Japanese medaka Olpax6.1 mutant as a potential model for spondylo-ocular syndrome.

pax6 is a canonic master gene for eye formation. Knockout of pax6 affects the development of craniofacial skeleton and eye in mice. Whether pax6 affects the development of spinal bone has not been reported yet. In the present study, we used CRISPR/Cas9 system to generate Olpax6.1 mutant in Japanese medaka. Phenotype analysis showed that ocular mutation caused by the Olpax6.1 mutation occurred in the homozygous mutant. The phenotype of heterozygotes is not significantly different from that of wild-type. In addition, knockout Olpax6.1 resulted in severe curvature of the spine in the homozygous F2 generation. Comparative transcriptome analysis and qRT-PCR revealed that the defective Olpax6.1 protein caused a decrease in the expression level of sp7, col10a1a, and bglap, while the expression level of xylt2 did not change significantly. The functional enrichment of differentially expressed genes (DEGs) using the Kyoto Encyclopedia of Genes and Genomes database showed that the DEGs between Olpax6.1 mutation and wild-type were enriched in p53 signaling pathway, extracellular matrix (ECM) -receptor interaction, et al. Our results indicated that the defective Olpax6.1 protein results in the reduction of sp7 expression level and the activation of p53 signaling pathway, which leads to a decrease in the expression of genes encoding ECM protein, such as collagen protein family and bone gamma-carboxyglutamate protein, which further inhibits bone development. Based on the phenotype and molecular mechanism of ocular mutation and spinal curvature induced by Olpax6.1 knockout, we believe that the Olpax6.1-/- mutant could be a potential model for the study of spondylo-ocular syndrome.

Organization of the gonadotropin-inhibitory hormone (Lpxrfa) system in the brain of zebrafish (Danio rerio).

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion in birds and mammals. However, the role of GnIH (Lpxrfa) in teleosts is unknown. In this study, a transgenic zebrafish (Danio rerio) line Tg(gnih:mCherry) was developed to determine the organization of GnIH neurons in the brain. Another transgenic line, Tg(gnih:mCherry; gnrh3:eGFP), was established to determine the positional relationships between GnIH and GnRH3 neurons. In these transgenic lines, the mCherry protein was specifically expressed in GnIH neurons, and eGFP was expressed exclusively in GnRH3 neurons. We found that GnIH cell somata were restricted to the posterior periventricular nucleus (NPPv). Most GnIH neuronal processes projected to the hypothalamus, but a few extended to the posterior tuberculum, telencephalon, and olfactory bulb. GnIH neuronal processes were in close apposition with GnRH3 cell somata and processes in the preoptic-hypothalamic area but were seldom in direct contact. However, in the olfactory bulb, GnIH neuronal processes were in proximity to the terminal nerve GnRH3 cell somata. Neither GnIH cell soma nor neuronal processes were detected in the pituitary, although GnIH receptor mRNAs (npffr1l1, npffr1l2, and npffr1l3) were detected. Intraperitoneal administration of GnIH-3 peptides promoted the transcription of brain gnrh3 as well as pituitary fshß but not lhß. Thus, GnIH cell somata were specifically distributed in the NPPv, and their fibers extended to the hypothalamus and advanced to the telencephalon and olfactory bulb. We conclude that GnIH may directly stimulate terminal nerve GnRH3 neurons in the zebrafish brain.

Gonadal, Not Maternal, Acquisition of Duplicated pax6 Orthologs in Megalobrama Amblycephala.

: The highly conserved transcription factor Pax6 is involved in the development of the eyes, brain, and pancreas in vertebrates and invertebrates, whereas the additional expression pattern in other organs is still elusive. In this study, we cloned and characterized two pax6 homologs in blunt snout bream (Megalobrama amblycephala), named Mapax6a and Mapax6b. The protein alignment and phylogenetic tree showed that Mapax6a and Mapax6b were highly conserved compared with their counterparts in other species. Genomic information analysis revealed that the synteny conservation of Wilms tumor, Aniridia, genitourinary abnormalities, and mental retardation loci was also maintained in this species. By reverse transcription polymerase chain reaction, the expression of Mapax6a was later than that of Mapax6b which was found in the blastula stage, while the expression of Mapax6a started from the somite stage, and both of them persisted in a subsequent stage during the embryonic development. By RNA and protein detection, Mapax6a and Mapax6b were detected in the eye and brain as canonic patterns, and most importantly, they were also enriched in germ cells of the testis and ovary. Therefore, our findings validate the duplication of pax6 in fish, confirm the classical expression patterns in the brain and eye, and, for the first time, present a new acquisition of Mapax6a and Mapax6b in gonadal germ cells in particular. Therefore, our results enrich the expression pattern and evolutionary relationship of pax6 by suggesting that duplicated Mapax6 is involved in gametogenesis in Megalobrama amblycephala.

Medaka (Oryzias latipes) Olpax6.2 acquires maternal inheritance and germ cells expression, but functionally degenerate in the eye.

