Puberty visualized: sexual maturation in the transparent Casper zebrafish.

Transparent Casper zebrafish allow studies of vertebrate sexual maturation and gonad development in vivo. Casper gonad dynamics can be observed longitudinally over time and non-invasively. Gonad maturation and reproduction are complex processes subject to disruption by endocrine-disrupting chemicals (EDCs), such as diethylstilbestrol (DES). DES was used as a 'proof of principle' to ascertain the usefulness of the Casper model to determine EDC effects on gonad maturation. Puberty onset in control juvenile Casper zebrafish (N = 43) averaged 13.2 weeks post fertilization (WPF) for females and included increased vent size, while in males puberty occurred at 11.7 WPF along with maintenance of small vents. DES treatment for 6 days in early juveniles (N = 20) induced an average delay in puberty of 5 weeks in females and 10 weeks in males. DES induced loss of breeding tubercles and vent enlargement in post-pubescent males. Puberty in control fish was correlated with an average body length of 1.7 cm for males and 1.8 cm for females. Increased testes opacity, small vent and breeding tubercles denoted male puberty. Puberty in females was defined as ovarian follicle diameters reaching 400 µm with increasingly opaque follicles and by an increased vent size. These results are like those for wild-type zebrafish and indicate that the Casper model is a useful system for studying gonad dynamics in vivo. Future use of transgenic reporter lines in Casper will allow new avenues of investigation into the reproductive biology of this vertebrate model.

In vivo assessment of gonad status, secondary sex characteristics and spawning in transparent Casper zebrafish.

Important aspects of vertebrate reproduction, such as gametogenesis, involve changes in organs found deep internally and thus not easily studied directly in most living vertebrates due to obscuring pigment and overlying tissues. Transparent lines of zebrafish, especially the Casper double mutant, allow direct observation and analysis of reproductive events in the gonads in vivo. The natural production of fertilized eggs in zebrafish is a complex process involving oogenesis, spermatogenesis, mating behavior, endocrine and neurological processes with inputs from the environment including light, temperature and nutrition. While these factors play important roles, the hypothalamic-pituitary-gonadal axis (HPGA) is central in the regulation of embryo output. Endocrine disrupting compounds (EDCs) include a variety of pollutants often present in the environment. EDCs may have direct effects on the HPGA or indirect effects through toxic action on supporting organs such as the liver or kidney. Estrogenic compounds such as diethylstilbestrol (DES) have been reported to affect reproduction in a variety of species including man. In this study, the effects of DES on reproduction were determined in a novel way by using transparent Casper zebrafish that allow direct visualization of gonad status over time. Changes in gonad status with DES treatment were correlated with effects on secondary sex characteristics (i.e., genital vent size and breeding tubercles) spawning and embryo production. The results suggest that the Casper zebrafish is a useful model for studying dynamics of reproductive events in vertebrate gonads in vivo and for determining effects of EDCs on zebrafish reproduction.

Centrosomes in the zebrafish (Danio rerio): a review including the related basal body.

Ever since Edouard Van Beneden and Theodor Boveri first formally described the centrosome in the late 1800s, it has captivated cell biologists. The name clearly indicated its central importance to cell functioning, even to these early investigators. We now know of its role as a major microtubule-organizing center (MTOC) and of its dynamic roles in cell division, vesicle trafficking and for its relative, the basal body, ciliogenesis. While centrosomes are found in most animal cells, notably it is absent in most oocytes and higher plant cells. Nevertheless, it appears that critical components of the centrosome act as MTOCs in these cells as well. The zebrafish has emerged as an exciting and promising new model organism, primarily due to the pioneering efforts of George Streisinger to use zebrafish in genetic studies and due to Christiane Nusslein-Volhard, Wolfgang Driever and their teams of collaborators, who applied forward genetics to elicit a large number of mutant lines. The transparency and rapid external development of the embryo allow for experiments not easily done in other vertebrates. The ease of producing transgenic lines, often with the use of fluorescent reporters, and gene knockdowns with antisense morpholinos further contributes to the appeal of the model as an experimental system. The added advantage of high-throughput screening of small-molecule libraries, as well as the ease of mass rearing together with low cost, makes the zebrafish a true frontrunner as a model vertebrate organism. The zebrafish has a body plan shared by all vertebrates, including humans. This conservation of body plan provides added significance to the existing lines of zebrafish as human disease models and adds an impetus to the ongoing efforts to develop new models. In this review, the current state of knowledge about the centrosome in the zebrafish model is explored. Also, studies on the related basal body in zebrafish and their relationship to ciliogenesis are reviewed.

