TrBase: A genome and transcriptome database of Temnopleurus reevesii.

Sea urchins have a long history as model organisms in biology, but their use in genetics is limited because of their long breeding cycle. In sea urchin genetics, genome editing technology was first established in Hemicentrotus pulcherrimus, whose genome has already been published. However, because this species also has a long breeding cycle, new model sea urchins that are more suitable for genetics have been sought. Here, we report a draft genome of another Western Pacific species, Temnopleurus reevesii, which we established as a new model sea urchin recently since this species has a comparable developmental process to other model sea urchins but a short breeding cycle of approximately half a year. The genome of T. reevesii was assembled into 28,742 scaffold sequences with an N50 length of 67.6 kb and an estimated genome size of 905.9 Mb. In the assembled genome, 27,064 genes were identified, 23,624 of which were expressed in at least one of the seven developmental stages. To provide genetic information, we constructed the genome database TrBase (https://cell-innovation.nig.ac.jp/Tree/). We also constructed the Western Pacific Sea Urchin Genome Database (WestPac-SUGDB) (https://cell-innovation.nig.ac.jp/WPAC/) with the aim of establishing a portal site for genetic information on sea urchins in the West Pacific. This site contains genomic information on two species, T. reevesii and H. pulcherrimus, and is equipped with homology search programs for comparing the two datasets. Therefore, TrBase and WestPac-SUGDB are expected to contribute not only to genetic research using sea urchins but also to comparative genomics and evolutionary research.

Inhibition of BCL2A1 by STAT5 inactivation overcomes resistance to targeted therapies of FLT3-ITD/D835 mutant AML.

Tyrosine kinase inhibitors (TKIs) are established drugs in the therapy of FLT3-ITD mutated acute myeloid leukemia (AML). However, acquired mutations, such as D835 in the tyrosine kinase domain (FLT3-ITD/D835), can induce resistance to TKIs. A cap analysis gene expression (CAGE) technology revealed that the gene expression of BCL2A1 transcription start sites was increased in primary AML cells bearing FLT3-ITD/D835 compared to FLT3-ITD. Overexpression of BCL2A1 attenuated the sensitivity to quizartinib, a type II TKI, and venetoclax, a selective BCL2 inhibitor, in AML cell lines. However, a type I TKI, gilteritinib, inhibited the expression of BCL2A1 through inactivation of STAT5 and alleviated TKI resistance of FLT3-ITD/D835. The combination of gilteritinib and venetoclax showed synergistic effects in the FLT3-ITD/D835 positive AML cells. The promoter region of BCL2A1 contains a BRD4 binding site. Thus, the blockade of BRD4 with a BET inhibitor (CPI-0610) downregulated BCL2A1 in FLT3-mutated AML cells and extended profound suppression of FLT3-ITD/D835 mutant cells. Therefore, we propose that BCL2A1 has the potential to be a novel therapeutic target in treating FLT3-ITD/D835 mutated AML.

Exogenous mitochondrial transfer and endogenous mitochondrial fission facilitate AML resistance to OxPhos inhibition.

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival, and they continually adapt to fluctuations in nutrient and oxygen availability in the bone marrow (BM) microenvironment. We investigated how the BM microenvironment affects the response to OxPhos inhibition in AML by using a novel complex I OxPhos inhibitor, IACS-010759. Cellular adhesion, growth, and apoptosis assays, along with measurements of expression of mitochondrial DNA and generation of mitochondrial reactive oxygen species indicated that direct interactions with BM stromal cells triggered compensatory activation of mitochondrial respiration and resistance to OxPhos inhibition in AML cells. Mechanistically, inhibition of OxPhos induced transfer of mitochondria derived from mesenchymal stem cells (MSCs) to AML cells via tunneling nanotubes under direct-contact coculture conditions. Inhibition of OxPhos also induced mitochondrial fission and increased functional mitochondria and mitophagy in AML cells. Mitochondrial fission is known to enhance cell migration, so we used electron microscopy to observe mitochondrial transport to the leading edge of protrusions of AML cells migrating toward MSCs. We further demonstrated that cytarabine, a commonly used antileukemia agent, increased mitochondrial transfer of MSCs to AML cells triggered by OxPhos inhibition. Our findings indicate an important role of exogenous mitochondrial trafficking from BM stromal cells to AML cells as well as endogenous mitochondrial fission and mitophagy in the compensatory adaptation of leukemia cells to energetic stress in the BM microenvironment.

Usage of the Sea Urchin Hemicentrotus pulcherrimus Database, HpBase.

HpBase ( http://cell-innovation.nig.ac.jp/Hpul/ ) is a database that provides genome and transcriptome resources of the sea urchin Hemicentrotus pulcherrimus. In addition to downloading the bulk data, several analysis tools for resource use are available: gene search, homology search, and genome browsing. HpBase also discloses the protocols for biological experiments using H. pulcherrimus that have been accumulated so far. Therefore, HpBase can assist efficient use of genome resources for researchers from various fields-evolutionary, developmental, and cell biology. In this chapter we present an overview and usage of tools in HpBase.

