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PubMedDate not supplied

Selective labeling of zebrafish thrombocytes: quantitation of thrombocyte function and detection during development.

Zebrafish thrombocytes, the nucleated equivalents of mammalian platelets, have been characterized morphologically, but knowledge about their developmental synthesis and biochemistry is limited. Given the increasing use of zebrafish as a genetic model to study hemostasis, it is important to isolate and study the function of zebrafish thrombocytes. Therefore, the objective of this study was to isolate thrombocytes, study their function in vitro, and identify the developmental stage at which they enter circulation. To achieve these goals, we developed a method for the selective labeling of thrombocytes and assayed these cells for activation by known mammalian platelet agonists. In both in vitro incubations of whole blood and blood labeled in vivo with the lipophilic dye DiI-C(18), we found labeling in only a single population of cells. These cells were identified as zebrafish thrombocytes by Wright-Giemsa staining. Using selective DiI-C(18) labeling, we showed the formation of thrombocyte aggregates, filopodia, and lipid rafts in response to platelet agonists. Additionally, we showed that aggregates are labeled by binding FITC-conjugated annexin V to exposed phosphatidylserine on the thrombocyte membrane. Using these fluorescent-labeling methods, we developed the first microquantitative assay for thrombocyte aggregation. With this assay, we provided evidence for the presence of an ADP receptor, P2Y(1), in the zebrafish thrombocytes. To study the developmental stage at which thrombocytes appear, we microinjected DiI-C(18) into the circulation of zebrafish embryos and identified the presence of DiI-C(18)-labeled thrombocytes at the 36 h postfertilization stage. These findings will prove helpful in dissecting the functions of thrombocytes in hemostasis and provide further insight into the role of platelets in thrombosis.

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PubMedDate not supplied

ERG assessment of zebrafish retinal development.

Research has shown that adult zebrafish have a complex visual system, with two possible opponent mechanisms. Anatomically, zebrafish retina develops in a sequential manner and is immature at hatching. The purpose of the present study was to assess zebrafish retinal development using the electroretinogram (ERG). ERG responses to visual stimuli were obtained from 4-5, 6-8, 13-15, and 21-24 days postfertilization (dpf) zebrafish. Individual waveforms were assessed and compared across the four age groups. Spectral-sensitivity functions were calculated for the a- and b-wave components of the ERG response. Results showed that the ERG waveforms and spectral-sensitivity functions varied with age. While the 21-24 dpf subjects had an ERG waveform that was similar to that of adults, the younger subjects did not. Although there were modest differences in the a-wave spectral sensitivity, substantial differences were found in the b-wave spectral sensitivities across the ages. There was a consistent strong response to ultraviolet wavelengths, while across the remaining parts of the spectrum, there was a gradual increase in sensitivity with age. Also, the 21-24 dpf subjects appear to have adult-like U- and S-cone functions, but were missing the L-M and the M-S opponent mechanisms found in the adult. These results support the findings of the anatomical studies and demonstrate that the zebrafish is a useful model for examining the development of retinal function.

Animals↗

PubMed2005

Homer 1b/c expression correlates with zebrafish olfactory system development.

The zebrafish, (Danio rerio) is an important model organism for the analysis of molecular mechanisms that govern neuronal circuit development. The neuronal circuitry that mediates olfaction is crucial for the development and survival of all teleost fishes. In concert with other sensory systems, olfaction is functional at early stages in zebrafish development and mediates important behavioral and survival strategies in the developing larva. Odorant cues are transduced by an array of signaling molecules from receptors in olfactory sensory neurons. The scaffolding protein family known as Homer is well placed to orchestrate this signaling cascade by interacting with and coupling membrane bound receptors to cytosolic signaling partners. To date, Homer has not been demonstrated in the zebrafish. Here we report that the Homer 1b/c isoform was prominent in the olfactory system from the earliest stages of differentiation. We describe the spatial and temporal distribution of Homer in the zebrafish olfactory system. At 24 hours post fertilization (hpf), Homer expression delineated the boundary of the presumptive olfactory placode. Subsequent expression steadily increased throughout the developing olfactory placode, with a prominent localization to the dendritic knobs of the olfactory sensory neurons. Homer expression in the developing olfactory bulb was punctate and prominent in the glomeruli, displaying an apparent synaptic localization. This work supports the hypothesis that Homer is an important molecule in neuronal circuit development, necessary for crucial behaviors required for development and survival.

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PubMed2000

Somite development in zebrafish.