The developing zebrafish (Danio rerio): a vertebrate model for high-throughput screening of chemical libraries.

The zebrafish, Danio rerio, a small, tropical freshwater species native to Pakistan and India, has become a National Institutes of Health-sanctioned model organism and, due to its many advantages as an experimental vertebrate, it has garnered intense interest from the world's scientific community. Some have labeled the zebrafish, the "vertebrate Drosophila," due to its genetic tractability, small size, low cost, and rapid development. The transparency of the embryo, external development, and the many hundreds of mutant and transgenic lines available add to the allure. Now it appears, the zebrafish can be used for high-throughput screening (HTS) of drug libraries in the discovery process of promising new therapeutics. In this review, various types of screening methods are briefly outlined, as are a variety of screens for different disease models, to highlight the range of zebrafish HTS possibilities. High-content screening (HCS) has been available for cell-based screens for some time and, very recently, HCS is being adapted for the zebrafish. This will allow analysis, at high resolution, of drug effects on whole vertebrates; thus, whole body effects as well as those on specific organs and tissues may be determined.

Use of flatbed transparency scanners in zebrafish research: versatile and economical adjuncts to traditional imaging tools for the Danio rerio laboratory.

Flatbed transparency scanners are typically relegated to routine office tasks, yet they do offer a variety of potentially useful imaging tools for the zebrafish laboratory. These include motility screens, oocyte maturation and egg activation assays as well as counting and measuring tasks. When coupled with Macroscheduler (http://www.mjtnet.com) and ImageJ (http://rsbweb.nih.gov/ij), the scanner becomes a stable platform for imaging large arrays of zebrafish oocytes, embryos, larvae, and adults. Such large arrays are a prerequisite to the development of high-throughput screens for small molecules as potential therapeutic drugs in the treatment of many diseases including cancer and epilepsy. Thus the scanner may have a role in adapting zebrafish to future drug and mutagenesis screening. In this chapter, some of the uses of scanners are outlined to bring attention to the potentials of this simple-to-use, flexible, inexpensive device for the zebrafish research community.

Oocyte maturation: converting the zebrafish oocyte to the fertilizable egg.

The process of oogenesis culminates in steroid-induced oocyte maturation to produce the fertilizable egg. A quintessential biological entity, the egg is central to the production of new individuals. The result of egg fertilization by a sperm cell is the production of the mother of all stem cells (i.e. the zygote). Furthermore, the egg cytoplasm is the only one known to support reprogramming a transplanted nucleus to give rise to an individual (i.e. animal cloning). Zebrafish oocyte maturation is a complex event encompassing a number of cellular changes including germinal vesicle migration (GVM) and dissolution or breakdown (GVD), ooplasmic clearing (OC) with correlated yolk protein changes (YP), development of osmoregulation (OR) in fresh water, the formation of the future embryonic pole, the blastodisc (BF) and activatibility (AC) or cortical maturation. In zebrafish, and many other teleosts, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha, 20beta-DP) has been shown to be the normal inducer of oocyte maturation. A 17alpha, 20beta-DP membrane-resident receptor mediates oocyte maturation via non-genomic mechanisms that are beginning to be understood. This paper will highlight some of the cellular markers resulting from the signaling initiated by 17alpha, 20beta-DP. By describing these markers, it is hoped that workers in the field will have additional tools to help further elucidate the signaling events of oocyte maturation.

Changes of gamma-tubulin expression and distribution in the zebrafish (Danio rerio) ovary, oocyte and embryo.

The tubulin gene family is important for individual zebrafish development from the oocyte through to hatching. This involves often rapid, complex changes in the gametes and embryonic cells that are reflected in underlying gene expression changes. Tubulin dynamics, i.e., the interchange of polymeric and soluble forms in zebrafish oogenesis and embryogenesis, is important for microtubule (MT) cellular functions. Nevertheless, our understanding of how tubulin gene expression changes during zebrafish development is not clear. Previous data showed that soluble alpha-tubulin and gamma-tubulin are associated with large molecular weight complexes (>2MDa) which are reduced by the blastula stage, with a concomitant decrease in soluble tubulin amount. Complexes (<2MDa) then increased in the gastrula with an increase in soluble tubulin. Microarray revealed similar patterns of tubulin gene product expression for zebrafish ovary and eggs while both differed from day 4 larva. In situ hybridization with gamma-tubulin oligonucleotide probes revealed diffuse label in oocytes, with a marked localization to the primordial blastodisc upon maturation. These findings, together with recent work on gamma-tubulin ring complexes in other species, suggest that gamma-tubulin (protein complexes) may be involved in regulating tubulin dynamics, thus is important for zebrafish oogenesis and embryogenesis.

