Adaptive Autoimmunity and Foxp3-Based Immunoregulation in Zebrafish

Background

Jawed vertebrates generate their immune-receptor repertoire by a recombinatorial mechanism that has the potential to produce harmful autoreactive lymphocytes. In mammals, peripheral tolerance to self-antigens is enforced by Foxp3⁺ regulatory T cells. Recombinatorial mechanisms also operate in teleosts, but active immunoregulation is thought to be a late incorporation to the vertebrate lineage.

Methods/Principal Findings

Here we report the characterization of adaptive autoimmunity and Foxp3-based immunoregulation in the zebrafish. We found that zebrafish immunization with an homogenate of zebrafish central nervous system (zCNS) triggered CNS inflammation and specific antibodies. We cloned the zebrafish ortholog for mammalian Foxp3 (zFoxp3) which induced a regulatory phenotype on mouse T cells and controlled IL-17 production in zebrafish embryos.

Conclusions/Significance

Our findings demonstrate the acquisition of active mechanisms of self-tolerance early in vertebrate evolution, suggesting that active regulatory mechanisms accompany the development of the molecular potential for adaptive autoimmunity. Moreover, they identify the zebrafish as a tool to study the molecular pathways controlling adaptive immunity.     

Lack of Developmental Redundancy between Unc45 Proteins in Zebrafish Muscle Development

Since the majority of protein-coding genes in vertebrates have intra-genomic homologues, it has been difficult to eliminate the potential of functional redundancy from analyses of mutant phenotypes, whether produced by genetic lesion or transient knockdown. Further complicating these analyses, not all gene products have activities that can be assayed in vitro, where the efficiency of the various family members can be compared against constant substrates. Two vertebrate UNC-45 homologues, unc45a and unc45b, affect distinct stages of muscle differentiation when knocked down in cell culture and are functionally redundant in vitro. UNC-45 proteins are members of the UCS (UNC-45/CRO1/She4p) protein family that has been shown to regulate myosin-dependent functions from fungi to vertebrates through direct interaction with the myosin motor domain. To test whether the same functional relationship exists between these unc45 paralogs in vivo, we examined the developmental phenotypes of doubly homozygous unc45b^−/−; unc45a^−/− mutant zebrafish embryos. We focused specifically on the combined effects on morphology and gene expression resulting from the zygotic lack of both paralogs. We found that unc45b^−/− and unc45b^−/−; unc45a^−/− embryos were phenotypically indistinguishable with both mutants displaying identical cardiac, skeletal muscle, and jaw defects. We also found no evidence to support a role for zygotic Unc45a function in myoblast differentiation. In contrast to previous in vitro work, this rules out a model of functional redundancy between Unc45a and Unc45b in vivo. Instead, our phylogenetic and phenotypic analyses provide evidence for the role of functional divergence in the evolution of the UCS protein family.     

Ethanol Affects the Development of Sensory Hair Cells in Larval Zebrafish (Danio rerio)

Children born to mothers with substantial alcohol consumption during pregnancy can present a number of morphological, cognitive, and sensory abnormalities, including hearing deficits, collectively known as fetal alcohol syndrome (FAS). The goal of this study was to determine if the zebrafish lateral line could be used to study sensory hair cell abnormalities caused by exposure to ethanol during embryogenesis. Some lateral line sensory hair cells are present at 2 days post-fertilization (dpf) and are functional by 5 dpf. Zebrafish embryos were raised in fish water supplemented with varying concentrations of ethanol (0.75%–1.75% by volume) from 2 dpf through 5 dpf. Ethanol treatment during development resulted in many physical abnormalities characteristic of FAS in humans. Also, the number of sensory hair cells decreased as the concentration of ethanol increased in a dose-dependent manner. The dye FM 1-43FX was used to detect the presence of functional mechanotransduction channels. The percentage of FM 1-43-labeled hair cells decreased as the concentration of ethanol increased. Methanol treatment did not affect the development of hair cells. The cell cycle markers proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) demonstrated that ethanol reduced the number of sensory hair cells, as a consequence of decreased cellular proliferation. There was also a significant increase in the rate of apoptosis, as determined by TUNEL-labeling, in neuromasts following ethanol treatment during larval development. Therefore, zebrafish are a useful animal model to study the effects of hair cell developmental disorders associated with FAS.     

