Animals can regenerate complex organs, yet this process 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.

Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether regeneration mirrors development is an open question in most regenerative species. Here, we take a transcriptomics approach to examine whether leg regeneration shows similar 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, the dynamics of gene expression during leg regeneration show a higher degree of variation related to the physiology of individual animals. A major driver of this variation is the molting cycle. We dissect the transcriptional signals of individual physiology and regeneration to obtain clearer temporal signals marking distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although the two processes use similar sets of genes, the temporal patterns in which these genes are deployed are different and cannot be systematically aligned.

Zika virus (ZIKV) infection can cause important developmental and neurological defects in Humans. Type I/III interferon responses control ZIKV infection and pathological processes, yet the virus has evolved various mechanisms to defeat these host responses. Here, we established a pipeline to delineate at high-resolution the genetic evolution of ZIKV in a controlled host cell environment. We uncovered that serially passaged ZIKV acquired increased infectivity and simultaneously developed a resistance to TLR3-induced restriction. We built a mathematical model that suggests that the increased infectivity is due to a reduced time-lag between infection and viral replication. We found that this adaptation is cell-type specific, suggesting that different cell environments may drive viral evolution along different routes. Deep-sequencing of ZIKV populations pinpointed mutations whose increased frequencies temporally coincide with the acquisition of the adapted phenotype. We functionally validated S455L, a substitution in ZIKV envelope (E) protein, recapitulating the adapted phenotype. Its positioning on the E structure suggests a putative function in protein refolding/stability. Taken together, our results uncovered ZIKV adaptations to the cellular environment leading to accelerated replication onset coupled with resistance to TLR3-induced antiviral response. Our work provides insights into Zika virus adaptation to host cells and immune escape mechanisms.

Supplementary data and code for the manuscript 'Distinct gene expression dynamics in developing and regenerating crustacean limbs', by Sinigaglia et al.

After demyelinating insult, the neuronal progenitors of the adult mouse sub-ventricular zone (SVZ) called neuroblasts convert into oligodendrocytes that participate to the remyelination process. We use this rare example of spontaneous fate conversion to identify the molecular mechanisms governing these processes. Using in vivo cell lineage and single cell RNA-sequencing, we demonstrate that SVZ neuroblasts fate conversion proceeds through formation of a non-proliferating transient cellular state co-expressing markers of both neuronal and oligodendrocyte identities. Transition between the two identities starts immediately after demyelination and occurs gradually, by a stepwise upregulation/downregulation of key TFs and chromatin modifiers. Each step of this fate conversion involves fine adjustments of the transcription and translation machineries as well as tight regulation of metabolism and migratory behaviors. Together, these data constitute the first in-depth analysis of a spontaneous cell fate conversion in the adult mammalian CNS.

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.

Arthropods are the most abundant and diverse animals on earth. Among them, pancrustaceans are an ancient and morphologically diverse group, comprising a wide range of aquatic and semi-aquatic crustaceans as well as the insects, which emerged from crustacean ancestors to colonize most terrestrial habitats. Within insects, Drosophila stands out as one of the most powerful animal models, making major contributions to our understanding of development, physiology and behavior. Given these attributes, crustaceans provide a fertile ground for exploring biological diversity through comparative studies. However, beyond insects, few crustaceans are developed sufficiently as experimental models to enable such studies. The marine amphipod Parhyale hawaiensis is currently the best established crustacean system, offering year-round accessibility to developmental stages, transgenic tools, genomic resources, and established genetics and imaging approaches. The Parhyale research community is small but diverse, investigating the evolution of development, regeneration, aspects of sensory biology, chronobiology, bioprocessing and ecotoxicology.

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.

Evidence is accumulating that evolutionary changes are not only common during biological invasions but may also contribute directly to invasion success. The genomic basis of such changes is still largely unexplored. Yet, understanding the genomic response to invasion may help to predict the conditions under which invasiveness can be enhanced or suppressed. Here we characterized the genome response of the spotted wing drosophila Drosophila suzukii during the worldwide invasion of this pest insect species, by conducting a genome-wide association study to identify genes involved in adaptive processes during invasion. Genomic data from 22 population samples were analyzed to detect genetic variants associated with the status (invasive versus native) of the sampled populations based on a newly developed statistic, we called C 2, that contrasts allele frequencies corrected for population structure. This new statistical framework has been implemented in an upgraded version of the program BayPass. We identified a relatively small set of single nucleotide polymorphisms (SNPs) that show a highly significant association with the invasive status of populations. In particular, two genes RhoGEF64C and cpo , the latter contributing to natural variation in several life-history traits (including diapause) in Drosophila melanogaster , contained SNPs significantly associated with the invasive status in the two separate main invasion routes of D. suzukii . Our methodological approaches can be applied to any other invasive species, and more generally to any evolutionary model for species characterized by non-equilibrium demographic conditions for which binary covariables of interest can be defined at the population level.

