Regulation of dynamic pigment cell states at single-cell resolution

Cells bearing pigment have diverse roles and are often under strict evolutionary selection. Here, we explore the regulation of pigmented cells in the purple sea urchin Strongylocentrotus purpuratus, an emerging model for diverse pigment function. We took advantage of single cell RNA-seq (scRNAseq) technology and discovered that pigment cells in the embryo segregated into two distinct populations, a mitotic cluster and a post-mitotic cluster. Gcm is essential for expression of several genes important for pigment function, but is only transiently expressed in these cells. We discovered unique genes expressed by pigment cells and test their expression with double fluorescence in situ hybridization. These genes include new members of the fmo family that are expressed selectively in pigment cells of the embryonic and in the coelomic cells of the adult - both cell-types having immune functions. Overall, this study identifies nodes of molecular intersection ripe for change by selective evolutionary pressures.

Analysis of chromosome karyotype and genome size in echiuran Urechis unicinctus Drasche, 1880 (Polychaeta, Urechidae)

Karyotype and genome size are two primary cytogenetic characteristics of species, which are of great significance to the study of cytogenetics, taxonomy, phylogenesis, evolution as well as molecular biology. However, this basic cytogenetic information in echiurans is lacking. Therefore, we analyzed characteristics of karyotype and genome size in the echiuran worm Urechisunicinctus Drasche, 1880. In this study, coelomic cells of U.unicinctus were used for analyzing the genome size by a flow cytometry with chicken erythrocytes as DNA standard, and the 2C DNA content was determined to be 1.85 pg, which was corresponded to the genome size of 904.58 Mbp approximately. Furthermore, trochophores of U.unicinctus were dissociated and cells were utilized for preparing the chromosomes stained with DAPI, and the karyotype was determined as 2n = 30 (10m + 6sm + 6st + 8t), FN=52. Our data provided the basic cytogenetic information of U.unicinctus, which could be utilized in taxonomic study and whole-genome sequencing in future.

Transcriptomic analysis in the leech Theromyzon tessulatum: involvement of cystatin B in innate immunity

At the present time, there is little information on mechanisms of innate immunity in invertebrate groups other than insects, especially annelids. In the present study, we have performed a transcriptomic study of the immune response in the leech Theromyzon tessulatum after bacterial challenge, by a combination of differential display RT (reverse transcriptase)–PCR and cDNA microarrays. The results show relevant modulations concerning several known and unknown genes. Indeed, threonine deaminase, malate dehydrogenase, cystatin B, polyadenylate-binding protein and α-tubulin-like genes are up-regulated after immunostimulation. We focused on cystatin B (stefin B), which is an inhibitor of cysteine proteinases involved in the vertebrate immune response. We have cloned the full-length cDNA and named the T. tessulatum gene as Tt-cysb. Main structural features of cystatins were identified in the derived amino acid sequence of Tt-cysb cDNA; namely, a glycine residue in the N-terminus and a consensus sequence of Gln-Xaa-Val-Xaa-Gly (QXVXG) corresponding to the catalytic site. Moreover, Tt-cysb is the first cystatin B gene characterized in invertebrates. We have determined by in situ hybridization and immunocytochemistry that Tt-cysb is only expressed in large coelomic cells. In addition, this analysis confirmed that Tt-cysb is up-regulated after bacterial challenge, and that increased expression occurs only in coelomic cells. These data demonstrate that the innate immune response in the leech involves a cysteine proteinase inhibitor that is not found in ecdysozoan models, such as Drosophila melanogaster or Caenorhabditis elegans, and so underlines the great need for information about innate immunity mechanisms in different invertebrate groups.