Caspase-3 Induced Apoptosis in Transgenic Zebrafish

Zebrafish is an attractive model organism for studying apoptosis development because of its genetic accessibility. Here we describe the induction of clonally derived apoptosis in transgenic zebrafish expressing mouse caspase-3 (CASP3) under control of the zebrafish β-actin promoter (βp). Visualization of apoptotic cells, expressing a chimeric transgene encoding CASP3 fused to green fluorescent protein (GFP) gene, revealed that apoptosis arose in the thymus, spread locally into gill arches and retro-orbital soft tissue, and then disseminated into abdominal organs like testis, kidney. This transgenic model provides a platform for over-expression of caspase-3 induced extensive apoptosis in embryos and adult. An erratum to this article is available at .     

Habitat Quality Affects Early Physiology and Subsequent Neuromotor Development of Juvenile Black-Capped Chickadees

In songbirds, the ability to learn and render the species-specific song is influenced by the development of both the song nuclei in the brain and the syrinx (bird''s vocal apparatus) early in the bird''s life. In black-capped chickadees (Poecille atricapillus), habitat quality is known to affect song structure, with birds in high-quality habitat (mature forest) having a higher song consistency than birds in low-quality habitat (young forest). Although this difference is suspected to stem from differences in development, the developmental status of juvenile birds in either habitat remains unexplored. In this study, we used ptilochronology and feather corticosterone to compare the conditional state of juvenile chickadees in young and mature forest during two distinct periods of song learning - the sensory phase, which occurs prior to settlement, and the sensorimotor phase, which occurs post-settlement. A sample of juvenile males was captured and euthanized several weeks prior to their first breeding season to compare the development of song center nuclei and syrinx in both habitats. The corticosterone levels of natally-grown feathers were greater among birds that settled in mature than young forests - as these feathers were grown pre-settlement, they reflect differences in physiology during the sensory phase. This difference in conditional state is reflected by differences in syrinx and song center nuclei development later during the sensorimotor phase - birds in young forest have smaller syrinx, and moderately-larger RA, than birds in mature forest. Those differences could be responsible for the difference in consistency in song structure observed across habitats. The difference in physiological state across habitats, combined with potential compounding effect of differences in winter resources between habitats, could influence the difference in syrinx and neural development seen in juvenile males during the early spring, and influence the male''s ability to learn and render their species-specific song.     

Loss of Ribosomal Protein L11 Affects Zebrafish Embryonic Development through a p53-Dependent Apoptotic Response

Ribosome is responsible for protein synthesis in all organisms and ribosomal proteins (RPs) play important roles in the formation of a functional ribosome. L11 was recently shown to regulate p53 activity through a direct binding with MDM2 and abrogating the MDM2-induced p53 degradation in response to ribosomal stress. However, the studies were performed in cell lines and the significance of this tumor suppressor function of L11 has yet to be explored in animal models. To investigate the effects of the deletion of L11 and its physiological relevance to p53 activity, we knocked down the rpl11 gene in zebrafish and analyzed the p53 response. Contrary to the cell line-based results, our data indicate that an L11 deficiency in a model organism activates the p53 pathway. The L11-deficient embryos (morphants) displayed developmental abnormalities primarily in the brain, leading to embryonic lethality within 6–7 days post fertilization. Extensive apoptosis was observed in the head region of the morphants, thus correlating the morphological defects with apparent cell death. A decrease in total abundance of genes involved in neural patterning of the brain was observed in the morphants, suggesting a reduction in neural progenitor cells. Upregulation of the genes involved in the p53 pathway were observed in the morphants. Simultaneous knockdown of the p53 gene rescued the developmental defects and apoptosis in the morphants. These results suggest that ribosomal dysfunction due to the loss of L11 activates a p53-dependent checkpoint response to prevent improper embryonic development.     

