SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
The zebrafish is rapidly increasing in popularity with developmental biologists. Driving this interest are the elegant methods for in vivo observations and recovery of early developmental mutations. The past year has seen the introduction of additional methods for in vivo manipulation of identified cells and the application of these methods to mutant analysis.
Zebrafish have become an important model organism to study the genetic control of vertebrate nervous system development. Here, we present an overview on the formation of dopaminergic neuronal groups in zebrafish and compare the positions of DA neurons in fish and mammals using the neuromere model of the vertebrate brain. Based on mutant analysis, we evaluate the role of several signaling pathways in catecholaminergic neuron specification. We further discuss the prospect of identifying novel genes involved in dopaminergic development through forward genetics mutagenesis screens.
Cyclin-dependent kinase 8 (cdk8) regulates transcription by phosphorylating RNA polymerase II and TFIIH. The mechanism of zygotic transcription activation during vertebrate embryonic development is poorly understood. Here we describe the cloning and developmental expression pattern of zebrafish cdk8 mRNA. It is highly conserved, sharing 79% DNA and 95% amino acid sequence identity with human cdk8, thereby indicating an important role for the protein. Northern blotting and whole mount in situ hybridisation revealed expression of zebrafish cdk8 maternally, following the onset of zygotic transcription at the mid-blastula transition (MBT) and throughout embryonic development.
In a large-scale screen for mutations affecting embryogenesis in zebrafish, we identified 48 mutations in 34 genetic loci specifically affecting craniofacial development. Mutants were analyzed for abnormalities in the cartilaginous head skeleton. Further, the expression of marker genes was studied to investigate potential abnormalities in mutant rhombencephalon, neural crest, and pharyngeal endoderm. The results suggest that the identified mutations affect three distinct aspects of craniofacial development. In one group, mutations affect the overall pattern of the craniofacial skeleton, suggesting that the genes are involved in the specification of these elements. Another large group of mutations affects differentiation and morphogenesis of cartilage, and may provide insight into the genetic control of chondrogenesis. The last group of mutations leads to the abnormal arrangement of skeletal elements and may uncover important tissue-tissue interactions underlying jaw development.
Large-scale mutant screens in zebrafish have led to the identification of more than 50 genes affecting various aspects of neural development and function, including neural induction, anteroposterior and dorsoventral regionalization, axon pathfinding, neuronal differentiation and survival, and behavior. Phenotypic analysis of mutants for some of these genes has already uncovered important genetic and cellular interactions during development. Ongoing molecular analyses promise to further elucidate the mechanisms underlying neural development in vertebrates.
Recent studies in zebrafish have contributed to our understanding of early endoderm formation in vertebrates. Specifically, they have illustrated the importance of Nodal signaling as well as three transcription factors, Faust/Gata5, Bonnie and Clyde, and Casanova, in this process. Ongoing genetic and embryological studies in zebrafish are also contributing to our understanding of later aspects of endoderm development, including the formation of the gut and its associated organs, the liver and pancreas. The generation of transgenic lines expressing GFP in these organs promises to be particularly helpful in such studies.
The vertebrate retina contains specialised regions for high-acuity vision, exemplified by the human fovea and its zebrafish analogue, the high-acuity zone (HAZ). Despite the widespread use of zebrafish to model retinal disease, a stage-resolved quantitative reference describing normal eye, photoreceptor layer (PRL) and lens growth has been lacking. Here, we apply contrast-enhanced micro-computed tomography (micro-CT) to construct the first three-dimensional micro-CT normative atlas of wild-type zebrafish eye development across five larval stages [3, 5, 7, 10 and 18 days post-fertilisation (dpf)], mapping circumferential PRL thickness, eye and lens morphology, and compartment growth rates. Regional PRL thickening within the temporo-ventral region of the expected HAZ emerged by 5 dpf and was sustained by a localised redistribution of growth, persisting and extending towards the optic nerve through 18 dpf. The PRL, lens and eye grew through four phases, alternating between disproportionate PRL expansion and coordinated growth, while the eye remodelled from a nasal-dominant to a temporo-ventral-dominant form. This regional specialisation was protracted relative to gross ocular growth and could proceed independently of it, paralleling the extended postnatal maturation of the human fovea. This atlas provides a quantitative baseline for distinguishing disease-induced changes from normal variation, supporting zebrafish models of foveal hypoplasia and related disorders.
