM1 and PRM2 necessary to complete spermatogenesis.

developmental biology↗

bioRxiv2018

Single-cell transcriptome analysis during cardiogenesis reveals basis for organ level developmental anomalies

Organogenesis involves integration of myriad cell types with reciprocal interactions, each progressing through successive stages of lineage specification and differentiation. Establishment of unique gene networks within each cell dictates fate determination, and mutations of transcription factors that drive such networks can result in birth defects. Congenital heart defects are the most common malformations and are caused by disruption of discrete subsets of progenitors1-3, however, determining the transcriptional changes in individual cells that lead to organ-level defects in the heart, or other organs, has not been tractable. Here, we employed single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular sub-populations can have catastrophic consequences. A network-based computational method for single-cell RNA-sequencing that predicts lineage specifying transcription factors4,5 identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite failure of right ventricular formation in Hand2-null mice6. Temporal single-cell transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium differentiated but failed to migrate into the anterior pole of the developing heart. Dysregulation of retinoic acid signaling, responsible for anterior-posterior patterning7, was associated with posteriorization of anterior cardiac progenitors in Hand2-null mutant hearts and ectopic atrial gene expression in outflow tract and right ventricle precursors. This work reveals transcriptional determinants in individual cells that specify cardiac progenitor cell fate and differentiation and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework to investigate congenital heart defects.

developmental biology↗

bioRxiv2018

C. elegans Mediator 15 permits low temperature-induced longevity via regulation of lipid and protein homeostasis

Low temperatures slow aging and extend lifespan in many organisms, including Caenorhabditis elegans. However, the metabolic and homeostatic aspects of low temperature-induced longevity remain poorly understood. Here, we show that changes in lipid composition regulated by MDT-15/Mediator 15, transcriptional co-regulator, is essential for low temperature-induced longevity and proteostasis in C. elegans. We find that inhibition of mdt-15 prevents animals from living long at low temperatures. We show that MDT-15 up-regulates fat-7, a fatty acid desaturase, at low temperatures, which increases the ratio of unsaturated to saturated fatty acids. We further demonstrate that maintaining this increased fatty acid ratio is essential for protein homeostasis and longevity at low temperatures. Thus, the homeostasis of lipid composition by MDT-15 appears to be a limiting factor for C. elegans proteostasis and longevity at low temperatures. Our findings highlight the crucial roles of fat regulation in maintaining normal organismal physiology under different environmental conditions.

developmental biology↗

bioRxiv2018

YAP/TAZ-TEAD Activity Links Mechanical Cues To Specific Cell Fate Within The Hindbrain Boundaries

Cells perceive their microenvironment through chemical and physical cues. However, how mechanical signals are interpreted during embryonic tissue deformation resulting in specific cell behaviors is largely unexplored. The Yap/Taz family of transcriptional co-activators has emerged as an important regulator of tissue growth and regeneration, responding to physical cues from the extracellular matrix, cell shape and actomyosin cytoskeleton. In this work, we unveiled the role of Yap/Taz-TEAD activity as sensor of mechanical signals in the regulation of the progenitor behavior of boundary cells during hindbrain compartmentalization. Monitoring in vivo Yap/Taz-activity during hindbrain segmentation we discovered that boundary cells respond to mechanical cues in a cell-autonomous manner through Yap/Taz-TEAD activity. Cell-lineage analysis revealed that Yap/Taz-TEAD boundary cells decrease their proliferative activity when Yap/Taz-TEAD ceased, preceding changes of cell fate: from proliferating progenitors to differentiated neurons. Functional experiments demonstrated the pivotal role of Yap/Taz-TEAD signaling in maintaining the progenitor features in the hindbrain boundary cell population.

developmental biology↗

bioRxiv2018

A cell cycle-coordinated nuclear compartment for Polymerase II transcription encompasses the earliest gene expression before global genome activation

Most metazoan embryos commence development with rapid cleavages without zygotic gene expression and their genome activation is delayed until the mid-blastula transition (MBT). However, a set of genes escape global repression during the extremely fast cell cycles, which lack gap phases and their transcription is activated before the MBT. Here we describe the formation and the spatio-temporal dynamics of a distinct transcription compartment, which encompasses the earliest detectable transcription during the first wave of genome activation. Simultaneous 4D imaging of expression of pri-miR430 and zinc finger genes by a novel, native transcription imaging approach reveals a pair of shared transcription compartments regulated by homolog chromosome organisation. These nuclear compartments carry the majority of nascent RNAs and transcriptionally active Polymerase II, are depleted of compact chromatin and represent the main sites for detectable transcription before MBT. We demonstrate that transcription occurs in the S-phase of the cleavage cycles and that the gradual slowing of these cell cycles are permissive to transcription before global genome activation. We propose that the demonstrated transcription compartment is part of the regulatory architecture of nucleus organisation, and provides a transcriptionally competent, supporting environment to facilitate early escape from the general nuclear repression before global genome activation.

developmental biology↗

bioRxiv2018

cis-regulatory architecture of a short-range EGFR organizing center in the Drosophila melanogaster leg.

We characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional ring enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE we identified many additional leg enhancers, suggesting that this logic is broadly used by many genes during Drosophila limb development.\n\nAuthor SummaryThe EGFR signaling pathway plays a major role in innumerable developmental processes in all animals and its deregulation leads to different types of cancer, as well as many other developmental diseases in humans. Here we explored the integration of inputs from the Wnt- and TGF-beta signaling pathways and the leg-specifying transcription factors Distal-less and Sp1 at enhancer elements of EGFR ligands. These enhancers trigger a specific EGFR-dependent developmental output in the fly leg that is limited to specifying distal-most fates. Our findings suggest that activation of the EGFR pathway during fly leg development occurs through the activation of multiple EGFR ligand enhancers that are active at different positions along the proximo-distal axis. Similar enhancer elements are likely to control EGFR activation in humans as well. Such DNA elements might be hot spots that cause formation of EGFR-dependent tumors if mutations in them occur. Thus, understanding the molecular characteristics of such DNA elements could facilitate the detection and treatment of cancer.

developmental biology↗

bioRxiv2018

The creation of sexual dimorphism in Drosophila gonad stem cell niches

Sex-specific development of the gonads is a key aspect of sexual dimorphism that is regulated by Doublesex/Mab3 Related Transcription Factors (DMRTs) in diverse animals species. We find that in mutants for Drosophila dsx, important components of the male and female gonad stem cell niches (hubs and terminal filaments/cap cells, respectively) still form. Initially, gonads in all dsx mutants (both XX and XY) initiate the male program of development, but later half of these gonads switch to form female stem cell niche structures. One individual can have both male-type and female-type gonad niches, however male and female niches are usually not observed in the same gonad, indicating that cells make a \"group decision\" about which program to follow. We conclude that dsx does not act in an instructive manner to regulate male vs. female niche formation, as these structures form in the absence of dsx function. Instead, dsx acts to \"tip the balance\" between the male or female programs, which are then executed independent of dsx. We show that bric a brac acts downstream of dsx to control the male vs. female niche decision. These results indicate that, in both flies and mammals, the sexual fate of the somatic gonad is remarkably plastic and is controlled by a combination of autonomous and non-autonomous cues.

developmental biology↗

bioRxiv2018

Myoglianin triggers the pre-metamorphosis stage in hemimetabolan insects

Insect metamorphosis is triggered by a decrease in juvenile hormone (JH) in the final juvenile instar. What induces this decline is therefore a very relevant question. Working with the cockroach Blattella germanica, we found that Myoglianin (Myo), a ligand in the TGF-{beta} signaling pathway, is highly expressed in the corpora allata (CA, the JH producing glands) and prothoracic glands (PG, which produce ecdysone) during the penultimate nymphal instar (N5). In the CA, high Myo levels during N5 repress the expression of jhamt, a JH biosynthesis gene. In the PG, decreasing JH levels trigger gland degeneration, mediated by the factors Kr-h1, FTZ-F1, E93 and IAP1. Also in the PG, a peak of myo expression in N5 stimulates the expression of ecdysone biosynthesis genes, such as nvd, thus enhancing the production of the metamorphic ecdysone pulse in N6. The myo expression peak in N5 also represses cell proliferation, which can contribute to enhance ecdysone production. The data indicate that Myo triggers the pre-metamorphic nymphal instar in B. germanica, and possibly in other hemimetabolan insects.

developmental biology↗

bioRxiv2018

Brd4 and P300 regulate zygotic genome activation through histone acetylation

The awakening of the zygote genome, signaling the transition from maternal transcriptional control to zygotic control, is a watershed in embryonic development, but the factors and mechanisms controlling this transition are still poorly understood. By combining CRISPR-Cas9-mediated live imaging of the first transcribed genes (miR-430), chromatin and transcription analysis during zebrafish embryogenesis, we observed that genome activation is gradual and stochastic, and the active state is inherited in daughter cells. We discovered that genome activation is regulated through both translation of maternal mRNAs and the effects of these factors on the chromatin. We show that chemical inhibition of H3K27Ac writer (P300) and reader (Brd4) block genome activation, while induction of a histone acetylation prematurely activates transcription, and restore genome activation in embryos where translation of maternal mRNAs is impaired, demonstrating that they are limiting factors for the activation of the genome. In contrast to current models, we do not observe triggering of genome activation by a reduction of the nuclear-cytoplasmic (N/C) ratio or slower cell division. We conclude that genome activation is controlled by a time-dependent mechanism involving the translation of maternal mRNAs and the regulation of histone acetylation through P300 and Brd4. This mechanism is critical to initiating zygotic development and developmental reprogramming.

developmental biology↗

bioRxiv2018

CBD-1 scaffolds two independent complexes required for eggshell vitelline layer formation and egg activation in C. elegans

Metazoan eggs have a specialized coat of extracellular matrix that aids in sperm-egg recognition. The coat is rapidly remodeled after fertilization to prevent polyspermy and establish a more permanent barrier to protect the developing embryo. In nematodes, this coat is called the vitelline layer, which is remodeled into the outermost layer of a rigid and impermeable eggshell. We have identified three key components of the vitelline layer structural scaffold - PERM-2, PERM-4 and CBD-1, the first such proteins to be described in the nematode C. elegans. CBD-1 recruited PERM-2 and PERM-4 to the nascent vitelline layer via two N-terminal chitin-binding domains. After fertilization, all three proteins redistributed from the zygote surface to the outer eggshell. Depletion of PERM-2 and PERM-4 from the scaffold led to a porous vitelline layer that permitted soluble factors to leak through the eggshell and resulted in embryonic death. In addition to its role in vitelline layer assembly, CBD-1 is also known to scaffold a protein complex required for fertilization and egg activation (EGG-1-5/CHS-1/MBK-2). We found the PERM complex and EGG complex to be structurally and functionally independent, and regulated through distinct domains of CBD-1. CBD-1 is thus a multifaceted regulator that promotes distinct aspects of vitelline layer assembly and egg activation. In sum, our findings identify the first vitelline layer components in nematodes, and provide a foundation through which to explore both conserved and species-specific strategies used by animals to build protective barriers following fertilization.

developmental biology↗