Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species.

Genome structural variations within species are rare. How selective constraints preserve gene order and chromosome structure is a central question in evolutionary biology that remains unsolved. Our sequencing of several genomes of the appendicularian tunicate Oikopleura dioica around the globe reveals extreme genome scrambling caused by thousands of chromosomal rearrangements, although showing no obvious morphological differences between these animals. The breakpoint accumulation rate is an order of magnitude higher than in ascidian tunicates, nematodes, Drosophila, or mammals. Chromosome arms and sex-specific regions appear to be the primary unit of macrosynteny conservation. At the microsyntenic level, scrambling did not preserve operon structures, suggesting an absence of selective pressure to maintain them. The uncoupling of the genome scrambling with morphological conservation in O. dioica suggests the presence of previously unnoticed cryptic species and provides a new biological system that challenges our previous vision of speciation in which similar animals always share similar genome structures.

Annelid functional genomics reveal the origins of bilaterian life cycles.

Indirect development with an intermediate larva exists in all major animal lineages1, which makes larvae central to most scenarios of animal evolution2-11. Yet how larvae evolved remains disputed. Here we show that temporal shifts (that is, heterochronies) in trunk formation underpin the diversification of larvae and bilaterian life cycles. We performed chromosome-scale genome sequencing in the annelid Owenia fusiformis with transcriptomic and epigenomic profiling during the life cycles of this and two other annelids. We found that trunk development is deferred to pre-metamorphic stages in the feeding larva of O. fusiformis but starts after gastrulation in the non-feeding larva with gradual metamorphosis of Capitella teleta and the direct developing embryo of Dimorphilus gyrociliatus. Accordingly, the embryos of O. fusiformis develop first into an enlarged anterior domain that forms larval tissues and the adult head12. Notably, this also occurs in the so-called 'head larvae' of other bilaterians13-17, with which the O. fusiformis larva shows extensive transcriptomic similarities. Together, our findings suggest that the temporal decoupling of head and trunk formation, as maximally observed in head larvae, facilitated larval evolution in Bilateria. This diverges from prevailing scenarios that propose either co-option9,10 or innovation11 of gene regulatory programmes to explain larva and adult origins.

Telomere-to-telomere assembly of the genome of an individual Oikopleura dioica from Okinawa using Nanopore-based sequencing.

BACKGROUND: The larvacean Oikopleura dioica is an abundant tunicate plankton with the smallest (65-70 Mbp) non-parasitic, non-extremophile animal genome identified to date. Currently, there are two genomes available for the Bergen (OdB3) and Osaka (OSKA2016) O. dioica laboratory strains. Both assemblies have full genome coverage and high sequence accuracy. However, a chromosome-scale assembly has not yet been achieved. RESULTS: Here, we present a chromosome-scale genome assembly (OKI2018_I69) of the Okinawan O. dioica produced using long-read Nanopore and short-read Illumina sequencing data from a single male, combined with Hi-C chromosomal conformation capture data for scaffolding. The OKI2018_I69 assembly has a total length of 64.3 Mbp distributed among 19 scaffolds. 99% of the assembly is contained within five megabase-scale scaffolds. We found telomeres on both ends of the two largest scaffolds, which represent assemblies of two fully contiguous autosomal chromosomes. Each of the other three large scaffolds have telomeres at one end only and we propose that they correspond to sex chromosomes split into a pseudo-autosomal region and X-specific or Y-specific regions. Indeed, these five scaffolds mostly correspond to equivalent linkage groups in OdB3, suggesting overall agreement in chromosomal organization between the two populations. At a more detailed level, the OKI2018_I69 assembly possesses similar genomic features in gene content and repetitive elements reported for OdB3. The Hi-C map suggests few reciprocal interactions between chromosome arms. At the sequence level, multiple genomic features such as GC content and repetitive elements are distributed differently along the short and long arms of the same chromosome. CONCLUSIONS: We show that a hybrid approach of integrating multiple sequencing technologies with chromosome conformation information results in an accurate de novo chromosome-scale assembly of O. dioica's highly polymorphic genome. This genome assembly opens up the possibility of cross-genome comparison between O. dioica populations, as well as of studies of chromosomal evolution in this lineage.

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica.

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact genome. For these reasons, it is considered a useful model organism for marine ecological studies, evolutionary developmental biology, and genomics. As research often requires a steady supply of animal resources, it is useful to establish a reliable, low-maintenance culture system. Here we describe a step-by-step method for establishing an O. dioica culture. We describe how to select potential sampling sites, collection methods, target animal identification, and the set-up of the culturing system. We provide troubleshooting advice based on our own experiences. We also highlight critical factors that help sustain a robust culture system. Although the culture protocol provided here is optimized for O. dioica, we hope our sampling technique and culture setup will inspire new ideas for maintaining other fragile pelagic invertebrates.

