Zebrafish in comparative context: A symposium

The Symposium "Zebrafish in Comparative Context" was organizedto bring together two largely separate but highly complementaryresearch traditions in order to make developmental and geneticinformation about a model species (Danio rerio, the zebrafish)more accessible to the comparative biology community. The meetingfocused on the relationship of this model organism to othervertebrates (particularly other fishes) using a comparativeand evolutionary approach. Topics included the phylogeny ofcypriniform fishes, genome evolution, the evolution of gastrulation,dentition, pigmentation, craniofacial development, and nervoussystem structure and function. Participants also met informallyto discuss ways to facilitate collaborative projects in areasof common interest and determine priorities for the developmentof shared resources. Continuing interactions between comparativebiologists, with their extensive body of knowledge of morphologicalvariation among fish species, and developmental biologists andgeneticists working with model species such as the zebrafishwill facilitate our understanding of the evolution of developmentalpatterns and processes in vertebrates.     

Considering the zebrafish in a comparative context

This article introduces a special issue on zebrafish biology that attempts to integrate developmental genetics with comparative studies of other fish species. For zebrafish researchers, comparative work offers a better understanding of the evolutionary history of their model system. Comparative biologists can gain many insights from the developmental and genetic mechanisms revealed in zebrafish that have contributed to the huge range of morphological variation among fishes that has arisen over millions of years. These ideas are considered here in various contexts, including systematics, genome organization and the development of the nervous system, pigmentation, craniofacial skeleton and dentition. Studies of the zebrafish in phylogenetic context provide an opportunity for synergy between communities using these two fundamentally different approaches.     

Cichlid genomics and phenotypic diversity in a comparative context

Cichlid fishes provide an excellent natural system for integrating studies of genomics and adaptive radiation. Cichlids are unique in comprising a substantial fraction of all vertebrate species, possessing unique jaw structures, displaying an exceptional range of breeding systems, and exhibiting rampant phenotypic convergence. The rate of divergence in cichlid jaws, teeth, color patterns, visual systems, reproductive biology, and mating behaviors is unparalleled among vertebrates. I discuss ways rapid divergence in cichlids and other adaptive radiations make understanding the genomic basis of adaptive divergence more tractable. Then, I briefly overview some major findings and insights into vertebrate adaptation that have been gained through cichlid genetic studies. Finally, I discuss the extensive evolutionary replication provided by cichlid adaptive radiations and their potential for studies of genotype-to-phenotype mapping.     

The human post‐fertile lifespan in comparative evolutionary context

There persist two widely held but mutually inconsistent views on the evolution of post‐fertile lifespan of human females. The first, prevalent within anthropology, sees post‐fertile lifespan (PFLS) in the light of adaptive processes, focusing on the social and economic habits of humans that selected for a lengthy PFLS. 1 - 3 This view rests on the assumption that human PFLS is distinct from that of other species, and focuses on quantifying the selective causes and consequences of that difference. The second view, prevalent within gerontology and comparative biology, emphasizes that PFLS is a phylogenetically widespread trait 4 - 6 or that human PFLS is predictable based on life‐history allometries. 7 In this view, human PFLS is part of a broad cross‐species pattern and its genesis cannot, therefore, rely on human‐specific traits. Those who advocate the second view have questioned the “special pleading” for human specific explanations of PFLS, 4 and have argued that human PFLS is quantitatively greater but not qualitatively different than PFLS in many other animals. 5 , 8 Papers asking whether human PFLS is explained by the importance of mothers more than grandmothers, whether paternal or maternal grandparents have more of an effect on child survival, or who is providing the excess calories are associated with the first view that assumes the need to explain the existence of human PFLS on the basis of a uniquely human socioecology. Anthropologists largely see human PFLS as derived, while comparative gerontologists point to evidence that it is one instance of a ubiquitous cross‐species pattern. The two groups generally occupy non‐overlapping research circles, in terms of conferences and journals, and therefore interact little enough to largely avoid the need to reconcile their views, allowing the persistence of misconceptions in each field. Our goal is to identify and address the most important of these misconceptions and thereby make clear that both of these seemingly incongruent views contain valid points. We argue that two distinct but related traits have been lumped together under the same concept of “post‐reproductive lifespan,” one (post‐fertile viability) that is tremendously widespread and another (a post‐fertile life stage) that is derived to hominins, and that the differences and connections between these two traits are necessary for understanding human life‐history evolution.     

