During embryogenesis, stereotypic vascular patterning requires guidance cues from neighboring tissues. However, key molecules involved in this process still remain largely elusive. Here, we report molecular cloning, expression, and functional studies of zebrafish max-1, a homolog of Caenorhabditis elegans max-1 that has been implicated in motor neuron axon guidance. During early embryonic development, zebrafish max-1 is specifically expressed in subsets of neuronal tissues, epithelial cells, and developing somites through which vascular endothelial cells migrate from large ventral axial vessels to form stereotypic intersegmental blood vessels (ISV). Blocking zebrafish max-1 mRNA splicing by morpholino injection led to aberrant ISV patterning, which could be rescued by injection of either C. elegans or zebrafish max-1 mRNA. Analysis of motor neurons in the same region showed normal neuronal axon pathfinding. Further studies suggested that the ISV defect caused by max-1 knockdown could be partially rescued by overexpression of ephrinb3 and that max-1 was involved in mediating membrane localization of ephrin proteins, which have been shown to provide guidance cues for endothelial cell migration. Our findings therefore suggest that max-1, acting upstream of the ephrin pathway, is critically required in vascular patterning in vertebrate species.

Noxa, a pro-apoptotic protein, plays an important role in cell apoptosis. The researches about noxa gene were concentrated in mammalians, whereas the role and transcriptional regulatory mechanism of noxa in fish were still unclear. In this study, the expression pattern and transcriptional regulatory mechanism of noxa gene in grass carp were analyzed. Noxa was constitutively expressed in all the examined tissues but the relative expression level differed. After exposure to grass carp reovirus (GCRV), mRNA expression level of noxa was down-regulated at the early phase whereas up-regulated at the late phase of infection. Luciferase assays showed that the promoter region -867 +107 of noxa had high activity and the region -678 -603 was important in the response to GCRV infection. By deleting the predicted transcription factor binding sites, transcription factors FOXO1 and CEBPbeta were found important for noxa in response to GCRV infection. Moreover, the noxa promoter was biotin-labeled and incubated with nuclear extracts from GCRV infected cells. Mass spectrometry analysis showed that transcription factors FOXO1 and CEBPbeta were also enriched in the combined proteins. Therefore, the results suggested that transcription factors FOXO1 and CEBPbeta may play an important role in the regulation of noxa. Our study would provide new insight into the transcriptional regulatory mechanism of noxa in teleost fish. Copyright =C2=A9 2015 Elsevier Ltd. All rights reserved.

During apoptosis, dying cells are quickly internalized by neighboring cells or phagocytes, and are enclosed in phagosomes that undergo a maturation process to generate the phagoslysosome, in which cell corpses are eventually degraded. It is not well understood how apoptotic cell degradation is regulated. Here we report the identification and characterization of the C. elegans tbc-2 gene, which is required for the efficient degradation of cell corpses. tbc-2 encodes a Rab GTPase activating protein (GAP) and its loss of function affects several events of phagosome maturation, including RAB-5 release, phosphatidylinositol 3-phosphate dynamics, phagosomal acidification, RAB-7 recruitment and lysosome incorporation, which leads to many persistent cell corpses at various developmental stages. Intriguingly, the persistent cell corpse phenotype of tbc-2 mutants can be suppressed by reducing gene expression of rab-5, and overexpression of a GTP-locked RAB-5 caused similar defects in phagosome maturation and cell corpse degradation. We propose that TBC-2 functions as a GAP to cycle RAB-5 from an active GTP-bound to an inactive GDP-bound state, which is required for maintaining RAB-5 dynamics on phagosomes and serves as a switch for the progression of phagosome maturation.

The Hippo-Warts pathway defines a novel signaling cascade involved in organ size control and tumor suppression. However, the developmental function of this pathway is less understood. Here we report that the Caenorhabditis elegans homolog of Warts, Ce-wts-1, plays important roles during worm development. The null allele of Ce-wts-1 causes L1 lethality. Partial loss of Ce-wts-1 function by RNAi reveals that Ce-wts-1 is involved in many developmental processes such as larval development, growth rate regulation, gut granule formation, pharynx development, dauer formation, life-span and body length control. Genetic analyses show that Ce-wts-1 functions synergistically with the TGF-beta Sma/Mab pathway to regulate body length. In addition, CE-WTS-1::GFP is enriched near the inner cell membrane, implying its possible membrane-related function. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

N6-methyladenosine (m6A), catalyzed by Mettl3 methyltransferase, is a highly conserved epigenetic modification in eukaryotic messenger RNA (mRNA). Previous studies have implicated m6A modification in multiple biological processes, but the in vivo function of m6A has been difficult to study, because mettl3 mutants are embryonic lethal in both mammals and plants. In this study, we have used transcription activator-like effector nucleases and generated viable zygotic mettl3 mutant, Zmettl3m/m , in zebrafish. We find that the oocytes in Zmettl3m/m adult females are stalled in early development and the ratio of full-grown stage (FG) follicles is significantly lower than that of wild type. Human chorionic gonadotropin-induced ovarian germinal vesicle breakdown in vitro and the numbers of eggs ovulated in vivo are both decreased as well, while the defects of oocyte maturation can be rescued by sex hormone in vitro and in vivo In Zmettl3m/m adult males, we find defects in sperm maturation and sperm motility is significantly reduced. Further study shows that 11-ketotestosterone (11-KT) and 17beta-estradiol (E2) levels are significantly decreased in Zmettl3m/m , and defective gamete maturation is accompanied by decreased overall m6A modification levels and disrupted expression of genes critical for sex hormone synthesis and gonadotropin signaling in Zmettl3m/m Thus, our study provides the first in vivo evidence that loss of Mettl3 leads to failed gamete maturation and significantly reduced fertility in zebrafish. Mettl3 and m6A modifications are essential for optimal reproduction in vertebrates. Copyright =C2=A9 2018 by the Genetics Society of America.