Author Contributions: V.G., T.H., B.B. and M.D. designed research; V.G., E.H., K.K., S.R.T., E.D., 23 C.G., P.V.M., M.P., S.M.-G. and L.S. performed research; V.G., E.H., K.K., S.R.T., E.D., C.G., P.V.M., 24 A.K., A.B., M.P., S.M.-G. and L.S. contributed new reagents/analytic tools; V.G., E.H., K.K., S.R.T., 25 E.D., C.G., P.V.M., A.K., M.P., A.B., S.M.-G., L.S., T.H., B.B. and M.D. analyzed data; and V.G., B.B., 26 and M.D. wrote the paper. 27

1AbstractThe SARS-CoV-2 epidemic in France has had a large death toll. It has not affected all regions similarly, since the death rate can vary several folds between regions where the epidemic has remained at a low level and regions where it got an early burst. The epidemic has been slowed down by a lockdown that lasted for almost eight weeks, and individuals can now move between metropolitan French regions without restriction. In this report we investigate the effect on the epidemic of summer holidays, during which millions of individuals will move between French regions. Additionally, we evaluate the effect of strong or weak seasonality and of several values for the reproduction number on the epidemic, in particular on the timing, the height and the spread of a second wave. To do so, we extend a SEIR model to simulate the effect of summer migrations between regions on the number and distribution of new infections. We find that the model predicts little effect of summer migrations on the epidemic, because the number of migrating infectious individuals are low as a consequence of the lockdown. However, all the reproduction numbers above 1.0 and the seasonality parameters we tried result in a second epidemic wave, with a peak date that can vary between October 2020 and April 2021. If the sanitary measures currently in place manage to keep the reproduction number below 1.0, the second wave will be avoided. If they keep the reproduction number at a low value, for instance at 1.1 as in one of our simulations, the second wave is flattened and could be similar to the first wave.

Over the past decade, the spotted wing Drosophila, Drosophila suzukii, has invaded Europe and America and has become a major agricultural pest in these areas, thereby prompting intense research activities to better understand its biology. Two draft genome assemblies already exist for this species but contain pervasive assembly errors and are highly fragmented, which limits their values. Our purpose here was to improve the assembly of the D. suzukii genome and to annotate it in a way that facilitates comparisons with D. melanogaster. For this, we generated PacBio long-read sequencing data and assembled a novel, high-quality D. suzukii genome assembly. It is one of the largest Drosophila genomes, notably because of the expansion of its repeatome. We found that despite 16 rounds of full-sib crossings the D. suzukii strain that we sequenced has maintained high levels of polymorphism in some regions of its genome. As a consequence, the quality of the assembly of these regions was reduced. We explored possible origins of this high residual diversity, including the presence of structural variants and a possible heterogeneous admixture pattern of North American and Asian ancestry. Overall, our assembly and annotation constitute a high-quality genomic resource that can be used for both high-throughput sequencing approaches, as well as manipulative genetic technologies to study D. suzukii.

Early embryogenesis is a unique developmental stage where genetic control of development is handed off from mother to zygote. Yet the contribution of this transition to the evolution of gene expression is poorly understood. Here we study two aspects of gene expression specific to early embryogenesis in Drosophila: sex-biased gene expression prior to the onset of canonical X chromosomal dosage compensation, and the contribution of maternally supplied mRNAs. We sequenced mRNAs from individual unfertilized eggs and precisely staged and sexed blastoderm embryos, and compared levels between D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. First, we find that mRNA content is highly conserved for a given stage and that studies relying on pooled embryos likely systematically overstate the degree of gene expression divergence. Unlike studies done on larvae and adults where most species show a larger proportion of genes with male-biased expression, we find that transcripts in Drosophila embryos are largely female-biased in all species, likely due to incomplete dosage compensation prior to the activation of the canonical dosage compensation mechanism. The divergence of sex-biased gene expression across species is observed to be often due to lineage-specific decrease of expression; the most drastic example of which is the overall reduction of male expression from the neo-X chromosome in D. pseudoobscura, leading to a pervasive female-bias on this chromosome. We see no evidence for a faster evolution of expression on the X chromosome in embryos (no “faster-X” effect), unlike in adults, and contrary to a previous study on pooled non-sexed embryos. Finally, we find that most genes are conserved in regard to their maternal or zygotic origin of transcription, and present evidence that differences in maternal contribution to the blastoderm transcript pool may be due to species-specific divergence of transcript degradation rates.

To better characterize how variation in regulatory sequences drives divergence in gene expression, we undertook a systematic study of transcription factor binding and gene expression in blastoderm embryos of four species, which sample much of the diversity in the 40 million-year old genus Drosophila: D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. We compared gene expression, measured by mRNA-seq, to the genome-wide binding, measured by ChIP-seq, of four transcription factors involved in early anterior-posterior patterning. We found that mRNA levels are much better conserved than individual transcription factor binding events, and that changes in a gene's expression were poorly explained by changes in adjacent transcription factor binding. However, highly bound sites, sites in regions bound by multiple factors and sites near genes are conserved more frequently than other binding, suggesting that a considerable amount of transcription factor binding is weakly or non-functional and not subject to purifying selection.

