Biology subjects

Averof, M.

Publications and source records attributed to Averof, M..

13 recordsLinked to original sources

bioRxiv2026

Genome-wide chromatin reset at the onset of leg regeneration in pancrustaceans

Regeneration requires injured tissues to selectively reactivate certain developmental programs while triggering regeneration-specific mechanisms and responding to the immediate consequences of tissue damage. How chromatin states are reorganized during this transition, and whether the underlying sequence of regulatory events is conserved across regenerating animals, remain poorly understood. We address these questions in the two-spotted cricket Gryllus bimaculatus, in which limb regeneration progressively declines during nymphal development and is absent in adults, and extend our analysis to the crustacean Parhyale hawaiensis. We first establish a chromosome-scale genomic and epigenomic framework for the cricket and then follow chromatin and transcriptional changes after limb amputation. Amputation triggers a striking genome-wide shutdown of enhancer-associated chromatin, marked by loss of accessible regulatory regions and depletion of H3K27ac and H3K4me1. This is followed, in regeneration-competent tissue, by selective reopening of a restricted population of regulatory elements and strong induction of the pioneer transcription factor Zelda (GbZelda). The GbZelda locus is unusually marked by both H3K27me3 and H3K9me3 in intact leg tissue; after injury, H3K9me3 is reduced broadly, whereas H3K27me3 is selectively relieved in regenerating nymphs. Finally, analysis of Parhyale reveals the same sequence of chromatin events during appendage regeneration: an initial enhancer shutdown followed by selective regulatory reopening concomitant with Zelda upregulation. Thus, rather than restoring the pre-injury regulatory landscape wholesale, appendage regeneration in these two pancrustacean models proceeds through a conserved sequence of genome-wide chromatin resetting, selective regulatory reopening and activation of a pioneer transcription factor.

developmental biology↗

bioRxiv2026

foxQ2 marks fast-acting brain interneurons including a subset of dopaminergic neurons innervating mushroom bodies and central complex in the beetle Tribolium castaneum

The brain is one of the most complex animal organs and the development of the many different neuron types remains enigmatic. A set of brain-specific transcription factors is involved in brain patterning but their specific contributions mostly remain to be elucidated, including foxQ2II. This transcription factor is conserved in anterior neuroectodermal patterning of most animals while it has been lost from vertebrates. The contribution of foxQ2II-positive neurons to the adult brain has remained enigmatic. Here, we use our enhancer trap, immunostainings and our newly established beetle brainbow system to categorize Tc-foxQ2II-positive neurons into nine clusters with different projection patterns. All clusters contain neurons with the fast-activating neurotransmitters acetylcholine and glutamate while no Tc-foxQ2II positive neuron is GABA-ergic or serotonin-positive. Interestingly, we found that many dopaminergic neurons were Tc-foxQ2II positive and we homologize them with dopaminergic neurons of the PPL2c, PPM1 and PPL1 cluster described in the Drosophila brain. Our results show that Tc-foxQ2II marks subsets of fast-acting interneurons contributing to the higher order brain centers mushroom bodies and central complex. Taken together, our work expands the known functional range of foxQ2 genes from sensory and neurosecretory cell specification to interneurons involved in the function of higher order brain centers.

neuroscience↗

bioRxiv2026

Genome-wide discovery of cis-regulatory elements in a large genome

Identifying non-coding regulatory elements in the genome poses a challenge in most organisms. Classical methods rely on trial and error to test the regulatory activities of DNA fragments using reporter constructs. In large eukaryotic genomes, where cis-regulatory elements can spread over long distances, separated by large stretches of non-functional DNA, this trial and error approach is particularly challenging. Here, we generate two types of resources that can be used to narrow the search for such cis-regulatory elements in the 3.6 Gbp genome of Parhyale hawaiensis (comparable in size to the human genome). First, we use bulk ATAC-seq to uncover genome-wide patterns of chromatin accessibility in embryonic and adult tissues of Parhyale (whole embryos and legs), and single-nucleus ATAC-seq to identify regions of open chromatin in diverse cell types recovered from adult legs, including epidermal, neuronal, muscle and blood cells. Second, by sequencing the genomes of three congeneric species of Parhyale hawaiensis - P. darvishi, P. aquilina and P. plumicornis - we identify islands of sequence conservation across the genome, corresponding to DNA elements that are functionally constrained during evolution. We present an approach by which low-coverage (10-15x) short-read genome sequencing, without genome assembly, is sufficient to provide reliable maps of sequence conservation. This approach cuts the cost and labour required to generate these maps, making the identification of cis-regulatory elements more widely accessible. We demonstrate the utility of these resources by identifying cis-regulatory elements that drive robust expression of fluorescent reporters ubiquitously and in specific cell types.

