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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Distinct roles and requirements for Ras pathway signaling in visceral versus somatic muscle founder specification

Pleiotropic signaling pathways must somehow engender specific cellular responses. In the Drosophila mesoderm, Ras pathway signaling specifies muscle founder cells from among the broader population of myoblasts. For somatic muscles, this is an inductive process mediated by the ETS-domain downstream Ras effectors Pointed and Aop (Yan). We demonstrate here that for the circular visceral muscles, despite superficial similarities, a significantly different specification mechanism is at work. Not only is visceral founder cell specification not dependent on Pointed or Aop, but Ras pathway signaling in its entirety can be bypassed. Our results show that de-repression, not activation, is the predominant role of Ras signaling in the visceral mesoderm and that accordingly, Ras signaling is not required in the absence of repression. The key repressor acts downstream of the transcription factor Lameduck and is likely a member of the ETS transcription factor family. Our findings fit with a growing body of data that point to a complex interplay between the Ras pathway, ETS transcription factors, and enhancer binding as a critical mechanism for determining unique responses to Ras signaling.\n\nSUMMARYA fundamentally different mechanism is shown for how Ras signaling governs cell fate specification in the Drosophila somatic versus visceral mesoderms, providing insight into how signaling specificity is achieved.

developmental biology↗

bioRxiv2018

TIAM-1/GEF can shape somatosensory dendrites independently of its GEF activity by regulating F-actin localization

Development of dendritic arbors is crucial for nervous system assembly, but the intracellular mechanisms that govern these processes remain incompletely understood. Here we show that the complex dendritic trees of PVD somatosensory neurons in Caenorhabditis elegans are patterned by distinct pathways downstream of the DMA-1 leucine rich transmembrane (LRR-TM) receptor. The guanine nucleotide exchange factor tiam-1/GEF and act-4/Actin function with the DMA-1/LRR-TM to pattern 4{degrees} higher order branches by localizing F-actin to the distal ends of developing dendrites. Biochemical experiments show that DMA-1/LRR-TM is part of a biochemical complex with TIAM-1/GEF and ACT-4/Actin. Surprisingly, TIAM-1/GEF appears to function independently of Rac1 guanine nucleotide exchange factor activity. Additionally, another pathway dependent on HPO-30/Claudin and TIAM-1/GEF is required for formation of 2{degrees} and 3{degrees} branches. Collectively, our experiments suggest that the DMA-1/LRR-TM receptor on PVD dendrites may control aspects of dendrite patterning by directly modulating F-actin dynamics, independently of TIAM-1/GEF enzymatic activity.

developmental biology↗

bioRxiv2018

Evolution and developmental diversity of skin spines in pufferfish

Teleost fishes develop a huge variety of skin ornaments. How these diverse skin structures develop in fishes is unknown. The teleost fish order Tetraodontiformes includes some of the most unusual fishes such as the ocean sunfish, triggerfish and pufferfish, and they all can develop a vast assortment of scale derivatives that cover their bodies. Pufferfish have some of the most extreme scale derivatives, dermal spines, which are erected during their characteristic puffing behavior. Here we show that pufferfish spines develop through conserved gene interactions essential for other vertebrate skin appendage formation, like hair and feathers. However, pufferfish spines form without EDA (ectodysplasin), an essential molecule for the development of most vertebrate skin appendages. Modifying signaling pathways lead to loss or reduction of spine coverage in pufferfish, suggesting a mechanism for skin appendage diversification. We suggest that pufferfish skin spines evolved from a basic teleost scale-type through derived gene network modification in Tetraodontiformes.

developmental biology↗

bioRxiv2018

Lineage segregation, pluripotency and X-chromosome inactivation in the pig pre-gastrulation embryo

High-resolution molecular programs delineating the cellular foundations of mammalian embryogenesis have emerged recently. Similar analysis of human embryos is limited to pre-implantation stages, since early post-implantation embryos are inaccessible. Notwithstanding, we previously suggested conserved principles of pig and human early development. For further insight on pluripotent states and lineage delineation, we analysed pig embryos at single cell resolution. Here we show progressive segregation of inner cell mass and trophectoderm in early blastocysts, and then of epiblast and hypoblast in late blastocysts. We detected distinct pluripotent states, first as a short naive state followed by a protracted primed state. Dosage compensation with respect to the X-chromosome in females is attained via X-inactivation in late epiblasts. Detailed human-pig comparison is a basis towards comprehending early human development and a foundation for further studies of human pluripotent stem cell differentiation in pig interspecies chimeras.

developmental biology↗

bioRxiv2018

Genome-wide analysis of H3K4me3 and H3K27me3 modifications throughout the mouse urogenital ridge at E11.5.