A descriptive and comparative study of the deciduous dentition of prehistoric Ohio Valley Amerindians

The deciduous dentition of 58 individuals from groups of prehistoric Ohio Valley Amerindians (2,000 B.C.-1,600 A.D.) was measured for antero-posterior and bucco-lingual dimensions and scored for morphological characteristics and macroscopic pathology. Only five dimensions of the posterior teeth and the frequency of severe linear enamel hypoplasia showed significant differences in the groups. In all cases focal agriculturalists exhibited smaller teeth and a higher frequency of severe linear enamel hypoplasia. These findings are explained as the result of changing diet and food preparation techniques, and/or sampling bias in the earlier burial cult groups where primarily higher status individuals may be the representatives. Comparison of metric and morphological characteristics of the deciduous dentition in the prehistoric Amerindians and roughly contemporaneous European groups indicates morphological characteristics are the better means of discrimination.     

Polynesian dentition

The chance discovery of a paper by Rottstock et al. (1983) comparing the individual diameters of 4,497 teeth from 711 skulls from anthropologically-different populations (Europeans, Negroids, Mongoloids, Melanesians) provided the impetus to add our measurements of 449 teeth from 89 Polynesians (mainland Maoris, Chatham Island, Wairau Bar) to the series. We confirm that dental measurements provide clear differences among the different population groups. We confirm that dental indices are useful by disclosing specific dental relationships among the different population groups.     

台湾地区斑马鱼研究现况

台湾地区斑马鱼研究起始于1996年,在经历了约8年的萌芽期(1996~2003年)之后,目前已进入到茁壮期(2004~现今),现今全台湾共有83个实验室使用斑马鱼作为实验材料,台湾地区斑马鱼研究社群的研究主题可大致分成3大类:(1)胚胎发育;(2)人类疾病;(3)生物技术。累积至今与斑马鱼相关论文发表总数已达到342篇。自2010年起,台湾也成立了两个斑马鱼种质资源库(TZCAS与ZeTH)。在种质资源库的充分协助下,目前许多医院的临床医师、工程与生物信息相关领域的研究人员,也开始加入斑马鱼研究社群进行跨领域的整合性研究,成为现今台湾地区斑马鱼研究的一大特色。     

斑马鱼在新药发现中的应用

在过去20年里,斑马鱼已成为一种重要的模式脊椎动物,在发育、遗传、免疫、肿瘤和毒理等诸多研究领域中被广泛应用。近年来,斑马鱼作为活体模型越来越多地应用于某些生物学过程的药物筛选。通过斑马鱼初步筛选,在药物研发初期可确定化合物的生物学活性、毒性以及副作用等。最近的研究还发现,斑马鱼不仅用于新药筛选,还可用于药物结构的优化。本文重点介绍斑马鱼在新药发现中的应用。     

Parvalbumin Isoforms in Zebrafish

By using an analysis of existing genomic information it is concluded that in zebrafish nine genes encode parvalbumin (PV). These genes possess introns that differ in size and show nucleotide variability but they contain the same number of exons, and for each corresponding exon, the number of nucleotides therein are identical in all the paralogs. This rule also applies to the multiple PV genes of other species e.g. mammals. Each of these genes displays, however, characteristic 5′ and 3′ UTRs which appear highly conserved between closely related species (so that orthologs among these species can be readily identified) but which show larger numbers of mutations between species that are more distant in evolution. A tree is presented which suggests that the traditional classification of PVs as alpha or beta (based mainly on charge of the protein molecule) is not sustainable. Numbers 1–9 are assigned to the various isoforms to facilitate their identification in future studies. A bifurcation of isoforms into 1 and 4; 2 and 3; 6 and 7; 8 and 9 appears to have occurred simultaneously in more recent time, i.e. perhaps approximately 60 mys ago when primates and rodents branched.     

