Chromosome-level genome assemblies of 2 hemichordates provide new insights into deuterostome origin and chromosome evolution.

Deuterostomes are a monophyletic group of animals that includes Hemichordata, Echinodermata (together called Ambulacraria), and Chordata. The diversity of deuterostome body plans has made it challenging to reconstruct their ancestral condition and to decipher the genetic changes that drove the diversification of deuterostome lineages. Here, we generate chromosome-level genome assemblies of 2 hemichordate species, Ptychodera flava and Schizocardium californicum, and use comparative genomic approaches to infer the chromosomal architecture of the deuterostome common ancestor and delineate lineage-specific chromosomal modifications. We show that hemichordate chromosomes (1N = 23) exhibit remarkable chromosome-scale macrosynteny when compared to other deuterostomes and can be derived from 24 deuterostome ancestral linkage groups (ALGs). These deuterostome ALGs in turn match previously inferred bilaterian ALGs, consistent with a relatively short transition from the last common bilaterian ancestor to the origin of deuterostomes. Based on this deuterostome ALG complement, we deduced chromosomal rearrangement events that occurred in different lineages. For example, a fusion-with-mixing event produced an Ambulacraria-specific ALG that subsequently split into 2 chromosomes in extant hemichordates, while this homologous ALG further fused with another chromosome in sea urchins. Orthologous genes distributed in these rearranged chromosomes are enriched for functions in various developmental processes. We found that the deeply conserved Hox clusters are located in highly rearranged chromosomes and that maintenance of the clusters are likely due to lower densities of transposable elements within the clusters. We also provide evidence that the deuterostome-specific pharyngeal gene cluster was established via the combination of 3 pre-assembled microsyntenic blocks. We suggest that since chromosomal rearrangement events and formation of new gene clusters may change the regulatory controls of developmental genes, these events may have contributed to the evolution of diverse body plans among deuterostomes.

An amphioxus neurula stage cell atlas supports a complex scenario for the emergence of vertebrate head mesoderm.

The emergence of new structures can often be linked to the evolution of novel cell types that follows the rewiring of developmental gene regulatory subnetworks. Vertebrates are characterized by a complex body plan compared to the other chordate clades and the question remains of whether and how the emergence of vertebrate morphological innovations can be related to the appearance of new embryonic cell populations. We previously proposed, by studying mesoderm development in the cephalochordate amphioxus, a scenario for the evolution of the vertebrate head mesoderm. To further test this scenario at the cell population level, we used scRNA-seq to construct a cell atlas of the amphioxus neurula, stage at which the main mesodermal compartments are specified. Our data allowed us to validate the presence of a prechordal-plate like territory in amphioxus. Additionally, the transcriptomic profile of somite cell populations supports the homology between specific territories of amphioxus somites and vertebrate cranial/pharyngeal and lateral plate mesoderm. Finally, our work provides evidence that the appearance of the specific mesodermal structures of the vertebrate head was associated to both segregation of pre-existing cell populations, and co-option of new genes for the control of myogenesis.

Unraveling the transcriptomic landscape of eye migration and visual adaptations during flatfish metamorphosis.

Flatfish undergo a remarkable metamorphosis from symmetrical pelagic larvae to fully asymmetrical benthic juveniles. The most distinctive features of this transformation is the migration of one eye. The molecular role of thyroid hormone in the metamorphosis process in flatfishes is well established. However, the regulatory network that facilitates eye movement remains enigmatic. This paper presents a morphological investigation of the metamorphic process in turbot eyes, using advanced imaging techniques and a global view of gene expression. The study covers migrant and non-migrant eyes and aims to identify the genes that are active during ocular migration. Our transcriptomic analysis shows a significant up-regulation of immune-related genes. The analysis of eye-specific genes reveals distinct patterns during the metamorphic process. Myosin is highlighted in the non-migrant eye, while ependymin is highlighted in the migrant eye, possibly involved in optic nerve regeneration. Furthermore, a potential association between the alx3 gene and cranial restructuring has been identified. Additionally, it confirmed simultaneous adaptation to low light in both eyes, as described by changes in opsins expression during the metamorphic process. The study also revealed that ocular migration activates systems asynchronously in both eyes, providing insight into multifaceted reorganization processes during metamorphosis of flatfish.

Rewiring of the epigenome and chromatin architecture by exogenously induced retinoic acid signaling during zebrafish embryonic development.