H3S28P Antibody Staining of Okinawan Oikopleura dioica Suggests the Presence of Three Chromosomes.

Oikopleura dioica is a ubiquitous marine tunicate of biological interest due to features that include dioecious reproduction, short life cycle, and vertebrate-like dorsal notochord while possessing a relatively compact genome. The use of tunicates as model organisms, particularly with these characteristics, offers the advantage of facilitating studies in evolutionary development and furthering understanding of enduring attributes found in the more complex vertebrates. At present, we are undertaking an initiative to sequence the genomes of Oikopleura individuals in populations found among the seas surrounding the Ryukyu Islands in southern Japan. To facilitate and validate genome assemblies, karyotyping was employed to count individual animals' chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. New imaging data of embryos and oocytes stained with two different antibodies were obtained; interpretation of these data lead us to conclude that the Okinawan Oikopleura dioica has three pairs of chromosomes, akin to previous results from genomic assemblies in Atlantic populations. The imaging data have been deposited to the open-access EBI BioImage Archive for reuse while additionally providing representative images of two commercially available anti-H3S28P antibodies' staining properties for use in epifluorescent and confocal based fluorescent microscopy.

Genomic analyses reveal mutational signatures and frequently altered genes in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide and the fourth most lethal cancer in China. However, although genomic studies have identified some mutations associated with ESCC, we know little of the mutational processes responsible. To identify genome-wide mutational signatures, we performed either whole-genome sequencing (WGS) or whole-exome sequencing (WES) on 104 ESCC individuals and combined our data with those of 88 previously reported samples. An APOBEC-mediated mutational signature in 47% of 192 tumors suggests that APOBEC-catalyzed deamination provides a source of DNA damage in ESCC. Moreover, PIK3CA hotspot mutations (c.1624G>A [p.Glu542Lys] and c.1633G>A [p.Glu545Lys]) were enriched in APOBEC-signature tumors, and no smoking-associated signature was observed in ESCC. In the samples analyzed by WGS, we identified focal (<100 kb) amplifications of CBX4 and CBX8. In our combined cohort, we identified frequent inactivating mutations in AJUBA, ZNF750, and PTCH1 and the chromatin-remodeling genes CREBBP and BAP1, in addition to known mutations. Functional analyses suggest roles for several genes (CBX4, CBX8, AJUBA, and ZNF750) in ESCC. Notably, high activity of hedgehog signaling and the PI3K pathway in approximately 60% of 104 ESCC tumors indicates that therapies targeting these pathways might be particularly promising strategies for ESCC. Collectively, our data provide comprehensive insights into the mutational signatures of ESCC and identify markers for early diagnosis and potential therapeutic targets.

Biochemical characterisation and assessment of fibril-forming ability of collagens extracted from Bester sturgeon Huso huso × Acipenser ruthenus.

Collagens purified from Bester sturgeon organs were characterised biochemically, and their fibril-forming abilities and fibril morphologies formed in vitro clarified. Yields of collagens were 2.1%, 11.9%, 0.4%, 18.1%, 0.4%, 0.8% and 0.03% (collagen dry weight/tissue wet weight) from scales, skin, muscle, swim bladder, digestive tract, notochord and snout cartilage, respectively. Using SDS-PAGE and amino acid composition analyses, collagens from scales, skin, muscle, the swim bladder and digestive tract were characterised as type I, and collagens from the notochord and snout cartilage as type II. Denaturation temperatures of the collagens, measured using circular dichroism, were 29.6, 26.8, 29.0, 32.9, 31.6 and 36.3 °C in scales, skin, muscle, swim bladder, digestive tract, and notochord, respectively. For fibril formation, swim bladder and skin collagen showed a more rapid rate of increase in turbidity, a shorter time to attain the maximum turbidity, and formed thicker fibrils compared with porcine tendon type I collagen.

Spatiotemporal expression of the dermatopontin gene in zebrafish Danio rerio.

There has been significant interest in the expression and function of dermatopontin (DPT) in mammals owing to recent evidence pointing to its critical role in collagen fibrillogenesis. Despite this interest, limited information is available about the site/s of DPT mRNA expression or changes in expression in vivo. We used reverse-transcription polymerase chain reaction and in situ hybridization to evaluate the spatial and temporal pattern of DPT mRNA expression in zebrafish, Danio rerio, a widely used vertebrate model. We observed that DPT transcripts were expressed in zebrafish embryos at all developmental stages in a range of tissues, including the brain and optic neuron cells. Based on our results, we hypothesize that DPT may also play a role in neural functions in vivo.