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.Download video file.^{(61M, mov)}     

Gonadal Development and Fertility of Triploid Grass Puffer Takifugu niphobles Induced by Cold Shock Treatment

Tiger puffer Takifugu rubripes is one of the most valuable fish species in Japan; however, there has not been much progress in their selective breeding until recently despite their potential in aquaculture. Their long generation time and the large body size of their broodstock make breeding difficult. Recently, we made a surrogate broodstock, which produced gametes of different species in salmonids. Therefore, by using closely related recipients, which have small body sizes and short generation times, it is possible to accelerate breeding of the tiger puffer. Thus, we considered the grass puffer Takifugu niphobles, which has a short generation time and a small maturation size, as a potential recipient for gamete production of the tiger puffer. Furthermore, if sterile triploid individuals are used as recipients, the resulting surrogate broodstock would produce only donor-derived gametes. Therefore, we examined conditions for inducing triploidy by suppressing meiosis II to retain the second polar body in grass puffer. We found that cold shock treatment, which is 5°C for 30 min starting from 5 min after fertilization, is optimal to obtain high triploidization and hatching rates. Although the resulting triploid grass puffers produced small amounts of gametes in both sexes, the offspring derived from the gametes could not live for over 3 days. Furthermore, we found that triploid grass puffer showed normal plasma sex steroid levels compared with diploids. These are important characteristics of triploid grass puffer as surrogate recipients used for germ cell transplantation.     

Heart Dissection in Larval,Juvenile and Adult Zebrafish,Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews^1-6), however, work examining post-embryonic through adult cardiac development has been limited^7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish''s lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis¹¹. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100μm ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)^{twu34 (12)}, which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.Download video file.^{(42M, mov)}     

Gonadal Mosaicism Induced by Chemical Treatment of Sperm in Drosophila melanogaster

Accurate interpretation of forward genetic screens of chromosomes exposed in mature spermatozoa to a mutagenic chemical requires understanding—incomplete to date—of how exposed chromosomes and their replicas proceed through early development stages from the fertilized ovum to establishment of the germline of the treated male’s offspring. We describe a model for early embryonic development and establishment of the germline of Drosophila melanogaster and a model-validating experiment. Our model proposes that, barring repair, DNA strands modified by treatment with alkylating agents are stable and mutagenic. Each replication of an alkylated strand can result in misreplication and a mutant-bearing daughter nucleus. Daughter nuclei thenceforth replicate faithfully and their descendants comprise the embryonic syncytium. Of the 256 nuclei present after the eighth division, several migrate into the polar plasm at the posterior end of the embryo to found the germline. Based upon distribution of descendants of the alkylated strands, the misreplication rate, and the number of nuclei selected as germline progenitors, the frequency of gonadal mosaicism is predictable. Experimentally, we tracked chromosomes 2 and 3 from EMS-treated sperm through a number of generations, to characterize autosomal recessive lethal mutations and infer gonadal genetic content of the sons of treated males. Over 50% of 106 sons bore germlines that were singly, doubly, or triply mosaic for chromosome 2 or chromosome 3. These findings were consistent with our model, assuming a rate of misreplication between 0.65 and 0.80 at each replication of an alkylated strand. Crossing treated males to mismatch-repair-deficient females had no apparent effect on mutation rate.     

Salt Stress Proteins Induced in Listeria monocytogenes

The ability of Listeria monocytogenes to tolerate salt stress is of particular importance, as this pathogen is often exposed to such environments during both food processing and food preservation. In order to understand the survival mechanisms of L. monocytogenes, an initial approach using two-dimensional polyacrylamide gel electrophoresis was performed to analyze the pattern of protein synthesis in response to salt stress. Of 400 to 500 visible proteins, the synthesis of 40 proteins (P < 0.05) was repressed or induced at a higher rate during salt stress. Some of the proteins were identified on the basis of mass spectrometry or N-terminal sequence analysis and database searching. Twelve proteins showing high induction after salt stress were similar to general stress proteins (Ctc and DnaK), transporters (GbuA and mannose-specific phosphotransferase system enzyme IIAB), and general metabolism proteins (alanine dehydrogenase, CcpA, CysK, EF-Tu, Gap, GuaB, PdhA, and PdhD).     