Retinoic acid (RA) is the ligand of RA receptors (RARs), transcription factors that bind to RA response elements. RA signaling is required for multiple processes during embryonic development, including body axis extension, hindbrain antero-posterior patterning and forelimb bud initiation. Although some RA target genes have been identified, little is known about the genome-wide effects of RA signaling during in vivo embryonic development. Here, we stimulate the RA pathway by treating zebrafish embryos with all-trans-RA (atRA) and use a combination of RNA-seq, ATAC-seq, ChIP-seq and HiChIP to gain insight into the molecular mechanisms by which exogenously induced RA signaling controls gene expression. We find that RA signaling is involved in anterior/posterior patterning, central nervous system development, and the transition from pluripotency to differentiation. AtRA treatment also alters chromatin accessibility during early development and promotes chromatin binding of RARαa and the RA targets Hoxb1b, Meis2b and Sox3, which cooperate in central nervous system development. Finally, we show that exogenous RA induces a rewiring of chromatin architecture, with alterations in chromatin 3D interactions involving target genes. Altogether, our findings identify genome-wide targets of RA signaling and provide a molecular mechanism by which developmental signaling pathways regulate target gene expression by altering chromatin topology.

The cellular landscape of the endochondral bone during the transition to extrauterine life.

The cellular complexity of the endochondral bone underlies its essential and pleiotropic roles during organismal life. While the adult bone has received significant attention, we still lack a deep understanding of the perinatal bone cellulome. Here, we have profiled the full composition of the murine endochondral bone at the single-cell level during the transition from fetal to newborn life and in comparison with the adult tissue, with particular emphasis on the mesenchymal compartment. The perinatal bone contains different fibroblastic clusters with blastema-like characteristics in organizing and supporting skeletogenesis, angiogenesis and hematopoiesis. Our data also suggest dynamic inter- and intra-compartment interactions, as well as a bone marrow milieu that seems prone to anti-inflammation, which we hypothesize is necessary to ensure the proper program of lymphopoiesis and the establishment of central and peripheral tolerance in early life. Our study provides an integrative roadmap for the future design of genetic and cellular functional assays to validate cellular interactions and lineage relationships within the perinatal bone.

Forkhead transcription factor FKH-8 cooperates with RFX in the direct regulation of sensory cilia in Caenorhabditis elegans.

Cilia, either motile or non-motile (a.k.a primary or sensory), are complex evolutionarily conserved eukaryotic structures composed of hundreds of proteins required for their assembly, structure and function that are collectively known as the ciliome. Ciliome gene mutations underlie a group of pleiotropic genetic diseases known as ciliopathies. Proper cilium function requires the tight coregulation of ciliome gene transcription, which is only fragmentarily understood. RFX transcription factors (TF) have an evolutionarily conserved role in the direct activation of ciliome genes both in motile and non-motile cilia cell-types. In vertebrates, FoxJ1 and FoxN4 Forkhead (FKH) TFs work with RFX in the direct activation of ciliome genes, exclusively in motile cilia cell-types. No additional TFs have been described to act together with RFX in primary cilia cell-types in any organism. Here we describe FKH-8, a FKH TF, as a direct regulator of the sensory ciliome genes in Caenorhabditis elegans. FKH-8 is expressed in all ciliated neurons in C. elegans, binds the regulatory regions of ciliome genes, regulates ciliome gene expression, cilium morphology and a wide range of behaviors mediated by sensory ciliated neurons. FKH-8 and DAF-19 (C. elegans RFX) physically interact and synergistically regulate ciliome gene expression. C. elegans FKH-8 function can be replaced by mouse FOXJ1 and FOXN4 but not by other members of other mouse FKH subfamilies. In conclusion, RFX and FKH TF families act jointly as direct regulators of ciliome genes also in sensory ciliated cell types suggesting that this regulatory logic could be an ancient trait predating functional cilia sub-specialization.

The little skate genome and the evolutionary emergence of wing-like fins.

Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments1,2. However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins-including gene expression, chromatin occupancy and three-dimensional conformation-we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with several hoxa genes, consistent with the redeployment of hox gene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

Genome-wide chromatin accessibility and gene expression profiling during flatfish metamorphosis.

Metamorphosis is a widely studied post-embryonic process in which many tissues undergo dramatic modifications to adapt to the new adult lifestyle. Flatfishes represent a good example of metamorphosis in teleost fishes. During metamorphosis of flatfish, organ regression and neoformation occur, with one of the most notable changes being the migration of one of the eyes to the other side of the body. In order to create a useful and reliable tool to advance the molecular study of metamorphosis in flatfish, we generated a chromatin accessible atlas as well as gene expression profile during four developmental stages ranging from a phylotypic to a post-metamorphic stage. We identified 29,019 differentially accessible chromatin regions and 3,253 differentially expressed genes. We found stage-specific regulatory regions and gene expression profiles, supporting the quality of the results. Our work provides strongly reproducible data for further studies to elucidate the regulatory elements that ensure successful metamorphosis in flatfish species.

Parallel evolution of amphioxus and vertebrate small-scale gene duplications.

BACKGROUND: Amphioxus are non-vertebrate chordates characterized by a slow morphological and molecular evolution. They share the basic chordate body-plan and genome organization with vertebrates but lack their 2R whole-genome duplications and their developmental complexity. For these reasons, amphioxus are frequently used as an outgroup to study vertebrate genome evolution and Evo-Devo. Aside from whole-genome duplications, genes continuously duplicate on a smaller scale. Small-scale duplicated genes can be found in both amphioxus and vertebrate genomes, while only the vertebrate genomes have duplicated genes product of their 2R whole-genome duplications. Here, we explore the history of small-scale gene duplications in the amphioxus lineage and compare it to small- and large-scale gene duplication history in vertebrates. RESULTS: We present a study of the European amphioxus (Branchiostoma lanceolatum) gene duplications thanks to a new, high-quality genome reference. We find that, despite its overall slow molecular evolution, the amphioxus lineage has had a history of small-scale duplications similar to the one observed in vertebrates. We find parallel gene duplication profiles between amphioxus and vertebrates and conserved functional constraints in gene duplication. Moreover, amphioxus gene duplicates show levels of expression and patterns of functional specialization similar to the ones observed in vertebrate duplicated genes. We also find strong conservation of gene synteny between two distant amphioxus species, B. lanceolatum and B. floridae, with two major chromosomal rearrangements. CONCLUSIONS: In contrast to their slower molecular and morphological evolution, amphioxus' small-scale gene duplication history resembles that of the vertebrate lineage both in quantitative and in functional terms.

Multi-omics approach dissects cis-regulatory mechanisms underlying North Carolina macular dystrophy, a retinal enhanceropathy.

North Carolina macular dystrophy (NCMD) is a rare autosomal-dominant disease affecting macular development. The disease is caused by non-coding single-nucleotide variants (SNVs) in two hotspot regions near PRDM13 and by duplications in two distinct chromosomal loci, overlapping DNase I hypersensitive sites near either PRDM13 or IRX1. To unravel the mechanisms by which these variants cause disease, we first established a genome-wide multi-omics retinal database, RegRet. Integration of UMI-4C profiles we generated on adult human retina then allowed fine-mapping of the interactions of the PRDM13 and IRX1 promoters and the identification of eighteen candidate cis-regulatory elements (cCREs), the activity of which was investigated by luciferase and Xenopus enhancer assays. Next, luciferase assays showed that the non-coding SNVs located in the two hotspot regions of PRDM13 affect cCRE activity, including two NCMD-associated non-coding SNVs that we identified herein. Interestingly, the cCRE containing one of these SNVs was shown to interact with the PRDM13 promoter, demonstrated in vivo activity in Xenopus, and is active at the developmental stage when progenitor cells of the central retina exit mitosis, suggesting that this region is a PRDM13 enhancer. Finally, mining of single-cell transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to synapse with retinal ganglion cells, supporting the hypothesis that altered PRDM13 or IRX1 expression impairs interactions between these cells during retinogenesis. Overall, this study provides insight into the cis-regulatory mechanisms of NCMD and supports that this condition is a retinal enhanceropathy.

Multidimensional chromatin profiling of zebrafish pancreas to uncover and investigate disease-relevant enhancers.

The pancreas is a central organ for human diseases. Most alleles uncovered by genome-wide association studies of pancreatic dysfunction traits overlap with non-coding sequences of DNA. Many contain epigenetic marks of cis-regulatory elements active in pancreatic cells, suggesting that alterations in these sequences contribute to pancreatic diseases. Animal models greatly help to understand the role of non-coding alterations in disease. However, interspecies identification of equivalent cis-regulatory elements faces fundamental challenges, including lack of sequence conservation. Here we combine epigenetic assays with reporter assays in zebrafish and human pancreatic cells to identify interspecies functionally equivalent cis-regulatory elements, regardless of sequence conservation. Among other potential disease-relevant enhancers, we identify a zebrafish ptf1a distal-enhancer whose deletion causes pancreatic agenesis, a phenotype previously found to be induced by mutations in a distal-enhancer of PTF1A in humans, further supporting the causality of this condition in vivo. This approach helps to uncover interspecies functionally equivalent cis-regulatory elements and their potential role in human disease.

