Annelid Comparative Genomics and the Evolution of Massive Lineage-Specific Genome Rearrangement in Bilaterians.

The organization of genomes into chromosomes is critical for processes such as genetic recombination, environmental adaptation, and speciation. All animals with bilateral symmetry inherited a genome structure from their last common ancestor that has been highly conserved in some taxa but seemingly unconstrained in others. However, the evolutionary forces driving these differences and the processes by which they emerge have remained largely uncharacterized. Here, we analyze genome organization across the phylum Annelida using 23 chromosome-level annelid genomes. We find that while many annelid lineages have maintained the conserved bilaterian genome structure, the Clitellata, a group containing leeches and earthworms, possesses completely scrambled genomes. We develop a rearrangement index to quantify the extent of genome structure evolution and show that, compared to the last common ancestor of bilaterians, leeches and earthworms have among the most highly rearranged genomes of any currently sampled species. We further show that bilaterian genomes can be classified into two distinct categories-high and low rearrangement-largely influenced by the presence or absence, respectively, of chromosome fission events. Our findings demonstrate that animal genome structure can be highly variable within a phylum and reveal that genome rearrangement can occur both in a gradual, stepwise fashion, or rapid, all-encompassing changes over short evolutionary timescales.

Spiralian genomics and the evolution of animal genome architecture.

Recent developments in sequencing technologies have greatly improved our knowledge of phylogenetic relationships and genomic architectures throughout the tree of life. Spiralia, a diverse clade within Protostomia, is essential for understanding the evolutionary history of parasitism, gene conversion, nervous systems and animal body plans. In this review, we focus on the current hypotheses of spiralian phylogeny and investigate the impact of long-read sequencing on the quality of genome assemblies. We examine chromosome-level assemblies to highlight key genomic features that have driven spiralian evolution, including karyotype, synteny and the Hox gene organization. In addition, we show how chromosome rearrangement has influenced spiralian genomic structures. Although spiralian genomes have undergone substantial changes, they exhibit both conserved and lineage-specific features. We recommend increasing sequencing efforts and expanding functional genomics research to deepen insights into spiralian biology.

Acoel single-cell atlas reveals expression dynamics and heterogeneity of adult pluripotent stem cells.

Adult pluripotent stem cell (aPSC) populations underlie whole-body regeneration in many distantly-related animal lineages, but how the underlying cellular and molecular mechanisms compare across species is unknown. Here, we apply single-cell RNA sequencing to profile transcriptional cell states of the acoel worm Hofstenia miamia during postembryonic development and regeneration. We identify cell types shared across stages and their associated gene expression dynamics during regeneration. Functional studies confirm that the aPSCs, also known as neoblasts, are the source of differentiated cells and reveal transcription factors needed for differentiation. Subclustering of neoblasts recovers transcriptionally distinct subpopulations, the majority of which are likely specialized to differentiated lineages. One neoblast subset, showing enriched expression of the histone variant H3.3, appears to lack specialization. Altogether, the cell states identified in this study facilitate comparisons to other species and enable future studies of stem cell fate potentials.

Acoel genome reveals the regulatory landscape of whole-body regeneration.

Whole-body regeneration is accompanied by complex transcriptomic changes, yet the chromatin regulatory landscapes that mediate this dynamic response remain unexplored. To decipher the regulatory logic that orchestrates regeneration, we sequenced the genome of the acoel worm Hofstenia miamia, a highly regenerative member of the sister lineage of other bilaterians. Epigenomic profiling revealed thousands of regeneration-responsive chromatin regions and identified dynamically bound transcription factor motifs, with the early growth response (EGR) binding site as the most variably accessible during Hofstenia regeneration. Combining egr inhibition with chromatin profiling suggests that Egr functions as a pioneer factor to directly regulate early wound-induced genes. The genetic connections inferred by this approach allowed the construction of a gene regulatory network for whole-body regeneration, enabling genomics-based comparisons of regeneration across species.

Dual Gene Repertoires for Larval and Adult Shells Reveal Molecules Essential for Molluscan Shell Formation.