Establishment of homozygous knock-out sea urchins.

Yaguchi et al. establish a homozygous knock-out sea urchin line by applying the CRISPR-Cas9 system to a new model species, Temnopleurus reevesii, whose breeding cycle takes about half a year.

Molecular phylogeny of ten intertidal hermit crabs of the genus Pagurus inferred from multiple mitochondrial genes, with special emphasis on the evolutionary relationship of Pagurus lanuginosus and Pagurus maculosus.

The hermit crab genus Pagurus exhibits high species diversity and a wide geological distribution. Despite the high species diversity of hermit crabs in the western Pacific coast of Japan, molecular phylogenetic studies of these species have yet to be conducted. To investigate their molecular phylogeny and genetic diversity, we obtained nearly complete mitochondrial genome sequences for ten Pagurus species found along the Pacific coast of Japan by next-generation sequencing, which were compared to other congeners deposited in the GenBank database. The genomes ranged from 13,458 to 16,401 base pairs in length, possessing 13 protein-coding genes, 2 rRNAs, and 22 tRNA genes. Based on the reconstructed phylogeny, we found that (1) Japanese Pagurus species separated into three groups, nested within the Northern Pacific species. (2) Pagurus lanuginosus and Pagurus maculosus, showed high morphological similarities, implying close kinship. Indeed, these two species were genetically closest to each other, compared to the remaining species studied. (3) An unspecified specimen sampled from the deep sea, which morphologically resembled Pagurus, might be a member of the Pagurus genus, but is genetically distant from the other Japanese Pagurus species. The novel data reported here may provide new perspectives for systematic studies of hermit crabs; these results provide important information that will facilitate population-level research and identifying intraspecific variation of these non-model, but ecologically important, decapod species.

Maser: one-stop platform for NGS big data from analysis to visualization.

Database URL: http://cell-innovation.nig.ac.jp/maser/.

HpBase: A genome database of a sea urchin, Hemicentrotus pulcherrimus.

To understand the mystery of life, it is important to accumulate genomic information for various organisms because the whole genome encodes the commands for all the genes. Since the genome of Strongylocentrotus purpratus was sequenced in 2006 as the first sequenced genome in echinoderms, the genomic resources of other North American sea urchins have gradually been accumulated, but no sea urchin genomes are available in other areas, where many scientists have used the local species and reported important results. In this manuscript, we report a draft genome of the sea urchin Hemincentrotus pulcherrimus because this species has a long history as the target of developmental and cell biology in East Asia. The genome of H. pulcherrimus was assembled into 16,251 scaffold sequences with an N50 length of 143 kbp, and approximately 25,000 genes were identified in the genome. The size of the genome and the sequencing coverage were estimated to be approximately 800 Mbp and 100×, respectively. To provide these data and information of annotation, we constructed a database, HpBase (http://cell-innovation.nig.ac.jp/Hpul/). In HpBase, gene searches, genome browsing, and blast searches are available. In addition, HpBase includes the "recipes" for experiments from each lab using H. pulcherrimus. These recipes will continue to be updated according to the circumstances of individual scientists and can be powerful tools for experimental biologists and for the community. HpBase is a suitable dataset for evolutionary, developmental, and cell biologists to compare H. pulcherrimus genomic information with that of other species and to isolate gene information.

Evolutionary history of larval skeletal morphology in sea urchin Echinometridae (Echinoidea: Echinodermata) as deduced from mitochondrial DNA molecular phylogeny.

The larval skeletons of sea urchins show considerable morphological diversity, even between closely related species, although the evolutionary history and functional significance of this diversity are poorly understood. To infer the evolutionary history of the skeletal morphology, we focused on echinometrid species for which the morphological variation in larval skeletons had been investigated qualitatively and quantitatively. We reconstructed the phylogenetic relationships among 14 echinometrid species based on mitochondrial ND1 and ND2 genes and mapped the morphological characters onto the resultant trees. The monophyly of each genus in the Echinometridae was well supported by our results, as was the close affinity between Colobocentrotus, Heterocentrotus, and Echinometra. The mapping of the morphological characters of the larval skeletons indicated that the length, direction, and density of spines on the postoral rods was well conserved in each group of Echinometridae and that the abundance of spines and the size and shape of the body skeleton changed relatively frequently and hence were less conserved. In Echinometrid species, morphological variation in relatively unconserved features tends to be associated with latitudinal distributions, rather than phylogenetic relationships, indicating that the morphological diversity of larval skeletons could have been caused by adaptation to the habitat environment. Some morphological differences, however, seem to be nonfunctional and generated by the constraints on larval skeletogenesis. Thus, echinometrid species can be a good model with which to study the evolutionary history from both ecological and developmental standpoints.