In mammals, the adrenal gland, testis and ovary arise from a common progenitor tissue known as the urogenital ridge (UGR). This small population of cells will adopt a number of different cell fates following sex determination, including forming the precursors of somatic cells (such as Sertoli and granulosa cells) and steroidogenic cells. In addition, this tissues also contains the Wolffian and Mullerian ducts that later form components of the reproductive tracts. A potential mechanism to maintain developmental plasticity of the UGR until gonad formation is through the epigenetic modification of histone proteins.\n\nIn order to provide a resource for future studies, we used chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) for two histone modifications, H3K4me3 and H3K27me3, in the E11.5 mouse UGR. These marks are both known to reflect the active, repressive or a poised chromatin state. We found that enrichment for each histone mark reflected transcriptional activity in precursor cells of the developing gonad. From the analysis of potential enhancer/regulator peak regions for DNA binding motifs, we identified several candidate transcription factors that may contribute to gonadal cell lineage specification. We additionally identified signaling pathway genes that are targeted by both chromatin modifications. Together, these datasets provide a useful resource for investigating gene regulatory networks functioning during UGR development at E11.5.

developmental biology↗

bioRxiv2018

A fat-tissue sensor couples growth to oxygen availability by remotely controlling insulin secretion

Organisms adapt their metabolism and growth to the availability of nutrients and oxygen, which are essential for normal development. This requires the ability to sense these environmental factors and respond by regulation of growth-controlling signals, yet the mechanisms by which this adaptation occurs are not fully understood. To identify novel growth-regulatory mechanisms, we conducted a global RNAi-based screen in Drosophila for size differences and identified 89 positive and negative regulators of growth. Among the strongest hits was the FGFR homologue breathless necessary for proper development of the tracheal airway system. Breathless deficiency results in tissue hypoxia (low oxygen), sensed primarily in this context by the fat tissue. The fat, in response, relays this information through release of one or more humoral factors that remotely inhibit insulin secretion from the brain, thereby restricting systemic growth. Thus our findings show that the fat tissue acts as an oxygen sensor that allows the organism to reduce its growth in adaptation to limited oxygen conditions.

developmental biology↗

bioRxiv2018

Altering the temporal regulation of one transcription factor drives sensory trade-offs

Size trade-offs of visual versus olfactory organs is a pervasive feature of animal evolution. Comparing Drosophila species, we find that larger eyes correlate with smaller antennae, where olfactory organs reside, and narrower faces. We demonstrate that this tradeoff arises through differential subdivision of the head primordium into visual versus non-visual fields. Specification of the visual field requires a highly-conserved eye development gene called eyeless in flies and Pax6 in humans. We discover that changes in the temporal regulation of eyeless expression during development is a conserved mechanism for sensory trade-offs within and between Drosophila species. We identify a natural single nucleotide polymorphism in the cis-regulatory region of eyeless that is sufficient to alter its temporal regulation and eye size. Because Pax6 is a conserved regulator of sensory placode subdivision, we propose that alterations in the mutual repression between sensory territories is a conserved mechanism for sensory trade-offs in animals.

developmental biology↗

bioRxiv2018

Dynamics of PAR proteins explain the oscillation and ratcheting mechanisms in dorsal closure

We present a vertex-based model for Drosophila dorsal closure that predicts the mechanics of cell oscillation and contraction from the dynamics of the PAR proteins. Based on experimental observations of how aPKC, Par-6 and Bazooka migrate from the circumference of the apical surface to the medial domain, and how they interact with each other and ultimately regulate the apicomedial actomyosin, we formulate a system of differential equations that capture the key features of the process. The oscillation in cell area in the early phase of dorsal closure results from an intracellular negative feedback loop that involves myosin, an actomyosin regulator, aPKC and Bazooka. In the slow phase, gradual sequestration of apicomedial aPKC into Bazooka clusters causes incomplete disassembly of the myosin network over each cycle of oscillation, thus producing the so-called ratchet. The fast phase of rapid cell and tissue contraction arises when medial myosin, no longer hindered by aPKC, builds up in time and produces sustained contraction. Thus, a minimal set of rules governing the dynamics of the PAR proteins, extracted from experimental observations, can account for all major mechanical outcomes of dorsal closure, including the transitions between its three distinct phases.\n\nInsert Received for publication Date and in final form Date

developmental biology↗

bioRxiv2018

Characterization of Drosophila Nidogen/entactin reveals roles in basement membrane stability, barrier function and nervous system plasticity