We cloned zebrafish runx3/aml2/cbfa3 and examined its expression and function during embryogenesis. In the developing embryo, runx3 is dynamically expressed in hematopoietic, neuronal, and cartilaginous tissues. Hematopoietic expression of runx3 commences late in embryogenesis in the ventral tail intermediate cell mass and later colocalizes with spi1 and lyz in circulating blood cells. In the cloche mutant, hematopoietic expression was absent, suggesting that Runx3 functions downstream of cloche in a hematopoietic pathway. Neuronal tissues expressing runx3 include the trigeminal ganglia and Rohon-Beard neurons. Runx3 appears to contribute to normal development of primitive and definitive hematopoietic cells. When Runx3 function was compromised using morpholino oligonucleotides, a reduction in the number of mature blood cells was observed. Furthermore, Runx3 depletion decreased runx1 expression in the ventral wall of the dorsal aorta and reduced the number of spi1- and lyz-containing blood cells. Conversely, ubiquitous overexpression of runx3 led to an increase in primitive blood cell numbers, together with an increase in runx1-expressing cells in the ventral wall of the dorsal aorta. We propose a role for Runx3 in the regulation of blood cell numbers.
MicroRNAs (miRNAs) are small noncoding RNAs, about 21 nucleotides in length, that can regulate gene expression by base-pairing to partially complementary mRNAs. Regulation by miRNAs can play essential roles in embryonic development. We determined the temporal and spatial expression patterns of 115 conserved vertebrate miRNAs in zebrafish embryos by microarrays and by in situ hybridizations, using locked-nucleic acid-modified oligonucleotide probes. Most miRNAs were expressed in a highly tissue-specific manner during segmentation and later stages, but not early in development, which suggests that their role is not in tissue fate establishment but in differentiation or maintenance of tissue identity.
The novel type I transforming growth factor-beta (TGF-beta) family member receptor Alk8 was previously identified in a degenerate RT-PCR screen for zebrafish type I and II TGF-beta family member receptors. Functional analyses revealed that Alk8 acts through Bmp signaling pathways in early embryonic dorsoventral patterning, in neural crest cell specification, and in patterning and differentiation of neural crest cell-derived pharyngeal arch cartilages. In addition, Alk8 forms active signaling complexes with TGF-beta1 and the TGF-beta RII receptor, suggesting that Alk8 mediates cross talk between Bmp and TGF-beta subfamily members. In this study, immunohistochemical analysis was performed on zebrafish aged 2 days postfertilization to 1 year, revealing immunolocalization of Alk8 to tissues of the tooth-bearing ceratobranchial 5 (cb5) arch including dental epithelial and mesenchymal tooth tissues of developing primary and replacement teeth, mucous-producing crypt epithelium, keratinized bite plate, and developing taste buds. These results suggest roles for Alk8 in patterning tooth-bearing pharyngeal epithelium, in the initiation of tooth development, in odontoblast and ameloblast differentiation, and in osteoblast maturation. The ability for zebrafish to continuously form teeth throughout their lives allows for the comparison of Alk8 expression in both primary and replacement tooth development, revealing identical Alk8 expression profiles. This study advances our current understanding of the functions of Alk8, particularly with respect to primary and replacement tooth formation, reveals additional roles for Alk8 in dental epithelial patterning and in odontoblast, ameloblast and osteoblast differentiation, and demonstrates the utility of the zebrafish as a model for primary and replacement tooth development.
BACKGROUND: Laminins represent major components of basement membranes and play various roles in embryonic and adult tissues. The functional laminin molecule consists of three chains, alpha, beta and gamma, encoded by separate genes. There are twelve different laminin genes identified in mammals to date that are highly homologous in their sequence but different in their tissue distribution. The laminin alpha -1 gene was shown to have the most restricted expression pattern with strong expression in ocular structures, particularly in the developing and mature lens. RESULTS: We identified the zebrafish lama1 gene encoding a 3075-amino acid protein (lama1) that possesses strong identity with the human LAMA1. Zebrafish lama1 transcripts were detected at all stages of embryo development with the highest levels of expression in the developing lens, somites, nervous and urogenital systems. Translation of the lama1 gene was inhibited using two non-overlapping morpholino oligomers that were complementary to sequences surrounding translation initiation. Morphant embryos exhibited an arrest in lens development and abnormalities in the body axis length and curvature. CONCLUSION: These results underline the importance of the laminin alpha 1 for normal ocular development and provide a basis for further analysis of its developmental roles.
