A genome resource for the marine annelid Platynereis dumerilii.

The marine annelid Platynereis dumerilii is a model organism used in many research areas including evolution and development, neurobiology, ecology and regeneration. Here we present the genomes of P. dumerilii and of the closely related P. massiliensis and P. megalops, to facilitate comparative genomic approaches and help explore Platynereis biology. We used long-read sequencing technology and chromosomal-conformation capture along with extensive transcriptomic resources to obtain and annotate a draft genome assembly of ~1.47 Gbp for P. dumerilii, of which more than half represent repeat elements. We predict around 29,000 protein-coding genes, with relatively large intron sizes, over 38,000 non-coding genes, and 580 miRNA loci. We further explore the high genetic variation (~3% heterozygosity) within the Platynereis species complex. Gene ontology reveals the most variable loci to be associated with pigmentation, development and immunity. The current work sets the stage for further development of Platynereis genomic resources.

Sex-biased gene expression precedes sexual dimorphism in the agonadal annelid Platynereis dumerilii.

Gametogenesis is the process by which germ cells differentiate into mature sperm and oocytes, cells essential for sexual reproduction. The sex-specific molecular programs that drive spermatogenesis and oogenesis can also serve as sex identification markers. Platynereis dumerilii is a research organism that has been studied in many areas of developmental biology. However investigations often disregard sex, as P. dumerilii juveniles lack sexual dimorphism. The molecular mechanisms of gametogenesis in the segmented worm P. dumerilii are also largely unknown. In this study, we used RNA sequencing to investigate the transcriptomic profiles of gametogenesis in P. dumerilii juveniles. Our analysis revealed that sex-biased gene expression becomes increasingly pronounced during the advanced developmental stages, particularly during the meiotic phases of gametogenesis. We identified conserved genes associated with spermatogenesis, such as dmrt1, and a novel gene psmt, that is associated with oogenesis. Additionally, putative long non-coding RNAs were upregulated in both male and female gametogenic programs. This study provides a foundational resource for germ cell research in P. dumerilii, markers for sex identification, and offers comparative data to enhance our understanding of the evolution of gametogenesis mechanisms across species.

Creation of an albino squid line by CRISPR-Cas9 and its application for in vivo functional imaging of neural activity.

Cephalopods are remarkable among invertebrates for their cognitive abilities, adaptive camouflage, novel structures, and propensity for recoding proteins through RNA editing. Due to the lack of genetically tractable cephalopod models, however, the mechanisms underlying these innovations are poorly understood. Genome editing tools such as CRISPR-Cas9 allow targeted mutations in diverse species to better link genes and function. One emerging cephalopod model, Euprymna berryi, produces large numbers of embryos that can be easily cultured throughout their life cycle and has a sequenced genome. As proof of principle, we used CRISPR-Cas9 in E. berryi to target the gene for tryptophan 2,3 dioxygenase (TDO), an enzyme required for the formation of ommochromes, the pigments present in the eyes and chromatophores of cephalopods. CRISPR-Cas9 ribonucleoproteins targeting tdo were injected into early embryos and then cultured to adulthood. Unexpectedly, the injected specimens were pigmented, despite verification of indels at the targeted sites by sequencing in injected animals (G0s). A homozygote knockout line for TDO, bred through multiple generations, was also pigmented. Surprisingly, a gene encoding indoleamine 2,3, dioxygenase (IDO), an enzyme that catalyzes the same reaction as TDO in vertebrates, was also present in E. berryi. Double knockouts of both tdo and ido with CRISPR-Cas9 produced an albino phenotype. We demonstrate the utility of these albinos for in vivo imaging of Ca2+ signaling in the brain using two-photon microscopy. These data show the feasibility of making gene knockout cephalopod lines that can be used for live imaging of neural activity in these behaviorally sophisticated organisms.

Segment number threshold determines juvenile onset of germline cluster expansion in Platynereis dumerilii.

Development of sexual characters and generation of gametes are tightly coupled with growth. Platynereis dumerilii is a marine annelid that has been used to study germline development and gametogenesis. P. dumerilii has germ cell clusters found across the body in the juvenile worms, and the clusters eventually form the gametes. Like other segmented worms, P. dumerilii grows by adding new segments at its posterior end. The number of segments reflect the growth state of the worms and therefore is a useful and measurable growth state metric to study the growth-reproduction crosstalk. To understand how growth correlates with progression of gametogenesis, we investigated germline development across several developmental stages. We discovered a distinct transition period when worms increase the number of germline clusters at a particular segment number threshold. Additionally, we found that keeping worms short in segment number, by manipulating environmental conditions or via amputations, supported a segment number threshold requirement for germline development. Finally, we asked if these clusters in P. dumerilii play a role in regeneration (as similar free-roaming cells are observed in Hydra and planarian regeneration) and found that the clusters were not required for regeneration in P. dumerilii, suggesting a strictly germline nature. Overall, these molecular analyses suggest a previously unidentified developmental transition dependent on the growth state of juvenile P. dumerilii leading to substantially increased germline expansion.

Developmental transition to bilaterally symmetric cell divisions is regulated by Pax-mediated transcription in embryos of the leech Helobdella austinensis.

The leech embryo develops by spiral cleavage, and establishes the symmetry properties of its adult body plan through the bilaterally symmetric divisions of mesodermal proteloblast DM″ and ectodermal proteloblast DNOPQ‴. We here show that transcriptional inhibitors α-amanitin and actinomycin D specifically disrupt the symmetry and orientation of these two proteloblast cell divisions while having no apparent effect on the timing or geometry of other divisions. Transcriptional inhibition had a similar effect on both proteloblasts, i.e. cytokinesis was highly asymmetric and the cleavage plane roughly orthogonal to that seen during normal development. These findings suggest that zygotic gene product(s) are required, either directly or indirectly, for the correct placement of the proteloblast cleavage furrow. The same phenotypes were also observed following in vivo expression of dominant-negative Pax gene constructs. These dominant-negative phenotypes depended on protein/DNA interaction, and could be rescued by coexpression of full length Pax proteins. However, symmetric cleavage of the mesodermal proteloblast was rescued by full length constructs of either Hau-Paxß1 or Hau-Pax2/5/8, while only Hau-Paxß1 rescued the symmetry of ectodermal cleavage. We conclude that both proteloblasts need Pax-mediated transcription to adopt their normally symmetric cleavage patterns, but differ in terms of the specific Pax proteins required. The implication of these findings for the evolution of spiral cleavage is discussed.