Molluscan shells, mainly composed of calcium carbonate, also contain organic components such as proteins and polysaccharides. Shell organic matrices construct frameworks of shell structures and regulate crystallization processes during shell formation. To date, a number of shell matrix proteins (SMPs) have been identified, and their functions in shell formation have been studied. However, previous studies focused only on SMPs extracted from adult shells, secreted after metamorphosis. Using proteomic analyses combined with genomic and transcriptomic analyses, we have identified 31 SMPs from larval shells of the pearl oyster, Pinctada fucata, and 111 from the Pacific oyster, Crassostrea gigas. Larval SMPs are almost entirely different from those of adults in both species. RNA-seq data also confirm that gene expression profiles for larval and adult shell formation are nearly completely different. Therefore, bivalves have two repertoires of SMP genes to construct larval and adult shells. Despite considerable differences in larval and adult SMPs, some functional domains are shared by both SMP repertoires. Conserved domains include von Willebrand factor type A (VWA), chitin-binding (CB), carbonic anhydrase (CA), and acidic domains. These conserved domains are thought to play crucial roles in shell formation. Furthermore, a comprehensive survey of animal genomes revealed that the CA and VWA-CB domain-containing protein families expanded in molluscs after their separation from other Lophotrochozoan linages such as the Brachiopoda. After gene expansion, some family members were co-opted for molluscan SMPs that may have triggered to develop mineralized shells from ancestral, nonmineralized chitinous exoskeletons.

Nemertean and phoronid genomes reveal lophotrochozoan evolution and the origin of bilaterian heads.

Nemerteans (ribbon worms) and phoronids (horseshoe worms) are closely related lophotrochozoans-a group of animals including leeches, snails and other invertebrates. Lophotrochozoans represent a superphylum that is crucial to our understanding of bilaterian evolution. However, given the inconsistency of molecular and morphological data for these groups, their origins have been unclear. Here, we present draft genomes of the nemertean Notospermus geniculatus and the phoronid Phoronis australis, together with transcriptomes along the adult bodies. Our genome-based phylogenetic analyses place Nemertea sister to the group containing Phoronida and Brachiopoda. We show that lophotrochozoans share many gene families with deuterostomes, suggesting that these two groups retain a core bilaterian gene repertoire that ecdysozoans (for example, flies and nematodes) and platyzoans (for example, flatworms and rotifers) do not. Comparative transcriptomics demonstrates that lophophores of phoronids and brachiopods are similar not only morphologically, but also at the molecular level. Despite dissimilar head structures, lophophores express vertebrate head and neuronal marker genes. This finding suggests a common origin of bilaterian head patterning, although different heads evolved independently in each lineage. Furthermore, we observe lineage-specific expansions of innate immunity and toxin-related genes. Together, our study reveals a dual nature of lophotrochozoans, where conserved and lineage-specific features shape their evolution.

Genetic and molecular basis of the immune system in the brachiopod Lingula anatina.

The extension of comparative immunology to non-model systems, such as mollusks and annelids, has revealed an unexpected diversity in the complement of immune receptors and effectors among evolutionary lineages. However, several lophotrochozoan phyla remain unexplored mainly due to the lack of genomic resources. The increasing accessibility of high-throughput sequencing technologies offers unique opportunities for extending genome-wide studies to non-model systems. As a result, the genome-based study of the immune system in brachiopods allows a better understanding of the alternative survival strategies developed by these immunologically neglected phyla. Here we present a detailed overview of the molecular components of the immune system identified in the genome of the brachiopod Lingula anatina. Our findings reveal conserved intracellular signaling pathways as well as unique strategies for pathogen detection and killing in brachiopods.

Possible co-option of engrailed during brachiopod and mollusc shell development.

In molluscs, two homeobox genes, engrailed (en) and distal-less (dlx), are transcription factors that are expressed in correlation with shell development. They are expressed in the regions between shell-forming and non-shell-forming cells, likely defining the boundaries of shell-forming fields. Here we investigate the expression of two transcription factors in the brachiopod Lingula anatina We find that en is expressed in larval mantle lobes, whereas dlx is expressed in larval tentacles. We also demonstrate that the embryonic shell marker mantle peroxidase (mpox) is specifically expressed in mantle lobes. Our results suggest that en and mpox are possibly involved in brachiopod embryonic shell development. We discuss the evolutionary developmental origin of lophotrochozoan biomineralization through independent gene co-option.

Cell division and cadherin-mediated adhesion regulate lens epithelial cell movement in zebrafish.

In vertebrates, lens epithelial cells cover the anterior half of the lens fiber core. During development, lens epithelial cells proliferate, move posteriorly and differentiate into lens fiber cells after passing through the equator. To elucidate the mechanisms underlying lens epithelial cell movement, we conducted time-lapse imaging of zebrafish lens epithelium. Lens epithelial cells do not intermingle but maintain their relative positions during development. Cell division induces epithelial rearrangement, which subsequently promotes cell movement towards the equator. These data suggest that cell division is the major driving force for cell movement. In zebrafish, E-cadherin is expressed in lens epithelium, whereas N-cadherin is required for lens fiber growth. E-cadherin reduced lens epithelial cell movement, whereas N-cadherin enhanced it. Laser ablation experiments revealed that lens epithelium is governed by pulling tension, which is modulated by these cadherins. Thus, cell division and cadherin-mediated adhesion regulate lens epithelial cell movement via modulation of epithelial tension.