Gene duplication provides raw material for the evolution of genetic and phenotypic complexity. It has remained a long-standing mystery how duplicated genes evolve into new genes by neofunctionalization via the acquisition of new expression and/or activity and simultaneous loss of the old expression and activity. Fishes have many gene duplicates from whole genome duplication, making them excellent for studying the evolution of gene duplicates. In the fish medaka (Oryzias latipes), an ancestral pax6 gene has given rise to Olpax6.1 and Olpax6.2. Here we report that medaka Olpax6.2 is evolving towards neofunctionalization. A chromosomal syntenic analysis indicated that Olpax6.1 and Olpax6.2 are structurally co-homologous to the single pax6 in other organisms. Interestingly, Olpax6.2 maintains all conserved coding exons but loses the non-coding exons of Olpax6.1, and has 4 promoters versus 8 in Olpax6.1. RT-PCR revealed that Olpax6.2 maintains expression in the brain eye, pancreas as Olpax6.1. Surprisingly, Olpax6.2 also exhibits maternal inheritance and gonadal expression by RT-PCR, in situ hybridization and RNA transcriptome analysis. The expression and distribution of Olpax6.2 is not different from Olpax6.1 in the adult brain, eye and pancreas, but exhibited overlapping and distinct expression in early embryogenesis. We show that ovarian Olpax6.2 expression occurs in female germ cells. Olpax6.2 knockout shows no obvious defect in eye development, while Olpax6.1 F0 mutant have severe defects in eye development. Thus, Olpax6.2 acquires maternal inheritance and germ cell expression, but functionally degenerates in the eye, making this gene as an excellent model to study the neofunctionalization of duplicated genes.

Mini-Review of Self-Healing Mechanism and Formulation Optimization of Polyurea Coating.

Self-healing polymers are categorized as smart materials that are capable of surface protection and prevention of structural failure. Polyurethane/polyurea, as one of the representative coatings, has also attracted attention for industrial applications. Compared with polyurethane, polyurea coating, with a similar formation process, provides higher tensile strength and requires shorter curing time. In this paper, extrinsic and intrinsic mechanisms are reviewed to address the efficiency of the self-healing process. Moreover, formulation optimization and strategic improvement to ensure self-healing within a shorter period of time with acceptable recovery of mechanical strength are also discussed. The choice and ratio of diisocyanates, as well as the choice of chain extender, are believed to have a crucial effect on the acceleration of the self-healing process and enhance self-healing efficiency during the preparation of polyurea coatings.

Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA.

Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka (Oryzias latipes) endogenous gene tyrosinase(tyr) or paired box 6.1 (pax6.1) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.

Morphological Characteristics and Comparative Transcriptome Analysis of Three Different Phenotypes of Pristella maxillaris.

Pristella maxillaris is known as the X-ray fish based on its translucent body. However, the morphological characteristics and the molecular regulatory mechanisms of these translucent bodies are still unknown. In this study, the following three phenotypes, a black-and-gray body color or wild-type (WT), a silvery-white body color defined as mutant I (MU1), and a fully transparent body with a visible visceral mass named as mutant II (MU2), were investigated to analyze their chromatophores and molecular mechanisms. The variety and distribution of pigment cells in the three phenotypes of P. maxillaris significantly differed by histological assessment. Three types of chromatophores (melanophores, iridophores, and xanthophores) were observed in the WT, whereas MU1 fish were deficient in melanophores, and MU2 fish lacked melanophores and iridophores. Transcriptome sequencing of the skin and peritoneal tissues of P. maxillaris identified a total of 166,089 unigenes. After comparing intergroup gene expression levels, more than 3,000 unigenes with significantly differential expression levels were identified among three strains. Functional annotation and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the differentially expressed genes (DEGs) identified a number of candidates melanophores and iridophores genes that influence body color. Some DEGs that were identified using transcriptome analysis were confirmed by quantitative real-time PCR. This study serves as a global survey of the morphological characteristics and molecular mechanism of different body colors observed in P. maxillaris and thus provides a valuable theoretical foundation for the molecular regulation of the transparent phenotype.

Genetic comparison of two color morphs of northern snakehead (Channa argus) and Chnannidae family.

Relationships of two species of Channa argus (northern snakeheads) named "Bicolor type" and "White type", as well as other six Channa species were investigated based on their partial sequence of mitochondrial DNA 16S rRNA genes in the present study. For the Channa family, the average genetic distance was 0.0863 with the inter-species genetic distance ranged from 0.0173 to 0.1384, and the average intra-species genetic distance in the genus channa was estimated as 0.00273 (range: 0.0019 to 0.0038). For the two C. argus species, the mean pair-wise genetic distance between "Bicolor type" and "White type" northern snakehead was estimated as 0.00218, which was within the intra-species genetic distance interval for Channa species, indicating that they belonged to the same species at molecular level. Moreover, these snakeheads can be divided into two distinct groups via 16S rRNA, which might be more accurate for Channa classification than traditional method based on the absence or presence of the pelvic fins of the fish.