A full understanding of somite development requires knowledge of the molecular genetic pathways for cell determination as well as the cellular behaviors that underlie segmentation, somite epithelialization, and somite patterning. The zebrafish has long been recognized as an ideal organism for cellular and histological studies of somite patterning. In recent years, genetics has proven to be a very powerful complementary approach to these embryological studies, as genetic screens for zebrafish mutants defective in somitogenesis have identified over 50 genes that are necessary for normal somite development. Zebrafish is thus an ideal system in which to analyze the role of specific gene products in regulating the cell behaviors that underlie somite development. We review what is currently known about zebrafish somite development and compare it where appropriate to somite development in chick and mouse. We discuss the processes of segmentation and somite epithelialization, and then review the patterning of cell types within the somite. We show directly, for the first time, that muscle cell and sclerotome migrations occur at the same time. We end with a look at the many questions about somitogenesis that are still unanswered.

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PubMed2001

Zebrafish E-cadherin: expression during early embryogenesis and regulation during brain development.

Zebrafish E-cadherin (cdh1) cell adhesion molecule cDNAs were cloned. We investigated spatial and temporal expression of cdh1 during early embryogenesis. Expression was observed in blastomeres, the anterior mesoderm during gastrulation, and developing epithelial structures. In the developing nervous system, cdh1 was detected at the pharyngula stage (24 hpf) in the midbrain-hindbrain boundary (MHB). Developmental regulation of MHB formation involves wnt1 and pax2.1. wnt1 expression preceded cdh1 expression during MHB formation, and cdh1 expression in the MHB was dependent on normal development of this structure.

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PubMed2000

Calcium signalling during zebrafish embryonic development.

Calcium signals appear throughout the first 24 hours of zebrafish development. These begin at egg activation, then continue to be generated throughout the subsequent zygote, cleavage, blastula, gastrula, and segmentation periods. They are thus associated with the major phases of pattern formation: cell proliferation, cell differentiation, axis determination, the generation of primary germ layers, the emergence of rudimentary organ systems, and therefore the establishment of the basic vertebrate body plan. When signals need to be transmitted across significant distances they take the form of waves, either intracellular waves when the cell size is large, or later in development when the cell size is reduced, intercellular waves. We will consider both types of calcium signals and their integration into signalling networks, and discuss their possible functions and developmental significance with regard to pattern formation. BioEssays 22:113-123, 2000.

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PubMed1989

Genetics and early development of zebrafish.

Zebrafish genes and development are being studied in a growing number of laboratories. Given that many other organisms are already being exploited by large numbers of investigators, and that our general knowledge about the zebrafish embryo and genome is at present rather sketchy, why should we now concern ourselves with how this tropical fish develops? Whereas the zebrafish embryo is similar in important ways to other vertebrate embryos, it is relatively simple and unusually accessible for both cellular and genetic analyses.

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PubMed2005

Critical roles of CD146 in zebrafish vascular development.

In this report, we use zebrafish as a model system to understand the importance of CD146 in vascular development. Endothelial-specific expression of CD146 was verified by whole-mount in situ hybridization. Suppression of CD146 protein expression by antisense morpholino oligonucleotides (MO) resulted in poorly developed intersomitic vessels (ISVs). In CD146 morphants, we observed a lack of blood flow through the ISV region, despite that fluorescence microangiography showed that the ISVs were present. This finding suggests that the lumens of the developing ISVs may be too narrow for proper circulation. Additionally, remodeling of the caudal vein plexus into functional vascular tubes appeared to be affected. Suppression of CD146 protein expression resulted in a circulation shunt that caused the circulation to by-pass part of the caudal artery/vein system. The same vascular defects were recapitulated by using a second morpholino oligonucleotide. This morphant expressed a truncated CD146 protein with amino acids V32 to T57 at the N terminus deleted in an in-frame manner. This region, therefore, is likely to contain elements critical for CD146 function. This study provides the first in vivo functional assessment of CD146 in embryonic development by showing that knockdown of CD146 protein expression severely hinders vascular development in zebrafish.

Amino Acid Sequence↗

PubMed2002

Forward and reverse genetic approaches to the analysis of eye development in zebrafish.

The zebrafish has been established as a mainstream research system, largely due to the immense success of genetic screens. Over 2000 mutant alleles affecting zebrafish's early development have been isolated in two large-scale morphological screens and several smaller efforts. So far, over 50 mutant strains display retinal defects and many more have been shown to affect the retinotectal projection. More recently, mutant isolation and characterization have been successfully followed by candidate and positional cloning of underlying genes. To supplement forward genetic mutational analysis, several reverse genetic techniques have also been developed. These recent advances, combined with the genome project, have established the zebrafish as one of the leading models for studies of visual system development.