Basement membranes (BMs) are specialized layers of extracellular matrix (ECM) mainly composed of Laminin, type IV Collagen, Perlecan and Nidogen/entactin (NDG). While the essential and evolutionary conserved functions of Laminin, Collagen and Perlecan are well documented in Drosophila and other species, the proposed role of NDG as the major ECM linker molecule has been challenged by several in vivo studies revealing that NDG is dispensable for viability and BM formation. Here, we report the characterization of the single Ndg gene in Drosophila. Embryonic Ndg expression differed from that of other BM components and was primarily observed in mesodermal tissues and the chordotonal organs, whereas NDG protein localized to all BMs. While loss of Laminin strongly affected BM-localization of NDG, Ndg null mutants exhibited no overt changes in the distribution of BM core components. However, loss of NDG led to ultrastructural BM defects compromising barrier function and stability in vivo. Although Ndg mutants were viable, loss of NDG led to decreased fecundity in flies as well as impaired crawling behavior and reduced response to vibrational stimuli in larvae. Further morphological analysis revealed accompanying defects in the larval peripheral nervous system especially in the chordotonal organs and the neuromuscular junction (NMJ), where Ndg genetically interacted with the Leukocyte-antigen-related-like (Lar) receptor gene to regulate NMJ extension and synaptic differentiation. Taken together, our analysis suggests that NDG is not essential for BM assembly but mediates BM stability and ECM-dependent neural plasticity during Drosophila development.\n\nSummary StatementIn this study we characterize Drosophila Nidogen/Entactin (Ndg) mutants revealing that loss of Ndg impairs basement membrane (BM) stability and permeability as well as proper function of the nervous system.

developmental biology↗

bioRxiv2018

Size control of the inner ear via hydraulic feedback

Animals make organs of precise size, shape, and symmetry despite noise in underlying molecular and cellular processes. How developing organs manage this noise is largely unknown. Here, we combine quantitative imaging, physical theory, and physiological measurement of hydrostatic pressure and fluid transport in zebrafish to study size control of the developing inner ear. We find that fluid accumulation creates hydrostatic pressure in the lumen leading to stress in the epithelium and expansion of the otic vesicle. Pressure, in turn, inhibits fluid transport into the lumen. This negative feedback loop between pressure and transport allows the otic vesicle to change growth rate to control natural or experimentally-induced size variation. Spatiotemporal patterning of contractility modulates pressure-driven strain for regional tissue thinning. Our work connects moleculardriven mechanisms, such as osmotic pressure driven strain and actomyosin tension, to the regulation of tissue morphogenesis via hydraulic feedback to ensure robust control of organ size.

developmental biology↗

bioRxiv2018

Switchable genome editing via genetic code expansion

Multiple applications of genome editing by CRISPR-Cas9 necessitate stringent regulation and Cas9 variants have accordingly been generated whose activity responds to small ligands, temperature or light. However, these approaches are often impracticable, for example in clinical therapeutic genome editing in situ or gene drives in which environmentally-compatible control is paramount. With this in mind, we have developed heritable Cas9-mediated mammalian genome editing that is acutely controlled by the cheap lysine derivative, Lys(Boc) (BOC). Genetic code expansion permitted non-physiological BOC incorporation such that Cas9 (Cas9BOC) was expressed in a full-length, active form in cultured somatic cells only after BOC exposure. Stringently BOC-dependent, heritable editing of transgenic and native genomic loci occurred when Cas9BOC was expressed at the onset of mouse embryonic development from cRNA or Cas9BOC transgenic females. The tightly controlled Cas9 editing system reported here promises to have broad applications and is a first step towards purposed, spatiotemporal gene drive regulation over large geographical ranges.