Autoimmunity against INS-IGF2 Protein Expressed in Human Pancreatic Islets

Insulin is a major autoantigen in islet autoimmunity and progression to type 1 diabetes. It has been suggested that the insulin B-chain may be critical to insulin autoimmunity in type 1 diabetes. INS-IGF2 consists of the preproinsulin signal peptide, the insulin B-chain, and eight amino acids of the C-peptide in addition to 138 amino acids from the IGF2 gene. We aimed to determine the expression of INS-IGF2 in human pancreatic islets and autoantibodies in newly diagnosed children with type 1 diabetes and controls. INS-IGF2, expressed primarily in beta cells, showed higher levels of expression in islets from normal compared with donors with either type 2 diabetes (p = 0.006) or high HbA1c levels (p < 0.001). INS-IGF2 autoantibody levels were increased in newly diagnosed patients with type 1 diabetes (n = 304) compared with healthy controls (n = 355; p < 0.001). Displacement with cold insulin and INS-IGF2 revealed that more patients than controls had doubly reactive insulin-INS-IGF2 autoantibodies. These data suggest that INS-IGF2, which contains the preproinsulin signal peptide, the B-chain, and eight amino acids of the C-peptide may be an autoantigen in type 1 diabetes. INS-IGF2 and insulin may share autoantibody-binding sites, thus complicating the notion that insulin is the primary autoantigen in type 1 diabetes.     

Pentylenetetrazole-Induced Chemoshock Affects Protein Kinase C and Substrate Proteins in Mouse Brain

Abstract: Protein kinase C (PKC) activity, western blot analysis of PKCα, β, γ, ε, and ζ by isozyme-specific antibodies, and in vitro phosphorylation of endogenous substrate proteins were studied in the mice brain after pentyl-enetetrazole-induced chemoshock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified by DE-52 columns eluted with buffer A containing 100 or 200 m M KCI. This method consistently separates cytosolic and membrane proteins and various PKC isoforms. The 100 m M KCI eluates from DE-52 columns contain more PKC α and β in both cytosol and membrane than the 200 m M KCI eluates, whereas PKCγ, ε, and ζappear in equal amounts in these two eluates. The kinase activity assayed by phosphorylation of exogenous histone was increased in the chemoshocked mice in both the cytosol and membrane of 200 m M KCI eluates. In further analysis by immunoblotting, this increased activity was found to be due to the increase in content of PKC7 isozyme. As for novel-type ε and ζ isozymes, they were not altered in the chemoshocked mice. From autoradiography, the endogenous substrate 17-kDa neurogranin, which was shown below 21 kDa, was mostly eluted by 100 m M KCI from the DE-52 column, whereas 43-kDa neuromodulin, which was also demonstrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, only appeared in the 200 m M KCI eluates. The in vitro phosphorylation of neuromodulin was found to be increased in the chemoshocked mice. Therefore, the increased phosphorylation of neuromodulin and increased content of the PKCγ isoform were involved in the pentylenetetrazole-induced chemoshock.     

Antibodies to Inhibitory Synaptic Proteins in Neurological Syndromes Associated with Glutamic Acid Decarboxylase Autoimmunity

Antibodies to glutamic acid decarboxylase (GAD-ab) associate to different neurological syndromes. It is unknown if the diversity in syndrome association represents epitopes in different immunodominant domains or co-existence of antibodies to other proteins of the inhibitory synapsis. We examined the serum and CSF of 106 patients with anti-GAD related syndromes (39 cerebellar ataxia, 32 stiff-person syndrome [SPS], 18 epilepsy, and 17 limbic encephalitis [LE]). GAD65-ab titres were quantified by ELISA. Immunoblot was used to determine if the antibody-targeted epitopes of GAD65 and GAD67 were linear. A cell-based assay (CBA) with HEK293 cells expressing the GAD65 N-terminal, central catalytic domain, or C-terminal was used to investigate the immunodominant domains. Antibodies to GAD67, gamma-aminobutyric acid A receptor (GABAaR), glycine receptor (GlyR), GABAaR-associated protein (GABARAP), and gephyrin were determined with CBA. GAD-ab internalization was investigated using cultured rat hippocampal neurons. CSF GAD65-ab titres were higher in patients with cerebellar ataxia and LE compared to those with SPS (p = 0.02). GAD67-ab were identified in 81% of sera and 100% of CSF. GAD65-ab recognized linear epitopes in 98% of the patients and GAD67-ab in 42% (p<0.001). The GAD65 catalytic domain was recognized by 93% of sera, and the three domains by 22% of sera and 74% of CSF (p<0.001). Six patients had GABAaR-ab and another 6 had GlyR-ab without association to distinctive symptoms. None of the patients had gephyrin- or GABARAP-ab. GAD65-ab were not internalized by live neurons. Overall, these findings show that regardless of the neurological syndrome, the CSF immune response against GAD is more widespread than that of the serum and that there is no specific association between clinical phenotype and the presence of antibodies against other proteins of the inhibitory synapsis.     