Fibroblast growth factor (Fgf) signaling plays important roles in brain development. Fgf3 and Fgf8 are crucial for the formation of the forebrain and hindbrain. Fgf8 is also required for the midbrain to form. Here, we identified zebrafish Fgf19 and examined its roles in brain development by knocking down Fgf19 function. We found that Fgf19 expressed in the forebrain, midbrain and hindbrain was involved in cell proliferation and cell survival during embryonic brain development. Fgf19 was also essential for development of the ventral telencephalon and diencephalon. Regional specification is linked to cell type specification. Fgf19 was also essential for the specification of gamma-aminobutyric acid (GABA)ergic interneurons and oligodendrocytes generated in the ventral telencephalon and diencephalon. The cross talk between Fgf and Hh signaling is critical for brain development. In the forebrain, Fgf19 expression was down-regulated on inhibition of Hh but not of Fgf3/Fgf8, and overexpression of Fgf19 rescued partially the phenotype on inhibition of Hh. The present findings indicate that Fgf19 signaling is crucial for forebrain development by interacting with Hh and provide new insights into the roles of Fgf signaling in brain development.
Ran, which functions in nucleocytoplasmic transport and mitosis, binds to and is regulated in part by RanBP1. We have identified a zebrafish RanBP1 cDNA and report that it encodes for a polypeptide of 233 amino acids with considerable similarity to human and Xenopus RanBP1, despite the fact that it is 10% longer due to an extension at its carboxy terminus. RanBP1 mRNA is present as a maternal transcript and is expressed ubiquitously throughout the developing embryo. At the protein level, RanBP1 is present at all embryonic stages. Surprisingly, the ectopic overexpression of the protein had no obvious effect on embryogenesis. Attempts were also made to down-regulate RanBP1 activity by RNA interference. Injecting double-stranded RNA augmented both the mortality rate and the frequency of induced defects. Specific defects accompanied by changes in RanBP1 expression were not seen, leading us to propose that RNAi is not a reliable method for deregulating the activity of constitutively expressed genes, like RanBP1, in zebrafish. Mol. Reprod. Dev. 59:235-248, 2001.
Fyn protein tyrosine kinase is present in the unfertilized and fertilized egg, becomes activated within minutes following fertilization, and has been localized to the cortical cytoplasm and spindle apparatus of the zygote. In order to establish the expression pattern of Fyn in the early embryo, we examined the distribution pattern of Fyn by immunofluorescence microscopy. Fyn protein is distributed evenly among cells of the cleavage stage zebrafish embryo and is concentrated in the cortical region of each cell. During blastula and gastrula stages, Fyn was expressed in all cells, however a subpopulation of cells exhibited strong nuclear staining for Fyn. Nuclear Fyn staining was not observed after the gastrula period of development, nor in the adult zebrafish. Immunoprecipitation of Fyn from isolated mid-blastula nuclei confirmed Fyn was present in the nucleus. This is, to our knowledge, the first demonstration of Fyn kinase, which lacks a nuclear localization signal, present in the nucleus. The transient compartmentalization of Fyn in the nucleus could be important in nuclear signaling.
Hypoxia-inducible factors (HIFs) regulate gene expression in response to hypoxia and in vertebrates they are known to participate in several developmental processes, including angiogenesis, vasculogenesis, heart and central nervous system development. Over the last decade, major progress in unraveling the molecular mechanisms that mediate regulation of HIF proteins by oxygen tension has been reported, but our knowledge on their developmental regulation during embryogenesis in model organisms is limited. Expression of hif-1alpha and hif-2alpha genes has been characterized during normal mouse development and they were found to be expressed from stages E7.5, later in E9.5 and E15.5 in several different tissues such as the brain, heart and blood vessels. However, there is no detailed temporal information on their expression at other embryonic stages, even though orthologous genes have been described in several different vertebrate species. In this study, we describe the cloning and detailed expression pattern of zebrafish hif-1alpha and hif-2alpha genes. Sequence analysis revealed that zebrafish Hif proteins are highly homologous to other vertebrate orthologues. Zebrafish hif-1alpha and hif-2alpha are both expressed throughout development in discrete territories in a dynamic pattern. Interestingly, in the notochord the expression of hif-1alpha is switched off, while hif-2alpha transcription is turned on, signifying that the two genes might have partially overlapping, although non-redundant functions in development. This is the first time that a detailed comparison of the expression of hif-1alpha and hif-2alpha is directly assessed in a vertebrate model system throughout development.