Soluble tubulin complexes, gamma-tubulin, and their changing distribution in the zebrafish (Danio rerio) ovary, oocyte and embryo.

Tubulin dynamics, i.e., the interchange of polymeric and soluble forms, is important for microtubule (MTs) cellular functions, and thus plays essential roles in zebrafish oogenesis and embryogenesis. A novel finding in this study revealed that there were soluble pools of tubulins in zebrafish oocytes that were sequestered and maintained in a temporary "oligomeric" state, which retained assembling and disassembling potential (suggested by undetected acetylated tubulin, marker of stable tubulin), but lacked abilities to assemble into MTs spontaneously in vivo. Using differential centrifugation, gel chromatography and DM1A-probed western blot, soluble alpha-tubulin was found to be associated with large molecular weight complexes (MW range to over 2 MDa) which were reduced in amount by the blastula stage, especially in some batches of embryos, with a concomitant decrease in soluble tubulin. Complexes (MW range less than 2 MDa) then increased in the gastrula with an increase in soluble alpha-tubulin. Two different anti-gamma-tubulin monoclonal antibodies, GTU 88 and TU 30, revealed the existence of soluble gamma-tubulin in both zebrafish oocytes and embryos, which also decreased by the blastula stage and increased in the gastrula stage. Soluble alpha-tubulin and gamma-tubulin extracted from zebrafish ovaries, oocytes and embryos co-localized in fractions on three different columns: S-200 Sephacryl, DEAE and Superose-6b. The soluble tubulin complexes were competent to assemble into MTs in vitro induced by taxol, and gamma-tubulin was co-localized with assembled MTs. These soluble tubulin complexes were stable during freeze-thaw cycles and resisted high ionic interaction (up to 1.5 M NaCl). Furthermore, some ovarian soluble alpha-tubulin could be co-immunoprecipitated with gamma-tubulin, and vice versa. Two antibodies specific for Xenopus gamma-tubulin ring complex proteins (Xgrip 109 and Xgrip 195) detected single bands from ovarian extracts in western blots, suggesting the existence of Xgrip 109 and Xgrip 195 homologues in zebrafish. These findings, together with recent work on gamma-tubulin ring complexes in oocytes, eggs and embryos of other species, suggest that soluble gamma-tubulin-associated protein complexes may be involved in regulating tubulin dynamics during zebrafish oogenesis and embryogenesis.

Computer-aided meiotic maturation assay (CAMMA) of zebrafish (danio rerio) oocytes in vitro.

We have developed a new technique called Computer-Aided Meiotic Maturation Assay (CAMMA) for imaging large arrays of zebrafish oocytes and automatically collecting image files at regular intervals during meiotic maturation. This novel method uses a transparency scanner interfaced to a computer with macro programming that automatically scans and archives the image files. Images are stacked and analyzed with ImageJ to quantify changes in optical density characteristic of zebrafish oocyte maturation. Major advantages of CAMMA include (1) ability to image very large arrays of oocytes and follow individual cells over time, (2) simultaneously image many treatment groups, (3) digitized images may be stacked, animated, and analyzed in programs such as ImageJ, NIH-Image, or ScionImage, and (4) CAMMA system is inexpensive, costing less than most microscopes used in traditional assays. We have used CAMMA to determine the dose response and time course of oocyte maturation induced by 17alpha-hydroxyprogesterone (HP). Maximal decrease in optical density occurs around 5 hr after 0.1 micro g/ml HP (28.5 degrees C), approximately 3 hr after germinal vesicle migration (GVM) and dissolution (GVD). In addition to changes in optical density, GVD is accompanied by streaming of ooplasm to the animal pole to form a blastodisc. These dynamic changes are readily visualized by animating image stacks from CAMMA; thus, CAMMA provides a valuable source of time-lapse movies for those studying zebrafish oocyte maturation. The oocyte clearing documented by CAMMA is correlated to changes in size distribution of major yolk proteins upon SDS-PAGE, and, this in turn, is related to increased cyclin B(1) protein.