developmental biology↗

bioRxiv2018

Vascular defects of DYRK1A knockouts are ameliorated by modulating calcium signaling in zebrafish

DYRK1A is a major causative gene in Down syndrome (DS). Reduced incidence of solid tumors and vascular anomalies in DS patients suggests a role of DYRK1A in angiogenesis, but in vivo evidence is lacking. Here, we used zebrafish dyrk1aa mutant embryos to understand DYRK1A function in the cerebral vasculature formation. Zebrafish dyrk1aa mutants exhibited cerebral hemorrhage and defects in angiogenesis of central arteries in the developing hindbrain. Such phenotypes were rescued by wild-type dyrk1aa mRNA, but not by a kinase-dead form, indicating the importance of DYRK1A kinase activity. Chemical screening using a bioactive small molecule library identified a calcium chelator, EGTA, as one of the hits that most robustly rescued the hemorrhage. Vascular defects of mutants were also rescued by independent modulation of calcium signaling by FK506. Furthermore, the transcriptomic analyses supported the alterations of calcium signaling networks in dyrk1aa mutants. Together, our results suggest that dyrk1aa plays an essential role in angiogenesis and in maintenance of the developing cerebral vasculature via regulation of calcium signaling, which may have therapeutic potential for DYRK1A-related vascular diseases.

developmental biology↗

bioRxiv2018

TORC1 modulation in adipose tissue is required for organismal adaptation to hypoxia in Drosophila.

Animals often develop in conditions where environmental conditions such as food, oxygen and temperature fluctuate. The ability to adapt their metabolism to these fluctuations is important to ensure normal development and viability. In most animals, low oxygen (hypoxia) is deleterious, however some animals can alter their physiology to thrive under hypoxia. Here we show that TORC1 modulation in adipose tissue is required for organismal adaptation to hypoxia in Drosophila. We find that hypoxia rapidly suppresses TORC1 kinase signalling in Drosophila larvae via TSC-mediated inhibition of Rheb. We show that this hypoxia-mediated inhibition of TORC1 specifically in the larval fat body is essential for viable development to adulthood. Moreover, we find that these effects of TORC1 inhibition on hypoxia tolerance are mediated through remodeling of fat body lipid droplets and lipid storage. These studies identify the larval adipose tissue as a key hypoxia sensing tissue that coordinates whole-body development and survival to changes in environmental oxygen by modulating TORC1 and lipid storage.

developmental biology↗

bioRxiv2018

Single-cell ID-seq identifies BMP signaling as a driver of a late stage epidermal differentiation program.

AbstractEpidermal homeostasis requires balanced and coordinated adult stem cell renewal and differentiation. These processes are controlled by both extracellular signaling and by cell intrinsic transcription regulatory networks, yet how these control mechanisms are integrated to achieve this unclear. Here, we developed single-cell ID-seq and measured 69 antibody-DNA conjugates (including 34 phospho-specific epitopes) to study the activation state of signaling pathways during epidermal differentiation at the single-cell level. Computational pseudo-timing inference revealed activation of the JAK-STAT, WNT and BMP pathways along the epidermal differentiation trajectory. During differentiation, cells start producing BMP2 ligands and activate the canonical intracellular effectors SMAD1/5/9. Mechanistically, the BMP pathway is responsible for directly activating a specific transcription program that includes the key differentiation transcription factors MAF and MAFB to allow terminal differentiation. We propose that incorporating autocrine signaling pathway activation into a transcription regulatory network enables regional coordination of transcription programs during epidermal differentiation.

developmental biology↗

bioRxiv2018

Staged developmental mapping and X chromosome transcriptional dynamics during mouse spermatogenesis

Understanding male fertility requires an in-depth characterisation of spermatogenesis, the developmental process by which male gametes are generated. Spermatogenesis occurs continuously throughout a males reproductive window and involves a complex sequence of developmental steps, both of which make this process difficult to decipher at the molecular level. To overcome this, we transcriptionally profiled single cells from multiple distinct stages during the first wave of spermatogenesis, where the most mature germ cell type is known. This naturally enriches for spermatogonia and somatic cell types present at very low frequencies in adult testes. Our atlas, available as a shiny app (https://marionilab.cruk.cam.ac.uk/SpermatoShiny), allowed us to reconstruct the three main processes of spermatogenesis: spermatogonial differentiation, meiosis, and spermiogenesis. Additionally, we profiled the chromatin changes associated with meiotic silencing of the X chromosome, revealing a set of genes specifically and strongly repressed by H3K9me3 in the spermatocyte stage, but which escape post-meiotic silencing in spermatids.