BACKGROUND: The preplacodal region (PPR) is a region of specialized ectoderm at the border of neural and nonneural ectoderm (NNE). Coordinated Bmp, Fgf, and Wnt signals are known to drive PPR development; however, the underlying mechanism is unknown.; RESULTS: We identified key components involved in PPR differentiation. The mesoderm/marginal Wnts at the early gastrula stage trigger differentiation by allowing the adjacent NNE border cells to start adopting caudal PPR fates; otherwise, the development of caudal PPR identity is hindered due to the persistent presence of gata3 mRNA. The caudal PPR fate dominates when foxi1 expression is enhanced at the late gastrula stage, and depleting Foxi1 after 6 hours postfertilization (hpf) reduces the otic-epibranchial placodal domain. When the Gata3 level is manipulated at the fertilized egg stage or near 6 hpf, the lens is always affected. In establishing PPR polarity, both Gata3 and Foxi1 inhibit Bmp signaling, whereas Foxi1 inhibits, but Gata3 enhances, Fgf sensitivity of the PPR cells.; CONCLUSIONS: Our study reveals that in zebrafish, (1) the PPR at the shield stage may enter a developmental state when the PPR cells preferentially adopt a particular placodal fate and (2) a network of genetically linked factors, including Wnt/beta-catenin, Fgfr, Bmp, Gata3, and Foxi1, direct the process of PPR differentiation. Copyright =C2=A9 2014 Wiley Periodicals, Inc.

We describe an enhancer trap transgenic zebrafish line, ETvmat2:GFP, in which most monoaminergic neurons are labeled by green fluorescent protein (GFP) during embryonic development. The reporter gene of ETvmat2:GFP was inserted into the second intron of vesicular monoamine transporter 2 (vmat2) gene, and the GFP expression pattern recapitulates that of the vmat2 gene. The GFP positive neurons include the large and pear-shaped tyrosine hydroxylase positive neurons (TH populations 2 and 4) in the posterior tuberculum of ventral diencephalon (PT neurons), which are thought to be equivalent to the midbrain dopamine neurons in mammals. We found that these PT neurons and two other GFP labeled non-TH type neuronal groups, one in the paraventricular organ of the posterior tuberculum and the other in the hypothalamus, were significantly reduced after exposure to MPTP, while the rest of GFP-positive neuronal clusters, including those in telencephalon, pretectum, raphe nuclei and locus coeruleus, remain largely unchanged. Furthermore, we showed that the effects of hedgehog signaling pathway inhibition on the development of monoaminergic neurons can be easily visualized in individual living ETvmat2:GFP embryos. This enhancer trap line should be useful for genetic and pharmacological analyses of monoaminergic neuron development and processes underlying Parkinson's disease. (C) 2007 Elsevier Inc. All rights reserved.

Multidomain proapoptotic Bcl-2-associated X (Bax) protein is an essential effector responsible for mitochondrial outer membrane permeabilization, resulting in cell death via apoptosis. In this study, two Bax genes of grass carp (Ctenopharyngodon idellus), designated as CiBax1 and CiBax2, were isolated and analyzed. The obtained CiBax1 cDNA is 2058 bp long, with a 579 bp open reading frame (ORF) coding a protein of 192 amino acid residues. The full-length cDNA of CiBax2 is 1161 bp, with a 618 bp ORF coding 205 amino acids. Both CiBax1 and CiBax2 are typical members of Bcl-2 family containing conserved Bcl and C-terminal domains, and they share conserved synteny with zebrafish Bax genes despite the grass carp Bax mapping to different linkage groups. Phylogenetic analysis showed that CiBax1 was clustered with Bax from most teleost fish, and CiBax2 was close to Bax2 from teleost fish but far separated from that of Salmo salar. Quantitative real-time PCR analysis revealed broad expression of CiBax1 and CiBax2 in tissues from healthy grass carp, but the relative expression level differed. The mRNA expression of CiBax1 and CiBax2 was both upregulated significantly and peaked in all examined tissues at days 5 or 6 post-infection with grass carp reovirus. Subcellular localization indicated that CiBax1 protein was localized in both nucleus and cytosol, while CiBax2 protein only in cytosol. Moreover, CiBax2, but not CiBax1 was colocalized with mitochondrion under normal condition. Taken together, the findings would be helpful for further understanding of the function of Bax in teleost fish.

Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts (average size, 139.7 kb) covering 7.4x haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3x haploid genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation for future physical mapping and whole-genome sequencing in grass carp.

Transgenic animals with improved qualities have the potential to upset the ecological balance of a natural environment. We investigated metabolic rates of 'all-fish' growth hormone (GH) transgenic common carp under routine conditions and during starvation periods to determine whether energy stores in transgenic fish would deplete faster than controls during natural periods of starvation. Before the oxygen uptake was measured, the mean daily feed intake of transgenic carp was 2.12 times greater than control fish during 4 days of feeding. The average oxygen uptake of GH transgenic fish was 1.32 times greater than control fish within 96 h of starvation, but was not significantly different from controls between 96 and 144 h of starvation. At the same time, GH transgenic fish did not deplete energy reserves at a faster rate than did the controls, as the carcass energy contents of the two groups following a 60-d starvation period were not significantly different. Consequently, we suggest that increased routine oxygen uptake in GH transgenic common carp over that of control fish may be mainly due to the effects of feeding, and not to an increase in basal metabolism. GH transgenic fish are similar to controls in the regulation of metabolism to normally distribute energy reserves during starvation. (c) 2008 Published by Elsevier B.V.