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PubMed2005

Exocrine pancreas development in zebrafish.

Although many of the genes that regulate development of the endocrine pancreas have been identified, comparatively little is known about how the exocrine pancreas forms. Previous studies have shown that exocrine pancreas development may be modeled in zebrafish. However, the timing and mechanism of acinar and ductal differentiation and morphogenesis have not been described. Here, we characterize zebrafish exocrine pancreas development in wild type and mutant larvae using histological, immunohistochemical and ultrastructural analyses. These data allow us to identify two stages of zebrafish exocrine development. During the first stage, the exocrine anlage forms from rostral endodermal cells. During the second stage, proto-differentiated progenitor cells undergo terminal differentiation followed by acinar gland and duct morphogenesis. Immunohistochemical analyses support a model in which the intrapancreatic ductal system develops from progenitors that join to form a contiguous network rather than by branching morphogenesis of the pancreatic epithelium, as described for mammals. Contemporaneous appearance of acinar glands and ducts in developing larvae and their disruption in pancreatic mutants suggest that common molecular pathways may regulate gland and duct morphogenesis and differentiation of their constituent cells. By contrast, analyses of mind bomb mutants and jagged morpholino-injected larvae suggest that Notch signaling principally regulates ductal differentiation of bipotential exocrine progenitors.

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PubMed2004

Fgf signaling is required for zebrafish tooth development.

We have investigated fibroblast growth factor (FGF) signaling during the development of the zebrafish pharyngeal dentition with the goal of uncovering novel roles for FGFs in tooth development as well as phylogenetic and topographic diversity in the tooth developmental pathway. We found that the tooth-related expression of several zebrafish genes is similar to that of their mouse orthologs, including both epithelial and mesenchymal markers. Additionally, significant differences in gene expression between zebrafish and mouse teeth are indicated by the apparent lack of fgf8 and pax9 expression in zebrafish tooth germs. FGF receptor inhibition with SU5402 at 32 h blocked dental epithelial morphogenesis and tooth mineralization. While the pharyngeal epithelium remained intact as judged by normal pitx2 expression, not only was the mesenchymal expression of lhx6 and lhx7 eliminated as expected from mouse studies, but the epithelial expression of dlx2a, dlx2b, fgf3, and fgf4 was as well. This latter result provides novel evidence that the dental epithelium is a target of FGF signaling. However, the failure of SU5402 to block localized expression of pitx2 suggests that the earliest steps of tooth initiation are FGF-independent. Investigations of specific FGF ligands with morpholino antisense oligonucleotides revealed only a mild tooth shape phenotype following fgf4 knockdown, while fgf8 inhibition revealed only a subtle down-regulation of dental dlx2b expression with no apparent effect on tooth morphology. Our results suggest redundant FGF signals target the dental epithelium and together are required for dental morphogenesis. Further work will be required to elucidate the nature of these signals, particularly with respect to their origins and whether they act through the mesenchyme.

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PubMed2006

Egr1 gene knockdown affects embryonic ocular development in zebrafish.

PURPOSE: To identify the changes in zebrafish embryonic ocular development after early growth response factor 1 (Egr1) gene knockdown by Egr1-specific translation inhibitor, morpholino oligonucleotides (MO). METHODS: Two kinds of Egr1-MO were microinjected separately with various dosages into one to four celled zebrafish embryos to find an optimal dose generating an acceptable mortality rate and high frequency of specific phenotype. Chordin-MO served as the positive control; a 5 mismatch MO of Egr1-MO1 and a nonspecific MO served as negative controls. We graded the Egr1 morphants according to their gross abnormalities, and measured their ocular dimensions accordingly. Western blot analysis and synthetic Egr1 mRNA rescue experiments confirmed whether the deformities were caused by Egr1 gene knockdown. Histological examination and three kinds of immunohistochemical staining were applied to identify glutamate receptor one expression in retinal ganglion cells and amacrine cells, to recognize acetylated alpha-tubulin expression which indicated axonogenesis, and to label photoreceptor cells with zpr-1 antibody. RESULTS: After microinjection of 8 ng Egr1-MO1 or 2 ng Egr1-MO2, 81.8% and 97.3% of larvae at 72 h postfertilization had specific defects, respectively. The gross phenotype included string-like heart, flat head, and deformed tail. The more severely deformed larvae had smaller eyes and pupils. Co-injection of 8 ng Egr1-MO1 and supplementary 12 pg synthetic Egr1 mRNA reduced the gross abnormality rate from 84.4% to 29.7%, and decreased the severity of deformities. Egr1 protein appeared in the wildtype and rescued morphants, but was lacking in the Egr1 morphants with specific deformities. Lenses of Egr1 morphants were smaller and had some residual nucleated lens fiber cells. Morphants' retinal cells arranged disorderly and compactly with thin plexiform layers. Immunohistochemical studies showed that morphants had a markedly decreased number of mature retinal ganglion cells, amacrine cells, and photoreceptor cells. Retinal axonogenesis was prominently reduced in morphants. CONCLUSIONS: The Egr1 gene plays an important role in zebrafish embryonic oculogenesis. Ocular structures including lens and retina were primitive and lacked appropriate differentiation. Such arrested retinal and lenticular development in Egr1 morphants resulted in microphthalmos.