developmental biology↗

bioRxiv2018

Timed collinear activation of Hox genes during gastrulation controls the avian forelimb position

Limb position along the body is highly consistent within one species but very variable among vertebrates. Despite major advances in our understanding of limb patterning in three dimensions, how limbs reproducibly form along the anteroposterior axis remains largely unknown. Hox genes have long been suspected to control limb position, however supporting evidences are mostly correlative and their role in this process remains unclear. Here we show that Hox genes determine the avian forelimb position in a two-step process: first, their sequential collinear activation during gastrulation controls the relative position of their own successive expression domains along the body axis. Then, within these collinear domains, Hox genes differentially activate or repress the genetic cascade responsible for forelimb initiation. Furthermore, we provide evidences that changes in the timing of collinear Hox gene activation might underlie natural variation in forelimb position between different birds. Altogether our results which characterize the cellular and molecular mechanisms underlying the regulation and natural variation of forelimb position in avians, show a direct and early role for Hox genes in this process.

developmental biology↗

bioRxiv2018

Single cell expression analysis uncouples transdifferentiation and reprogramming

Many somatic cell types are plastic, having the capacity to convert into other specialized cells (transdifferentiation)(1) or into induced pluripotent stem cells (iPSCs, reprogramming)(2) in response to transcription factor over-expression. To explore what makes a cell plastic and whether these different cell conversion processes are coupled, we exposed bone marrow derived pre-B cells to two different transcription factor overexpression protocols that efficiently convert them either into macrophages or iPSCs and monitored the two processes over time using single cell gene expression analysis. We found that even in these highly efficient cell fate conversion systems, cells differ in both their speed and path of transdifferentiation and reprogramming. This heterogeneity originatesin two starting pre-B cell subpopulations,large pre-BII and the small pre-BII cells they normally differentiate into. The large cells transdifferentiate slowly but exhibit a high efficiency of iPSC reprogramming. In contrast, the small cells transdifferentiate rapidly but are highly resistant to reprogramming. Moreover, the large B cells induce a stronger transient granulocyte/macrophage progenitor (GMP)-like state, while the small B cells undergo a more direct conversion to the macrophage fate. The large cells are cycling and exhibit high Myc activity whereas the small cells are Myc low and mostly quiescent. The observed heterogeneity of the two cell conversion processes can therefore be traced to two closely related cell types in the starting population that exhibit different types of plasticity. These data show that a somatic cells propensity for either transdifferentiation and reprogramming can be uncoupled.\n\nOne sentence summarySingle cell transcriptomics of cell conversions

developmental biology↗

bioRxiv2018

Fbxo2VHC mouse and embryonic stem cell reporter lines delineate in vitro-generated inner ear sensory epithelia cells and enable otic lineage selection and Cre-recombination

While the mouse has been a productive model for inner ear studies, the lack of highly specific genes and tools have presented challenges, specifically for in vitro studies of otic development, where innate cellular heterogeneity and disorganization increase the reliance on lineage-specific markers. To address this challenge in mice and embryonic stem (ES) cells, we targeted the lineage-specific otic gene Fbxo2 with a multicistronic reporter cassette (Venus/Hygro/CreER = VHC). In otic organoids derived from ES cells, Fbxo2VHC specifically delineates otic progenitors and inner ear sensory epithelia. In mice, Venus expression and CreER activity reveal a cochlear developmental gradient, label the prosensory lineage, show enrichment in a subset of type I vestibular hair cells, and expose strong expression in adult cerebellar granule cells. We provide a toolbox of multiple spectrally distinct reporter combinations to the community for studies that require use of fluorescent reporters, hygromycin selection, and conditional Cre-mediated recombination.\n\nSUMMARY STATEMENTA multifunctional Fbxo2-targeted reporter in mice and stem cells was developed and characterized as a resource for inner ear studies, along with a toolbox of plasmids to facilitate the use of this technique for other users.

developmental biology↗