Glial Cell Development and Function in Zebrafish

The zebrafish is a premier vertebrate model system that offers many experimental advantages for in vivo imaging and genetic studies. This review provides an overview of glial cell types in the central and peripheral nervous system of zebrafish. We highlight some recent work that exploited the strengths of the zebrafish system to increase the understanding of the role of Gpr126 in Schwann cell myelination and illuminate the mechanisms controlling oligodendrocyte development and myelination. We also summarize similarities and differences between zebrafish radial glia and mammalian astrocytes and consider the possibility that their distinct characteristics may represent extremes in a continuum of cell identity. Finally, we focus on the emergence of zebrafish as a model for elucidating the development and function of microglia. These recent studies have highlighted the power of the zebrafish system for analyzing important aspects of glial development and function.Following the pioneering work of George Streisinger in the early 1980s, the zebrafish has emerged as a premier vertebrate model system (Streisinger et al. 1981). A key strength of the zebrafish is that the embryos and early larvae are transparent, allowing exquisite cellular analysis of many dynamic processes, including cell migration, axonal pathfinding, and myelination, among many others (e.g., Gilmour et al. 2002; Lyons et al. 2005; Czopka et al. 2013). The zebrafish also has many advantages for large-scale genetic studies, including relatively small size and rapid development, high fecundity, and the ability to manipulate the ploidy of gametes and early embryos (Kimmel 1989). Through the 1980s and early 1990s, insightful studies of several interesting mutations elegantly exploited these experimental advantages (e.g., Kimmel et al. 1989; Ho and Kane 1990; Hatta et al. 1991; Grunwald and Eisen 2002), attracting many researchers from other fields to the zebrafish system. Following the explosion of interest in the zebrafish in the 1990s, advances in many areas have added to the strengths of the system, including large-scale screens that identified thousands of new mutations (Driever et al. 1996; Haffter et al. 1996), rapid transgenesis (Kawakami et al. 2004), new methods for imaging and tracking all cells during development (Huisken 2012), genetic mapping and sequencing to identify genes and mutated loci (Postlethwait et al. 1994; Howe et al. 2013), optogenetic methods to control neural activity (Portugues et al. 2013), the advent of targeted nucleases to create mutations in genes of interest (Huang et al. 2011; Sander et al. 2011; Bedell et al. 2012; Chang et al. 2013; Hwang et al. 2013), and small molecule screening approaches to isolate compounds with novel biological activities in vivo (Peterson and Fishman 2011).Many fundamental similarities in physiology and body plan unite the zebrafish and other vertebrates (Kimmel 1989). In addition, analysis of genes and genomes has revealed that sequence, expression, and function of many genes are conserved among zebrafish and other vertebrates (Postlethwait and Talbot 1997; Howe et al. 2013). Thus, insights from studies in zebrafish will apply broadly to other vertebrates, including humans. On the other hand, there are important genetic, genomic, and physiological differences among vertebrates. It is, therefore, important to keep possible differences in mind and to recognize that analyzing the diversity among different species may enhance overall understanding of important processes. For example, zebrafish and other teleosts have a much more extensive regenerative ability than mammals, so that studies of fin, heart, and spinal cord regeneration in zebrafish may suggest avenues toward new therapeutic approaches in humans (Gemberling et al. 2013; Becker and Becker 2014).In this review, we provide an overview of different types of glia in the zebrafish, with a focus on some recent studies that highlight the power of the zebrafish system to analyze different aspects of glial development and function.     