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PubMed2006

Late onset of NMDA receptor-mediated ventilatory control during early development in zebrafish (Danio rerio).

Increased ventilation frequency (fV) in response to hypoxia in adult fish depends on ionotropic N-methyl-D-aspartate (NMDA) receptors. Nonetheless, the ontogeny of central control mechanisms mediating hypoxic ventilatory chemoreflexes in lower vertebrates has not been studied. Therefore, the aim of this study was to determine when the hypoxic ventilatory response during zebrafish (Danio rerio) development is mediated via NMDA receptors, by performing physiological experiments and western blot analysis of NMDA receptor subunits. Zebrafish larvae at stages 4-16 days post-fertilisation (dpf) were exposed to an hypoxic pulse in control groups and in groups treated with MK801 (NMDA receptor antagonist). The hypoxic increase in fV was present at all larval stages, and it matured during development. The reflex became MK801 sensitive at 8 dpf, but did not completely rely on a glutamatergic transmission until 13 dpf. This, together with changing subunit composition during the different stages (increasing amounts of NMDAR1 subunits and appearance of NMDAR2A subunits in adults), suggests that the amount of functional NMDA receptors needed to achieve a fully developed reflex is not attained until later stages. Furthermore, our results suggest that other non-NMDA receptor mechanisms are responsible for the hypoxia-induced increase in fV during the earlier developmental stages.

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PubMed2005

Differential expression of the duplicated cellular retinoic acid-binding protein 2 genes (crabp2a and crabp2b) during zebrafish embryonic development.

The cellular retinoic acid-binding protein 2 (CRABP2) is believed to be involved in regulating access of retinoic acid to nuclear retinoic acid receptors. We have determined the cDNA sequence and the genomic organization of the duplicated crabp2 gene (crabp2b) in zebrafish. The crabp2b cDNA was 522bp in length and encodes a polypeptide consisting of 146 amino acids. Radiation hybrid mapping assigned the crabp2b gene to zebrafish linkage group 19. The comparison of the mapped human CRABP2 gene, zebrafish crabp2a and zebrafish crabp2b genes revealed that human chromosome 1 has a syntenic relationship to zebrafish linkage groups 16 and 19. Reverse transcription-polymerase chain reaction (RT-PCR) detected crabp2b mRNA in total RNA extracted from whole adult zebrafish, but not in any of the adult zebrafish tissues examined. The crabp2a mRNA was detected in total RNA extracted from whole adult zebrafish, adult zebrafish muscle, testes, and skin and to a lesser extent in heart, ovary and brain. No crabp2a mRNA-specific product was detected in kidney, liver or intestine of the adult zebrafish. Whole mount in situ hybridization detected crabp2b and crabp2a mRNA in a number of structures known to require retinoic acid signaling during embryonic development. The crabp2b mRNA was detected in the central nervous system, branchial arches, pectoral fins, retina (dorsal to the lens), epidermis and otic vesicle of the developing zebrafish. The crabp2a transcripts were detected by whole mount in situ hybridization in the central nervous system, epidermis, proliferative zone of the retina, intestinal bulb, oesophagus, pectoral fins and branchial arches during zebrafish embryonic development.

Amino Acid Sequence↗

PubMed2002

A critical period for functional vestibular development in zebrafish.