Mitochondrial gene order variation in the brachiopod Lingula anatina and its implications for mitochondrial evolution in lophotrochozoans.

Vertebrate mitochondrial (mt) genomes display highly conserved gene order and relatively low evolutionary rates. However, these features are variable in marine invertebrates. Here we present the mt genome of the lingulid brachiopod, Lingula anatina, from Amami Island, Japan, as part of the nuclear genome project. We obtain ~2000-fold coverage of the 17.9-kb mt genome using Illumina sequencing, and we identify hypervariable regions within the same individual. Transcriptome analyses show that mt transcripts are polycistronic and expressed differentially. Unexpectedly, we find that the mt gene order of Amami Lingula is completely shuffled compared to that of a specimen from Yanagawa, suggesting that there may be cryptic species. Using breakpoint distance analyses with 101 metazoan mt genomes, we show that the evolutionary history of mt gene order among lophotrochozoans is unique. Analyses of non-synonymous substitution rates reveal that mt protein-coding genes of Lingula have experienced rapid evolution comparable to that expected for interspecific comparisons. Whole genome phylogenetic analyses suggest that mt genomes have limited value for inferring the phylogenetic positions of lophotrochozoans because of their high evolutionary rates in brachiopods and bivalves.

The Lingula genome provides insights into brachiopod evolution and the origin of phosphate biomineralization.

The evolutionary origins of lingulid brachiopods and their calcium phosphate shells have been obscure. Here we decode the 425-Mb genome of Lingula anatina to gain insights into brachiopod evolution. Comprehensive phylogenomic analyses place Lingula close to molluscs, but distant from annelids. The Lingula gene number has increased to ∼34,000 by extensive expansion of gene families. Although Lingula and vertebrates have superficially similar hard tissue components, our genomic, transcriptomic and proteomic analyses show that Lingula lacks genes involved in bone formation, indicating an independent origin of their phosphate biominerals. Several genes involved in Lingula shell formation are shared by molluscs. However, Lingula has independently undergone domain combinations to produce shell matrix collagens with EGF domains and carries lineage-specific shell matrix proteins. Gene family expansion, domain shuffling and co-option of genes appear to be the genomic background of Lingula's unique biomineralization. This Lingula genome provides resources for further studies of lophotrochozoan evolution.

Expression of germline markers in three species of amphioxus supports a preformation mechanism of germ cell development in cephalochordates.

BACKGROUND: In a previous study, we showed that the cephalochordate amphioxus Branchiostoma floridae has localized maternal transcripts of conserved germ cell markers Vasa and Nanos in its early embryos. These results provided strong evidence to support a preformation mechanism for primordial germ cell (PGC) development in B. floridae. RESULTS: In this study, we further characterize the expression of B. floridae homologs of Piwi and Tudor, which play important roles in germline development in diverse metazoan animals. We show that maternal mRNA of one of the identified Piwi-like homologs, Bf-Piwil1, also colocalizes with Vasa in the vegetal germ plasm and has zygotic expression in both the putative PGCs and the tail bud, suggesting it may function in both germline and somatic stem cells. More interestingly, one Tudor family gene, Bf-Tdrd7, is only expressed maternally and colocalizes with Vasa in germ plasm, suggesting that it may function exclusively in germ cell specification. To evaluate the conservation of the preformation mechanism among amphioxus species, we further analyze Vasa, Nanos, Piwil1, and Tdrd7 expression in two Asian amphioxus species, B. belcheri and B. japonicum. Their maternal transcripts all localize in similar patterns to those seen in B. floridae. In addition, we labeled putative PGCs with Vasa antibody to trace their dynamic distribution in developing larvae. CONCLUSIONS: We identify additional germ plasm components in amphioxus and demonstrate the molecular distinction between the putative germline stem cells and somatic stem cells. Moreover, our results suggest that preformation may be a conserved mechanism for PGC specification among Branchiostoma species. Our Vasa antibody staining results suggest that after the late neurula stage, amphioxus PGCs probably proliferate with the tail bud cells during posterior elongation and are deposited near the forming myomere boundaries. Subsequently, these PGCs would concentrate at the ventral tip of the myoseptal walls to form the gonad anlagen.

Opposing nodal and BMP signals regulate left-right asymmetry in the sea urchin larva.

