The regulatory origin of cephalopod limbs

2019 Fiscal Year Research-status Report

• Project/Area Number: 19K06620
• Research Institution: Okinawa Institute of Science and Technology Graduate University
• Principal Investigator: MARLETAZ Ferdinand 沖縄科学技術大学院大学, 分子遺伝学ユニット, 研究員 (90833955)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: cephalopod / gene expression / regulatory profiling / limbs

Outline of Annual Research Achievements

During the first year of the Kakenhi project, we finished the sequencing of the genome of our bobtail squid model E. berryi using a combination of short and long reads (pacbio) as well as HiC scaffolding. We obtained a 5.91Gb assembly with 46 large chromosomes consistent with chromosome number in other members of the clade Decapoda. The genome was thoroughly annotated, 57259 genes were identified that were subsequently used for gene family reconstruction.Key development gene families (Pax, Wnt, Homeodomain) were analysed using phylogenetic reconstruction to determine the orthodoxy relationships. Our genome assembly harbours a complete Hox cluster spanning over 17Mb which confirms and expands previous observation in other cephalopod species and suggests unusual principles of Hox genes regulation.
We performed preliminary assays of ATAC-seq and RNA-seq for embryonic stages and organs, which is an important resource for the project. During the season 2020, we obtained many embryos and successfully dissected limb buds as planned in the grant. Some samples were preserved for RNA-seq and with others, we tested ATAC-seq experiments using the omni-atac protocol. We managed to obtain ATAC-seq with a good size profile using paired limb pairs (right and left size). There samples are due for sequencing. In parallel, efforts have been made in the lab to establish antibody staining and fluorescent imaging. We are quite confident about the next steps of the project.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Genome is sequencing and protocol established. Some samples were preserved for RNA-seq and with others, we tested ATAC-seq experiments using the omni-atac protocol. We managed to obtain ATAC-seq with a good size profile using paired limb pairs (right and left size). There samples are due for sequencing.

Strategy for Future Research Activity

We are quite confident about the next steps of the project. Samples will be sequenced and bioinformatic analyses start soon.

Causes of Carryover

Main sequencing costs incurring next year.