Gene Regulatory Networks of Epidermal and Neural Fate Choice in a Chordate.

Neurons are a highly specialized cell type only found in metazoans. They can be scattered throughout the body or grouped together, forming ganglia or nerve cords. During embryogenesis, centralized nervous systems develop from the ectoderm, which also forms the epidermis. How pluripotent ectodermal cells are directed toward neural or epidermal fates, and to which extent this process is shared among different animal lineages, are still open questions. Here, by using micromere explants, we were able to define in silico the putative gene regulatory networks (GRNs) underlying the first steps of the epidermis and the central nervous system formation in the cephalochordate amphioxus. We propose that although the signal triggering neural induction in amphioxus (i.e., Nodal) is different from vertebrates, the main transcription factors implicated in this process are conserved. Moreover, our data reveal that transcription factors of the neural program seem to not only activate neural genes but also to potentially have direct inputs into the epidermal GRN, suggesting that the Nodal signal might also contribute to neural fate commitment by repressing the epidermal program. Our functional data on whole embryos support this result and highlight the complex interactions among the transcription factors activated by the signaling pathways that drive ectodermal cell fate choice in chordates.

Gain of gene regulatory network interconnectivity at the origin of vertebrates.

SignificanceIn this manuscript, we address an essential question in developmental and evolutionary biology: How have changes in gene regulatory networks contributed to the invertebrate-to-vertebrate transition? To address this issue, we perturbed four signaling pathways critical for body plan formation in the cephalochordate amphioxus and in zebrafish and compared the effects of such perturbations on gene expression and gene regulation in both species. Our data reveal that many developmental genes have gained response to these signaling pathways in the vertebrate lineage. Moreover, we show that the interconnectivity between these pathways is much higher in zebrafish than in amphioxus. We conclude that this increased signaling pathway complexity likely contributed to vertebrate morphological novelties during evolution.

Duplications disrupt chromatin architecture and rewire GPR101-enhancer communication in X-linked acrogigantism.

X-linked acrogigantism (X-LAG) is the most severe form of pituitary gigantism and is characterized by aggressive growth hormone (GH)-secreting pituitary tumors that occur in early childhood. X-LAG is associated with chromosome Xq26.3 duplications (the X-LAG locus typically includes VGLL1, CD40LG, ARHGEF6, RBMX, and GPR101) that lead to massive pituitary tumoral expression of GPR101, a novel regulator of GH secretion. The mechanism by which the duplications lead to marked pituitary misexpression of GPR101 alone was previously unclear. Using Hi-C and 4C-seq, we characterized the normal chromatin structure at the X-LAG locus. We showed that GPR101 is located within a topologically associating domain (TAD) delineated by a tissue-invariant border that separates it from centromeric genes and regulatory sequences. Next, using 4C-seq with GPR101, RBMX, and VGLL1 viewpoints, we showed that the duplications in multiple X-LAG-affected individuals led to ectopic interactions that crossed the invariant TAD border, indicating the existence of a similar and consistent mechanism of neo-TAD formation in X-LAG. We then identified several pituitary active cis-regulatory elements (CREs) within the neo-TAD and demonstrated in vitro that one of them significantly enhanced reporter gene expression. At the same time, we showed that the GPR101 promoter permits the incorporation of new regulatory information. Our results indicate that X-LAG is a TADopathy of the endocrine system in which Xq26.3 duplications disrupt the local chromatin architecture forming a neo-TAD. Rewiring GPR101-enhancer interaction within the new regulatory unit is likely to cause the high levels of aberrant expression of GPR101 in pituitary tumors caused by X-LAG.

Coordination between cell proliferation and apoptosis after DNA damage in Drosophila.

Exposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These "life" or "death" cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. In this work, we demonstrate that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation-induced apoptosis. We show that p53 binding to the regulatory elements of the proapoptotic genes and its ability to activate their expression is compromised in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.

Brains in Metamorphosis: Temporal Transcriptome Dynamics in Hatchery-Reared Flatfishes.