developmental biology↗

bioRxiv2024

A toolkit for testing membrane localisation tags across species

Transgenic markers and tools have revolutionised how we study cells and developing organisms. Some of the elements needed to construct those tools are universally applicable (e.g. fluorescent proteins), while others are species-specific (e.g. cis-regulatory elements driving transcription). Membrane-localising signals that target proteins to the plasma membrane have been identified in several model organisms. Unfortunately, the efficacy of these signals varies greatly across species. To address this problem, we generated a toolkit of 11 membrane-localising tags that can be screened rapidly in diverse organisms. The toolkit includes tags that target the plasma membrane through different mechanisms, including signal peptides, the attachment of lipids, and fusion with lipid-binding domains. Each tag has been fused to the red fluorescent protein mScarlet3 and placed downstream of a T7 promoter, allowing the in vitro production of mRNA that can be readily delivered in a wide range of embryos and cells of interest. Through a collaborative effort, we tested this toolkit in ten species of animals spanning diverse phyla, including chordates, echinoderms, arthropods, nematodes, annelids, flatworms and cnidarians. We identify robust membrane-localising tags in each of these animals, and in one of animals closest relatives, the choanoflagellates. Three tags (KRas, GAP43 and Src64B) work in all of the species tested.

developmental biology↗

bioRxiv2024

Long-term live imaging, cell identification and cell tracking in regenerating crustacean legs

High resolution live imaging of regeneration presents unique challenges, due to the nature of the specimens (large mobile animals), the duration of the process (spanning days or weeks), and the fact that cellular resolution must be achieved without damage caused by lengthy exposures to light. Building on previous work that allowed us to image different parts of the process of leg regeneration in the crustacean Parhyale hawaiensis, we present here a method for live imaging that captures the entire process of leg regeneration, spanning up to 10 days, at cellular resolution. Our method includes (1) mounting and long-term live imaging of regenerating legs under conditions that yield high spatial and temporal resolution but minimise photodamage, (2) fixing and in situ staining of the regenerated legs that were imaged, to identify cell fates, and (3) computer-assisted cell tracking to determine the cell lineages and progenitors of identified cells. The method is optimised to limit light exposure while maximising tracking efficiency. Combined with appropriate cell-type-specific markers, this method may allow the description of cell lineages for every regenerated cell type in the limb.

developmental biology↗

bioRxiv2021

Regenerated crustacean limbs are precise replicas

Animals can regenerate complex organs, yet this frequently results in imprecise replicas of the original structure. In the crustacean Parhyale, embryonic and regenerating legs differ in gene expression dynamics but produce apparently similar mature structures. We examine the fidelity of Parhyale leg regeneration using complementary approaches to investigate microanatomy, sensory function, cellular composition and cell molecular profiles. We find that regeneration precisely replicates the complex microanatomy and spatial distribution of external sensory organs, and restores their sensory function. Single-nuclei sequencing shows that regenerated and uninjured legs are indistinguishable in terms of cell type composition and transcriptional profiles. This remarkable fidelity highlights the ability of organisms to achieve identical outcomes via distinct processes.

