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The cellular and molecular mechanisms underlying lymphocyte development are diverse among teleost species. Although recent scRNA-seq analyses of zebrafish hematopoietic cells have advanced our understanding of teleost hematopoiesis, comparative studies using another genetic model, medaka, which is evolutionarily distant among teleosts, is useful for understanding commonality and species-specificity in teleosts. In order to gain insight into how different molecular and cellular mechanisms of lymphocyte development in medaka and zebrafish, we established a recombination activating gene 1 (rag1) mutant medaka, which exhibited defects in V(D)J rearrangement of lymphocyte antigen receptor genes, accordingly lacking mature B and T cells. scRNA-seq analysis of wild type and rag1 mutant lymphocytes in the thymus and kidney characterized the developing stages of T and B cells, and found that most developed cd4+cd8– and cd4–cd8+ single-positive (SP) T-cell populations are absent in the thymus, and identified lymphoid progenitor cells already committed to the T lineage in kidney, implying unique features of medaka lymphocyte development.

Abstract The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

Abstract Unconventional T cells, such as innate natural killer T cells (iNKT) cells, are an important part of vertebrate immune defences. iNKT recognise glycolipids through a T cell receptor (TCR) that is composed of a semi-invariant TCR α chain, paired with a restricted set of TCR β chains. Here, we show that splicing of the cognate Trav11-Traj18-Trac pre-mRNA encoding the characteristic Vα14Jα18 variable region of this semi-invariant TCR depends on the presence of Tnpo3. The Tnpo3 gene encodes a nuclear transporter of the β-karyopherin family whose cargo includes various splice regulators. The block of iNKT cell development in the absence of Tnpo3 can be overcome by transgenic provision of a rearranged Trav11-Traj18-Trac cDNA, indicating that Tnpo3 deficiency does not interfere with the development of iNKT cells per se. Our study thus identifies a role for Tnpo3 in regulating the splicing of the pre-mRNA encoding the cognate TCRα chain of iNKT cells.

The interactions between the host immune system and intestinal microorganisms have been studied in many animals, including fish. However, a detailed analysis has not been performed in medaka, an established fish model for biological studies. Here, we investigated the effect of immunodeficiency on the microbiota composition and the effect of gut bacteria on intestinal epithelial development and immune responses in medaka. Chronological analysis of the intestinal microbiota of interleukin 2 receptor subunit gamma (il2rg) mutant medaka showed a gradual decrease in the evenness of operational taxonomic units, mainly caused by the increased abundance of the Aeromonadaceae family. Exposure of wild-type medaka to high doses of an intestine-derived opportunistic bacterium of the Aeromonadaceae family induced an inflammatory response, suggesting a harmful effect on adult il2rg mutants. In addition, we established germ-free conditions in larval medaka and observed large absorptive vacuoles in intestinal epithelial cells, indicating a block in epithelial maturation. Transcriptome analysis revealed a decrease in the expression of genes involved in the defense response, including the antimicrobial peptide gene hepcidin, whose expression is induced by lipopolysaccharide stimulation in normal larvae. These results show that reciprocal interactions between the microbiome and the intestinal tract are required for the maturation of the medaka immune system.

CRSIPR-mediated barcoding of pre-TCR-expressing T cells is used to investigate the clonal dynamics of thymic T cells and reveals new fundamental understanding of how the skewed CD4/CD8 ratio arises during thymic selection.

Abstract The development of the parathyroid and the thymus from the third pharyngeal pouch depends on the activities of the Gcm2 and Foxn1 transcription factors, respectively, whose expression domains sharply demarcate two regions in the developing third pharyngeal pouch. Here, we have generated novel mouse models to examine whether ectopic co-expression of Gcm2 in the thymic epithelium and of Foxn1 in the parathyroid perturbs the establishment of organ fates in vivo. Expression of Gcm2 in the thymic rudiment does not activate a parathyroid-specific expression programme, even in the absence of Foxn1 activity. Co-expression of Foxn1 in the parathyroid fails to impose thymopoietic capacity. We conclude that the actions of Foxn1 and Gcm2 transcription factors are cell context-dependent and that they each require permissive transcription factor landscapes in order to successfully interfere with organ-specific cell fate.

The vertebral column is a characteristic structure of vertebrates. Genetic studies in mice have shown that Hox-mediated patterning plays a key role in specifying discrete anatomical regions of the vertebral column. Expression pattern analyses in several vertebrate embryos have provided correlative evidence that the anterior boundaries of Hox expression coincide with distinct anatomical vertebrae. However, because functional analyses have been limited to mice, it remains unclear which Hox genes actually function in vertebral patterning in other vertebrates. In this study, various zebrafish Hox mutants were generated for loss-of-function phenotypic analysis to functionally decipher the Hox code responsible for the zebrafish anterior vertebrae between the occipital and thoracic vertebrae. We found that Hox genes in HoxB- and HoxC-related clusters participate in regulating the morphology of the zebrafish anterior vertebrae. In addition, medaka hoxc6a was found to be responsible for anterior vertebral identity, as in zebrafish. Based on phenotypic similarities with Hoxc6 knockout mice, our results suggest that the Hox patterning system, including at least Hoxc6, may have been functionally established in the vertebral patterning of the common ancestor of ray-finned and lobe-finned fishes. [ABSTRACT FROM AUTHOR]

The dorsal and anal fins can vary widely in position and length along the anterior-posterior axis in teleost fishes. However, the molecular mechanisms underlying the diversification of these fins remain unknown. Here, we used genetic approaches in zebrafish and medaka, in which the relative positions of the dorsal and anal fins are opposite, to demonstrate the crucial role of hox genes in the patterning of the teleost posterior body, including the dorsal and anal fins. By the CRISPR-Cas9-induced frameshift mutations and positional cloning of spontaneous dorsalfinless medaka, we show that various hox mutants exhibit the absence of dorsal or anal fins, or a stepwise posterior extension of these fins, with vertebral abnormalities. Our results indicate that multiple hox genes, primarily from hoxc-related clusters, encompass the regions responsible for the dorsal and anal fin formation along the anterior-posterior axis. These results further suggest that shifts in the anterior boundaries of hox expression which vary among fish species, lead to diversification in the position and size of the dorsal and anal fins, similar to how modulations in Hox expression can alter the number of anatomically distinct vertebrae in tetrapods. Furthermore, we show that hox genes responsible for dorsal fin formation are different between zebrafish and medaka. Our results suggest that a novel mechanism has occurred during teleost evolution, in which the gene network responsible for fin formation might have switched to the regulation downstream of other hox genes, leading to the remarkable diversity in the dorsal fin position. [ABSTRACT FROM AUTHOR]