斑马鱼母源因子在胚胎发育中的作用

动物受精时,精子主要是将雄原核释放到卵子中,形成的合子中雌、雄原核融合为合子核,但受精卵基因组在前几次有丝分裂过程中不转录,合理的逻辑性推测是其早期发育完全依赖于卵质中储存的RNA和蛋白质,即母源因子.上世纪80年代对无脊椎动物的正向遗传研究发现,母源因子在卵子和胚胎极性的决定、早期胚胎的图式形成等方面发挥了决定性作用.过去10多年来,通过对斑马鱼和小鼠突变体的研究,也证明母源因子在脊椎动物胚胎早期发育中起着重要作用.本文主要综述斑马鱼母源因子在卵母细胞的极性、卵子的激活、早期细胞分裂、母源mRNA的清除、合子基因转录激活以及胚层的形成和分化、体轴的建立等方面的作用,相关知识对于研究人类生育障碍和先天性疾病的发生机制和诊治有借鉴意义.     

Pannexin1 Channel Proteins in the Zebrafish Retina Have Shared and Unique Properties

In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo.     

Ror2 Receptor Mediates Wnt11 Ligand Signaling and Affects Convergence and Extension Movements in Zebrafish

The receptor-tyrosine kinase Ror2 acts as an alternative receptor or co-receptor for Wnt5a and mediates Wnt5a-induced convergent extension movements during embryogenesis in mice and Xenopus as well as the polarity and migration of several cell types during development. However, little is known about whether Ror2 function is conserved in other vertebrates or is involved in other non-canonical Wnt ligands in vivo. In this study we demonstrated that overexpression of dominant-negative ror2 (ror2-TM) mRNA in zebrafish embryos resulted in convergence and extension defects and incompletely separated eyes, which is consistent with observations from slb/wnt11 mutants or wnt11 knockdown morphants. Moreover, the co-injection of ror2-TM mRNA and a wnt11 morpholino or the coexpression of ror2 and wnt11 in zebrafish embryos synergetically induced more severe convergence and extension defects. Transplantation studies further demonstrated that the Ror2 receptor responded to the Wnt11 ligand and regulated cell migration and cell morphology during gastrulation. DnRor2 inhibited the action of Wnt11, which was revealed by a decreased percentage of Wnt11-induced convergence and extension defects. Ror2 physically interacts with Wnt11. The intracellular Tyr-647 and Ser-863 sites of Ror2 are essential for mediating the action of Wnt11. Dishevelled and RhoA act downstream of Wnt11-Ror2 to regulate convergence and extension movements. Overall, our data suggest an important role of Ror2 in mediating Wnt11 signaling and in regulating convergence and extension movements in zebrafish.     

Nucleotide Composition Bias Affects Amino Acid Content in Proteins Coded by Animal Mitochondria

We show that in animal mitochondria homologous genes that differ in guanine plus cytosine (G + C) content code for proteins differing in amino acid content in a manner that relates to the G + C content of the codons. DNA sequences were analyzed using square plots, a new method that combines graphical visualization and statistical analysis of compositional differences in both DNA and protein. Square plots divide codons into four groups based on first and second position A + T (adenine plus thymine) and G + C content and indicate differences in amino acid content when comparing sequences that differ in G + C content. When sequences are compared using these plots, the amino acid content is shown to correlate with the nucleotide bias of the genes. This amino acid effect is shown in all protein-coding genes in the mitochondrial genome, including cox I, cox II, and cyt b, mitochondrial genes which are commonly used for phylogenetic studies. Furthermore, nucleotide content differences are shown to affect the content of all amino acids with A + T- and G + C-rich codons. We speculate that phylogenetic analysis of genes so affected may tend erroneously to indicate relatedness (or lack thereof) based only on amino acid content. Received: 3 July 1996 / Accepted: 6 November 1996