Nodal and BMP signals are important for establishing left-right (LR) asymmetry in vertebrates. In sea urchins, Nodal signaling prevents the formation of the rudiment on the right side. However, the opposing pathway to Nodal signaling during LR axis establishment is not clear. Here, we revealed that BMP signaling is activated in the left coelomic pouch, specifically in the veg2 lineage, but not in the small micromeres. By perturbing BMP activities, we demonstrated that BMP signaling is required for activating the expression of the left-sided genes and the formation of the left-sided structures. On the other hand, Nodal signals on the right side inhibit BMP signaling and control LR asymmetric separation and apoptosis of the small micromeres. Our findings show that BMP signaling is the positive signal for left-sided development in sea urchins, suggesting that the opposing roles of Nodal and BMP signals in establishing LR asymmetry are conserved in deuterostomes.

BMP and Delta/Notch signaling control the development of amphioxus epidermal sensory neurons: insights into the evolution of the peripheral sensory system.

The evolution of the nervous system has been a topic of great interest. To gain more insight into the evolution of the peripheral sensory system, we used the cephalochordate amphioxus. Amphioxus is a basal chordate that has a dorsal central nervous system (CNS) and a peripheral nervous system (PNS) comprising several types of epidermal sensory neurons (ESNs). Here, we show that a proneural basic helix-loop-helix gene (Ash) is co-expressed with the Delta ligand in ESN progenitor cells. Using pharmacological treatments, we demonstrate that Delta/Notch signaling is likely to be involved in the specification of amphioxus ESNs from their neighboring epidermal cells. We also show that BMP signaling functions upstream of Delta/Notch signaling to induce a ventral neurogenic domain. This patterning mechanism is highly similar to that of the peripheral sensory neurons in the protostome and vertebrate model animals, suggesting that they might share the same ancestry. Interestingly, when BMP signaling is globally elevated in amphioxus embryos, the distribution of ESNs expands to the entire epidermal ectoderm. These results suggest that by manipulating BMP signaling levels, a conserved neurogenesis circuit can be initiated at various locations in the epidermal ectoderm to generate peripheral sensory neurons in amphioxus embryos. We hypothesize that during chordate evolution, PNS progenitors might have been polarized to different positions in various chordate lineages owing to differential regulation of BMP signaling in the ectoderm.

Asymmetric localization of germline markers Vasa and Nanos during early development in the amphioxus Branchiostoma floridae.

The origin of germline cells was a crucial step in animal evolution. Therefore, in both developmental biology and evolutionary biology, the mechanisms of germline specification have been extensively studied over the past two centuries. However, in many animals, the process of germline specification remains unclear. Here, we show that in the cephalochordate amphioxus Branchiostoma floridae, the germ cell-specific molecular markers Vasa and Nanos become localized to the vegetal pole cytoplasm during oogenesis and are inherited asymmetrically by a single blastomere during cleavage. After gastrulation, this founder cell gives rise to a cluster of progeny that display typical characters of primordial germ cells (PGCs). Blastomeres separated at the two-cell stage grow into twin embryos, but one of the twins fails to develop this Vasa-positive cell population, suggesting that the vegetal pole cytoplasm is required for the formation of putative PGCs in amphioxus embryos. Contrary to the hypothesis that cephalochordates may form their PGCs by epigenesis, our data strongly support a preformation mode of germ cell specification in amphioxus. In addition to the early localization of their maternal transcripts in the putative PGCs, amphioxus Vasa and Nanos are also expressed zygotically in the tail bud, which is the posterior growth zone of amphioxus. Thus, in addition to PGC specification, amphioxus Vasa and Nanos may also function in highly proliferating somatic stem cells.

The dynamic gene expression patterns of transcription factors constituting the sea urchin aboral ectoderm gene regulatory network.

The temporal and spatial expression patterns of regulatory genes are required for building a gene regulatory network (GRN). The current ectoderm GRN model for the sea urchin embryo includes pregastrular specification functions in the oral (OE) and aboral ectoderm (AE). Unlike the OE, which is resolved into several subdomains, the AE is considered a simpler territory due to the lack of detailed gene expression studies in this territory. Here, we perform temporal and spatial gene expression studies on the eight transcription factor genes constituting the AE GRN. Based on the differential gene expression patterns, we conclude that the AE contains at least three subdomains at the mesenchyme blastula stage. We also performed immunostaining for pSmad1/5/8 to monitor the activation of the BMP signaling pathway. The dynamic changes in the expression patterns of these transcription factor genes and the nuclearization of pSmad1/5/8 may provide a foundation for resolving the AE GRN.