Thyroid hormones (THs), and more precisely the 3,3',5-triiodo-l-thyronine (T(3)) acetic derivative 3,3',5-triiodothyroacetic acid (TRIAC), have been shown to activate metamorphosis in amphioxus. However, it remains unknown whether TRIAC is endogenously synthesized in amphioxus and more generally whether an active TH metabolism is regulating metamorphosis. Here we show that amphioxus naturally produces TRIAC from its precursors T(3) and l-thyroxine (T(4)), supporting its possible role as the active TH in amphioxus larvae. In addition, we show that blocking TH production inhibits metamorphosis and that this effect is compensated by exogenous T(3), suggesting that a peak of TH production is important for advancement of proper metamorphosis. Moreover, several amphioxus genes encoding proteins previously proposed to be involved in the TH signaling pathway display expression profiles correlated with metamorphosis. In particular, thyroid hormone receptor (TR) and deiodinases gene expressions are either up- or down-regulated during metamorphosis and by TH treatments. Overall, these results suggest that an active TH metabolism controls metamorphosis in amphioxus, and that endogenous TH production and metabolism as well as TH-regulated metamorphosis are ancestral in the chordate lineage.

Retinoid X nuclear receptors (RXRs), as well as their insect orthologue, ultraspiracle protein (USP), play an important role in the transcription regulation mediated by the nuclear receptors as the common partner of many other nuclear receptors. Phylogenetic and structural studies have shown that the several evolutionary shifts have modified the ligand binding ability of RXRs. To understand the vertebrate-specific character of RXRs, we have studied the RXR ligand-binding domain of the cephalochordate amphioxus (Branchiostoma floridae), an invertebrate chordate that predates the genome duplication that produced the three vertebrates RXRs (alpha, beta, and gamma). Here we report the crystal structure of a novel apotetramer conformation of the AmphiRXR ligand-binding domain, which shows some similarity with the structures of the arthropods RXR/USPs. AmphiRXR adopts an apo antagonist conformation with a peculiar conformation of helix H11 filling the binding pocket. In contrast to the arthropods RXR/USPs, which cannot be activated by any RXR ligands, our functional data show that AmphiRXR, like the vertebrates/mollusk RXRs, is able to bind and be activated by RXR ligands but less efficiently than vertebrate RXRs. Our data suggest that amphioxus RXR is, functionally, an intermediate between arthropods RXR/USPs and vertebrate RXRs.

In amphioxus larvae, the club-shaped gland is a tube connecting the pharyngeal lumen with the external environment. The functions of the gland and its fate during the larva-to-juvenile metamorphosis have long been controversial. Here we use a fixative including ruthenium red to preserve extracellular secretions (presumably glycoproteins) in late pre-metamorphic larvae. This procedure reveals reddish, fibrogranular material in the lumen of the club-shaped gland and in the pharynx adjacent to the gland's inner opening. This finding strengthens the idea that secretions of the club-shaped gland are exported to the pharyngeal lumen to help form a mucous trap for capturing food particles entering the mouth. We also use the terminal desoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay to study apoptosis in the tissues of metamorphosing larvae. One of the earliest events of metamorphosis is the massive apoptotic destruction of the club-shaped gland. Therefore, despite some previous opinions to the contrary, the cells of the gland do not survive to participate in the genesis of the definitive endostyle or any other post-larval structures.

Thyroid hormones (THs) have pleiotropic effects on vertebrate development, with amphibian metamorphosis as the most spectacular example. However, developmental functions of THs in non-vertebrate chordates are largely hypothetical and even TH endogenous production has been poorly investigated. In order to get better insight into the evolution of the thyroid hormone signaling pathway in chordates, we have taken advantage of the recent release of the amphioxus genome. We found amphioxus homologous sequences to most of the genes encoding proteins involved in thyroid hormone signaling in vertebrates, except the fast-evolving thyroglobulin: sodium iodide symporter, thyroid peroxidase, deiodinases, thyroid hormone receptor, TBG, and CTHBP. As only some genes encoding proteins involved in TH synthesis regulation were retrieved (TRH, TSH receptor, and CRH receptor but not their corresponding receptors and ligands), there may be another mode of upstream regulation of TH synthesis in amphioxus. In accord with the notion that two whole genome duplications took place at the base of the vertebrate tree, one amphioxus gene often corresponded to several vertebrate homologs. However, some amphioxus specific duplications occurred, suggesting that several steps of the TH pathway were independently elaborated in the cephalochordate and vertebrate lineages. The present results therefore indicate that amphioxus is capable of producing THs. As several genes of the TH signaling pathway were also found in the sea urchin genome, we propose that the thyroid hormone signaling pathway is of ancestral origin in chordates, if not in deuterostomes, with specific elaborations in each lineage, including amphioxus.