developmental biology↗

bioRxiv2021

Distinct gene expression dynamics in developing and regenerating limbs

Regenerating animals have the ability to reproduce organs that were originally generated in the embryo and subsequently lost due to injury. Understanding whether the process of regeneration mirrors development is an open question in most regenerative species. Here we take a transcriptomics approach to examine to what extent leg regeneration shows the same temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, global patterns of gene expression during leg regeneration show a high degree of variation, related to the physiology of individual animals. A major driver of this variation is the molting cycle. After dissecting the transcriptional signals of individual physiology from regeneration, we obtain temporal signals that mark distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although both processes use largely the same genes, the temporal patterns in which these gene sets are deployed are different and cannot be systematically aligned. HIGHLIGHTSO_LISingle-limb data on transcriptional dynamics of leg development and regeneration C_LIO_LIDeveloping embryonic legs show stereotypic transcriptional profiles C_LIO_LIRegenerating leg transcriptomes show a high degree on individual variation C_LIO_LIRegenerating leg transcriptomes are influenced by adult physiology, especially molting C_LIO_LIRegenerating leg transcriptomes reveal distinct phases of leg regeneration C_LIO_LILeg development and regeneration use overlapping sets of genes in different temporal patterns C_LI

developmental biology↗

bioRxiv2021

Tracking cell lineages in 3D by incremental deep learning

Deep learning is emerging as a powerful approach for bioimage analysis. Its use in cell tracking is limited by the scarcity of annotated data for the training of deep learning models. Moreover, annotation, training, prediction and proofreading currently lack a unified user interface. We present ELEPHANT, an interactive platform for 3D cell tracking that addresses these challenges by taking an incremental approach to deep learning. ELEPHANT provides an interface that seamlessly integrates cell track annotation, deep learning, prediction and proofreading. This enables users to implement cycles of incremental learning starting from a few annotated nuclei. Successive prediction-validation cycles enrich the training data, leading to rapid improvements in tracking performance. We test the softwares performance against state-of-the-art methods and track lineages spanning the entire course of leg regeneration in a crustacean over 1 week (504 timepoints). ELEPHANT yields accurate, fully-validated cell lineages with a modest investment in time and effort.

developmental biology↗

bioRxiv2020

CeLaVi: An Interactive Cell Lineage Visualisation Tool

Recent innovations in genetics and imaging are providing the means to reconstruct cell lineages, either by tracking cell divisions using live microscopy, or by deducing the history of cells using molecular recorders. A cell lineage on its own, however, is simply a description of cell divisions as branching events. A major goal of current research is to integrate this description of cell relationships with information about the spatial distribution and identities of the cells those divisions produce. Visualising, interpreting and exploring these complex data in an intuitive manner requires the development of new tools. Here we present CeLaVi, a web-based visualisation tool that allows users to navigate and interact with a representation of cell lineages, whilst simultaneously visualising the spatial distribution, identities and properties of cells. CeLaVis principal functions include the ability to explore and manipulate the cell lineage tree; to visualise the spatial distribution of cell clones at different depths of the tree; to colour cells in the 3D viewer based on lineage relationships; to visualise various cell qualities on the 3D viewer (e.g. gene expression, cell type, tissue layer) and to annotate selected cells/clones. All these capabilities are demonstrated with four different example data sets. CeLaVi is available at http://www.celavi.pro.

developmental biology↗

bioRxiv2019

The visual system of the genetically tractable crustacean Parhyale hawaiensis: diversification of eyes and visual circuits associated with low-resolution vision

BackgroundArthropod eyes have diversified during evolution to serve multiple needs, such as finding mates, hunting prey, and navigating in complex surroundings under varying light conditions. This diversity is reflected in the optical apparatus, photoreceptors and neural circuits that underpin vision. While this diversity has been extensively documented, our ability to genetically manipulate the visual system to investigate its function is largely limited to a single species, the fruitfly Drosophila melanogaster. Here, we describe the visual system of Parhyale hawaiensis, an amphipod crustacean for which we have established tailored genetic tools. ResultsAdult Parhyale have apposition-type compound eyes made up of [~]50 ommatidia. Each ommatidium contains four photoreceptor cells with large rhabdomeres (R1-4), expected to be sensitive to the polarisation of light, and one photoreceptor cell with a smaller rhabdomere (R5). The two types of photoreceptors express different opsins, belonging to families with distinct wavelength sensitivities. Using the cis.-regulatory regions of opsin genes, we established transgenic reporters expressed in each photoreceptor cell type. Based on these reporters, we show that R1-4 and R5 photoreceptors extend axons to the first optic lobe neuropil, revealing striking differences compared with the photoreceptor projections found in related crustaceans and insects. Investigating visual function, we show that Parhyale has a positive phototactic response and is capable of adapting its eyes to different levels of light intensity. ConclusionsWe propose that the visual system of Parhyale serves low-resolution visual tasks, such as orientation and navigation, based on broad gradients of light intensity and polarisation. Optic lobe structure and photoreceptor projections point to significant divergence from the conserved visual circuits found in other malacostracan crustaceans and insects, which could be associated with a shift to low-resolution vision. Our study provides the foundation for research in the visual system of this genetically tractable species.