Metamorphosis is a captivating process of change during which the morphology of the larva is completely reshaped to face the new challenges of adult life. In the case of fish, this process initiated in the brain has traditionally been considered to be a critical rearing point and despite the pioneering molecular work carried out in other flatfishes, the underlying molecular basis is still relatively poorly characterized. Turbot brain transcriptome of three developmental stages (pre-metamorphic, climax of metamorphosis and post-metamorphic) were analyzed to study the gene expression dynamics throughout the metamorphic process. A total of 1570 genes were differentially expressed in the three developmental stages and we found a specific pattern of gene expression at each stage. Unexpectedly, at the climax stage of metamorphosis, we found highly expressed genes related to the immune response, while the biological pathway enrichment analysis in pre-metamorphic and post-metamorphic were related to cell differentiation and oxygen carrier activity, respectively. In addition, our results confirm the importance of thyroid stimulating hormone, increasing its expression during metamorphosis. Based on our findings, we assume that immune system activation during the climax of metamorphosis stage could be related to processes of larval tissue inflammation, resorption and replacement, as occurs in other vertebrates.

CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression.

Coordinated chromatin interactions between enhancers and promoters are critical for gene regulation. The architectural protein CTCF mediates chromatin looping and is enriched at the boundaries of topologically associating domains (TADs), which are sub-megabase chromatin structures. In vitro CTCF depletion leads to a loss of TADs but has only limited effects over gene expression, challenging the concept that CTCF-mediated chromatin structures are a fundamental requirement for gene regulation. However, how CTCF and a perturbed chromatin structure impacts gene expression during development remains poorly understood. Here we link the loss of CTCF and gene regulation during patterning and organogenesis in a ctcf knockout zebrafish model. CTCF absence leads to loss of chromatin structure and affects the expression of thousands of genes, including many developmental regulators. Our results demonstrate the essential role of CTCF in providing the structural context for enhancer-promoter interactions, thus regulating developmental genes.

Topologically Associating Domains and Regulatory Landscapes in Development, Evolution and Disease.

Animal genomes are folded in topologically associating domains (TADs) that have been linked to the regulation of the genes they contain by constraining regulatory interactions between cis-regulatory elements and promoters. Therefore, TADs are proposed as structural scaffolds for the establishment of regulatory landscapes (RLs). In this review, we discuss recent advances in the connection between TADs and gene regulation, their relationship with gene RLs and their dynamics during development and differentiation. Moreover, we describe how restructuring TADs may lead to pathological conditions, which explains their high evolutionary conservation, but at the same time it provides a substrate for the emergence of evolutionary innovations that lay at the origin of vertebrates and other phylogenetic clades.

Genome-Wide Mapping of Histone H3 Lysine 4 Trimethylation (H3K4me3) and Its Involvement in Fatty Acid Biosynthesis in Sunflower Developing Seeds.

Histone modifications are of paramount importance during plant development. Investigating chromatin remodeling in developing oilseeds sheds light on the molecular mechanisms controlling fatty acid metabolism and facilitates the identification of new functional regions in oil crop genomes. The present study characterizes the epigenetic modifications H3K4me3 in relationship with the expression of fatty acid-related genes and transcription factors in developing sunflower seeds. Two master transcriptional regulators identified in this analysis, VIV1 (homologous to Arabidopsis ABI3) and FUS3, cooperate in the regulation of WRINKLED 1, a transcriptional factor regulating glycolysis, and fatty acid synthesis in developing oilseeds.

Functional Conservation of Divergent p63-Bound cis-Regulatory Elements.

The transcription factor p63 is an essential regulator of vertebrate ectoderm development, including epidermis, limbs, and craniofacial tissues. Here, we have investigated the evolutionary conservation of p63 binding sites (BSs) between zebrafish and human. First, we have analyzed sequence conservation of p63 BSs by comparing ChIP-seq data from human keratinocytes and zebrafish embryos, observing a very poor conservation. Next, we compared the gene regulatory network orchestrated by p63 in both species and found a high overlap between them, suggesting a high degree of functional conservation during evolution despite sequence divergence and the large evolutionary distance. Finally, we used transgenic reporter assays in zebrafish embryos to functionally validate a set of equivalent p63 BSs from zebrafish and human located close to genes involved in epidermal development. Reporter expression was driven by human and zebrafish BSs to many common tissues related to p63 expression domains. Therefore, we conclude that the gene regulatory network controlled by p63 is highly conserved across vertebrates despite the fact that p63-bound regulatory elements show high divergence.