Estrogen synthesized in the brain itself by the action of cytochrome P450 aromatase (P450arom) is known to have permanent organizing effects on the developing CNS. In fish, estrogen upregulates the predominant brain isoform (P450aromB), implying that xenoestrogens (XE) could act as neurodevelopmental toxicants by altering P450aromB. To test this hypothesis, zebrafish embryos were exposed to 17beta-estradiol (E(2)), diethylstilbestrol (DES, a potent agonist), and bisphenol A (BPA, a weak agonist). RT-PCR/Southern transfer analysis showed that E(2) (0.01-10 microM) upregulated P450aromB in a dose-response manner. The effect of DES (0.01 microM) was similar to 1 microM E(2) (three- to four-fold higher than control), but BPA was less effective (<threefold increase at 10 microM). mRNA levels of the predominant ovarian isoform (P450aromA) were unchanged by estrogen. Treatment with E(2) (0.1-10 microM) between 2 and 72 hpf had dose-response effects on mortality and hatching and induced a 'curved tail down' phenotype characteristic of mutants with defects of early CNS development. The critical period of estrogen sensitivity for effects on mortality and curved tails was 2-24 hpf, whereas hatching effects were both stage- and duration-dependent. Developmental effects of DES and BPA were similar to E(2) but testosterone, and 5alpha-dihydrotestosterone were ineffective. 17alpha-Estradiol showed a small but significant effect on curved tails. We conclude that P450aromB mRNA is a sensitive marker of XE effect during embryogenesis, but further studies are required to determine whether changes in neural aromatase expression and estrogen biosynthesis have consequences for CNS development.
Gelatinase A, also called matrix metalloproteinase 2 (MMP-2), belongs to the matrix metalloproteinase (MMP) family. MMP-2 cleaves type IV collagen, denatured collagen (gelatin), and other extracellular matrix (ECM) components. MMP-2 has been reported to be involved in a number of biological and pathological processes, but previous studies have not indicated that its expression is essential for early embryogenesis. In the current study, we have utilized zebrafish as a developmental model to study the role of MMP-2 during embryogenesis. We have successfully isolated a zebrafish MMP-2 (zMMP-2) homologue showing over 80% identity and over 90% similarity to its human counterpart. In situ analysis showed that zMMP-2 was expressed as early as the one-cell stage implying a maternal origin during oogenesis, and embryos continued to express zMMP-2 through at least the 72-h stage of development. RT-PCR analysis confirmed the in situ expression pattern and gelatin zymography indicated that a metalloproteinase with the same gel mobility as vertebrate MMP-2 was present in zebrafish embryos. Injection of zMMP-2 antisense morpholino oligonucleotides into 1- to 4-cell embryos resulted in a truncated axis, monitored through 72 h of development indicating that this metalloproteinase plays an important role in zebrafish embryogenesis. Monpholino-induced alterations in development began to be observed at 12 h of embryogenesis based on morphological and axis marker studies. The results obtained in zebrafish are in contrast to murine knockout studies that indicate that MMP-2 does not have a major role in mouse embryogenesis.
To determine the critical stage of zebrafish development where exposure to xenoestrogens can affect sex ratio and vitellogenin induction, zebrafish (Danio rerio) were exposed to 17alpha-ethinylestradiol (actual concentration 15.4+/-1.4 ng EE2/l) during early development: from fertilisation to hatch; hatch to 10 days post hatch (dph); 10-20 dph; 20-30 dph; 20-40 dph; 20-60 dph; fertilisation to 25 dph; or hatch to 60 dph. Vitellogenin was measured in whole body homogenate 30 dph by ELISA and sex ratio was determined 60 dph by histological examination of the gonads. All exposure periods significantly induced vitellogenin synthesis and affected the sex differentiation leading to development of ovo-testis or complete feminisation of the exposed fish depending on exposure period. Complete sex reversal was obtained in groups exposed from 20 to 60 dph and hatch to 60 dph. The half-life for degradation of vitellogenin was calculated. Juvenile zebrafish were exposed to 15.4+/-1.4 ng EE2/l (actual concentration) from fertilisation to 25 dph and transferred to clean water, after which weekly measurements of vitellogenin concentration in whole body homogenate were performed until day 46 post hatch. The half-life of vitellogenin was 2.4 days.