zoology↗

bioRxiv2018

Clonal analysis by tunable CRISPR-mediated excision

Clonal marking techniques based on the Cre/lox and Flp/FRT systems are widely used in multicellular model organisms to mark individual cells and their progeny, in order to study their morphology, growth properties and developmental fates. The same tools can be adapted to introduce specific genetic changes in a subset of cells within the body, i.e. to perform mosaic genetic analysis. Marking and manipulating distinct cell clones requires control over the frequency of clone induction, which is sometimes difficult to achieve. Here we present Valcyrie, a new method that replaces the conventional Cre or Flp recombinase-mediated excision of a marker cassette by CRISPR-mediated excision. A major advantage of this approach is that CRISPR efficiency can be tuned in a predictable fashion by manipulating the degree of sequence complementarity between the CRISPR guide RNA and its targets. We establish the method in the beetle Tribolium castaneum. We demonstrate that clone marking frequency can be tuned to generate embryos carrying single marked clones. The Valcyrie approach can be applied to a wide range of experimental settings, for example to modulate clone frequency with existing tools in established model organisms and to introduce clonal analysis in emerging experimental models.

genetics↗

bioRxiv2018

Is it possible to reconstruct an accurate cell lineage using CRISPR recorders?

Cell lineages provide the framework for understanding how multicellular organisms are built and how cell fates are decided during development. Describing cell lineages in most organisms is challenging, given the number of cells involved; even a fruit fly larva has ~50,000 cells and a small mammal has more than 1 billion cells. Recently, the idea of using CRISPR to induce mutations during development as heritable markers for lineage reconstruction has been proposed and trialled by several groups. While an attractive idea, its practical value depends on the accuracy of the cell lineages that can be generated by this method. Here, we use computer simulations to estimate the performance of this approach under different conditions. Our simulations incorporate empirical data on CRISPR-induced mutation frequencies in Drosophila. We show significant impacts from multiple biological and technical parameters - variable cell division rates, skewed mutational outcomes, target dropouts and different mutation sequencing strategies. Our approach reveals the limitations of recently published CRISPR recorders, and indicates how future implementations can be optimised to produce accurate cell lineages.

developmental biology↗

bioRxiv2017

Repeated co-option of a conserved gene regulatory module underpins the evolution of the crustacean carapace, insect wings and other flat outgrowths

Summary statementThe genes vestigial, scalloped and wingless comprise a conserved regulatory module that was co-opted repeatedly for the evolution of flat structures, such as insect wings, and crustacean carapace, tergites and coxal plates.\n\nSummaryHow novelties arise is a key question in evolutionary developmental biology. The crustacean carapace is a novelty that evolved in the early Cambrian. In an extant crustacean, Daphnia magna, the carapace grows from the body wall as a double-layered sheet with a specialized margin. We show that the growing margin of this carapace expresses vestigial, scalloped and wingless, genes that are known to play key roles in regulating growth at the insect wing margin. RNAi-mediated knockdown of scalloped and wingless impair carapace development, indicating that carapace and wing might share a common mechanism for margin outgrowth. However, carapace and wings arise in different parts of the body and their margins have different orientations, arguing that these structures have independent evolutionary origins. We show that scalloped is also expressed at the margin of unrelated flat outgrowths (tergites and coxal plates) in the distantly related crustacean Parhyale hawaiensis. Based on these observations, we propose that the vestigial-scalloped-wingless gene module has a common role in the margin of diverse flat structures, originating before the divergence of major crustacean lineages and the emergence of insects. Repeated co-option of this module occurred independently in the carapace, wing and other flat outgrowths, underpinning the evolution of distinct novelties in different arthropod lineages.

developmental biology↗