We have determined a critical period for vestibular development in zebrafish by using a bioreactor designed by NASA to simulate microgravity for cells in culture. A critical period is defined as the briefest period of time during development when stimulus deprivation results in long lasting or permanent sensory deficits. Zebrafish eggs were collected within 3 hours of being laid and fertilized. In experiment 1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours postfertilization (hPF) and maintained in the bioreactor until 96 hPF. In experiment 2, eggs were placed in the bioreactor immediately after they were collected and maintained in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF. Beginning at 96 hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once each day for 5 days. Only larvae that hatched from eggs that were placed in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor later than 66 hPF in experiment 2 had VOR deficits that persisted for at least 5 days. These data suggest a critical period for vestibular development in the zebrafish that begins before 30 hPF and ends after 66 hPF. To confirm this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed at 72 hPF. VORs were evaluated in these larvae once each day for 5 days beginning at 96 hPF. These larvae had VOR deficits that persisted for at least 5 days. In addition, larvae that had been maintained in the bioreactor from 24 to 66 hPF or from 30 to 72 hPF, had only temporary VOR deficits. In a final experiment, zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF but the bioreactor was turned off from 24 hPF to 72 hPF. These larvae had normal VORs when they were removed from the bioreactor at 96 hPF. Taken as a whole, these data support the idea that there is a critical period for functional maturation of the zebrafish vestibular system. The developmental period identified includes the timeframe during which the vestibular primary afferent neurons are born, innervate their central and peripheral targets, and remodel their central projections.

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PubMed1995

Transparent things: cell fates and cell movements during early embryogenesis of zebrafish.

Development of an animal embryo involves the coordination of cell divisions, a variety of inductive interactions and extensive cellular rearrangements. One of the biggest challenges in developmental biology is to explain the relationships between these processes and the mechanisms that regulate them. Teleost embryos provide an ideal subject for the study of these issues. Their optical lucidity combined with modern techniques for the marking and observation of individual living cells allow high resolution investigations of specific morphogenetic movements and the construction of detailed fate maps. In this review we describe the patterns of cell divisions, cellular movements and other morphogenetic events during zebrafish early development and discuss how these events relate to the formation of restricted lineages.

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PubMed2004

Adenomatous polyposis coli control of retinoic acid biosynthesis is critical for zebrafish intestinal development and differentiation.

Mutations in the APC (adenomatous polyposis coli) tumor suppressor gene cause uncontrolled proliferation and impaired differentiation of intestinal epithelial cells. Recent studies indicate that human colon adenomas and carcinomas lack retinol dehydrogenases (RDHs) and that APC regulates the expression of human RDHL. These data suggest a model wherein APC controls enterocyte differentiation by controlling retinoic acid production. However, the importance of APC and retinoic acid in mediating control of normal enterocyte development and differentiation remains unclear. To examine the relationship between APC and retinoic acid biosynthesis in normal enterocytes, we have identified two novel zebrafish retinol dehydrogenases, termed zRDHA and zRDHB, that show strong expression within the gut of developing zebrafish embryos. Morpholino knockdown of either APC or zRDHB in zebrafish embryos resulted in defects in structures known to require retinoic acid. These defects included cardiac abnormalities, pericardial edema, failed jaw and pectoral fin development, and the absence of differentiated endocrine and exocrine pancreas. In addition, APC or zRDHB morphant fish developed intestines that lacked columnar epithelial cells and failed to express the differentiation marker intestinal fatty acid-binding protein. Treatment of either APC or zRDHB morphant embryos with retinoic acid rescued the defective phenotypes. Downstream of retinoic acid production, we identified hoxc8 as a retinoic acid-induced gene that, when ectopically expressed, rescued phenotypes of APC- and zRDHB-deficient zebrafish. Our data establish a genetic link supporting a critical role for retinoic acid downstream of APC and confirm the importance of retinoic acid in enterocyte differentiation.

Adenomatous Polyposis Coli Protein↗

PubMed1995

Genetic control of adult pigment stripe development in zebrafish.

Adult zebrafish stripes are formed from stripes of gold iridophores alternating with stripes of black melanocytes lying beneath silver stripes. Analysis of defects in pigment pattern development caused by sparse (spa, rose (ros), and leopard (leo) single and double mutant combinations suggests that spa+ and ros+ functions are required for development of separate populations of pigment cells in the adult and that leo+ functions to control assembly of melanocytes into stripes. Thus, between 2 and 3 weeks of zebrafish development, spa-dependent melanocytes differentiate throughout the flank, followed by leo-dependent assembly of these cells into stripes. Beginning at 3 weeks of development, a distinct ros-dependent population of melanocytes differentiates in the stripe. Both early and late differentiating melanocytes then affect the formation of the silver stripes, ensuring registration of melanocyte and iridophore stripes.

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