Profiling Sex-Specific piRNAs in Zebrafish

Piwi proteins and their partner small RNAs play an essential role in fertility, germ-line stem cell development, and the basic control and evolution of animal genomes. However, little knowledge exists regarding piRNA biogenesis. Utilizing microfluidic chip analysis, we present a quantitative profile of zebrafish piRNAs expressed differentially between testis and ovary. The sex-specific piRNAs are derived from separate loci of repeat elements in the genome. Ovarian piRNAs can be categorized into groups that reach up to 92 members, indicating a sex-specific arrangement of piRNA genes in the genome. Furthermore, precursor piRNAs preferentially form a hairpin structure at the 3′end, which seem to favor the generation of mature sex-specific piRNAs. In addition, the mature piRNAs from both the testis and the ovary are 2′-O-methylated at their 3′ ends.SMALL RNAs, ranging from 19 to 30 nucleotides (nt) in length, constitute a large family of regulatory molecules with diverse functions in invertebrates, vertebrates, plants, and fungi (Bartel 2004; Nakayashiki 2005). Two major classes of small RNAs are microRNAs (miRNAs) and small interfering RNAs (siRNAs). The functions of small RNAs have been conserved through evolution; they have been shown to inhibit gene expression at the levels of mRNA degradation, translational repression, chromatin modification, heterochromatin formation, and DNA elimination (Mochizuki et al. 2002; Bartel 2004; Kim et al. 2005; Brodersen and Voinnet 2006; Lee and Collins 2006; Vaucheret 2006).Over the past few years, focus on the genetics of small RNAs has helped clarify the mechanisms behind the regulation of these molecules. While hundreds of small RNAs have been identified from mammalian somatic tissues, relatively little is known about small RNAs in germ cells. A recent breakthrough has been the identification of small RNAs that associate with Piwi proteins (piRNAs) from Drosophila and mammalian gonads (Aravin et al. 2001, 2006; Girard et al. 2006; Grivna et al. 2006; Vagin et al. 2006; Watanabe et al. 2006). piRNAs and their interacting proteins Ziwi/Zili have also been identified in zebrafish (Houwing et al. 2007, 2008). Increasing evidence indicates that piRNAs play roles mainly in germ cell differentiation and genomic stability (Carthew 2006; Lau et al. 2006; Vagin et al. 2006; Brennecke et al. 2007; Chambeyron et al. 2008; Klattenhoff and Theurkauf 2008; Kuramochi-Miyagawa et al. 2008; Kim et al. 2009; Lim et al. 2009; Unhavaithaya et al. 2009). Moreover, although piRNAs are mostly expressed in germ line cells, recent studies showed piRNA expression in nongerm cells, for example, T-cell lines (Jurkat cells and MT4) (Azuma-Mukai et al. 2008; Yeung et al. 2009), indicating other functions such as in the immune system. piRNAs do not appear to be derived from double-stranded RNA precursors, and their biogenesis mechanisms, although unclear, may be distinct from those of siRNA and miRNA. Recently, two distinct piRNA production pathways were further proposed: the “ping-pong” model (Brennecke et al. 2007; Gunawardane et al. 2007) and the Ago3-independent piRNA pathway centered on Piwi in somatic cells (Li et al. 2009; Malone et al. 2009). However, the mechanistic pathways of piRNA activity and their biogenesis are still largely unknown.Teleost fishes comprise >24,000 species, accounting for more than half of extant vertebrate species, displaying remarkable variation in morphological and physiological adaptations (see review in Zhou et al. 2001). Recently, Houwing et al. (2007, 2008) reported findings on Ziwi/Zili and associated piRNAs, implicating roles in germ cell differentiation, meiosis, and transposon silencing in the germline of the zebrafish. However, some of the identified zebrafish piRNAs are nonrepetitive and nontransposon-related piRNAs, suggesting that piRNAs may have additional unknown roles. In this study, we show that for males and females, piRNAs are specifically derived from separate loci of the repeat elements, and that ovarian piRNAs are far more often associated in groups. Genomic analysis of piRNAs indicates a tendency to folding at the 3′ end of the piRNA precursor, which may favor cleavage of the piRNA precursor to generate mature sex-specific piRNAs. Furthermore, methylation modification occurs at the 2′-O-hydroxyl group on the ribose of the final 3′ nucleotide in both the testis and the ovary.     

Baculovirus-Mediated Gene Expression in Zebrafish

Abstract In an effort to misexpress genes in zebrafish, we tested the ability of baculovirus to infect and drive gene expression in embryos. By injecting virus into specific tissues and using appropriate promoters, both the location and time of gene expression could be controlled. Using a virus with 2 different promoters, LacZ and GFP could be expressed independently. The efficiency of expression appears to depend on the promoter used. As a test of this system, baculovirus was used to ectopically express ephrinB2a in the presomitic mesoderm. EphrinB2a is normally expressed in the posterior region of developing somites, and baculovirus-mediated misexpression caused abnormal somite boundary formation. Baculovirus can thus be used as a tool for gene misexpression experiments in the zebrafish, especially when localized misexpression is required late in development.     

Thrombin Generation in Zebrafish Blood

To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis.     

Temporally-Controlled Site-Specific Recombination in Zebrafish

Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreER^T2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms.