Inhibition of skiA and skiB gene expression ventralizes zebrafish embryos.
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Animals↗
Biomedical subjects
P B Hackett
Publications and source records attributed to P B Hackett.
Ectopic expression of c-ski disrupts gastrulation and neural patterning in zebrafish.
The c-ski proto-oncogene encodes a transcriptional regulator that has been implicated in the development of different tissues at different times during vertebrate development. We identified two novel paralogues of the c-ski gene family, skiA and skiB in zebrafish (Danio rerio). The skiA protein is maternal and ubiquitous while skiB is zygotic. Overexpression of SkiA or SkiB disrupted gastrulation and resulted in a dorsalized phenotype. In situ analyses suggested that overexpression of Ski leads to a slight expansion of dorsal-axial mesoderm, diminishment or loss of ventral mesoderm and radialization of dorsal neuroectoderm. The dorsalized phenotype could be rescued by the ventral specifying factor, BMP4. These results provide evidence that Ski proteins participate in dorsal-ventral specification of both neuroectoderm and mesoderm.
Amino Acid Sequence↗
Alternative transcriptional initiation and splicing define the translational efficiencies of zebrafish mRNAs encoding eukaryotic initiation factor 4E.
Translation initiation factor 4E (eIF4E) binds to the m7GTP cap structure of eukaryotic mRNAs and influences the overall rates of translation. The eIF4E protein is subject to regulation at a number of levels that allow it to modulate translation of maternal mRNAs in early embryos before the onset of zygotic transcription. In zebrafish eIF4E (zeIF4E) mRNA levels are elevated in specific tissues and at specific times during embryogenesis. We have characterized the organization of the zeIF4E gene to facilitate elucidation of the molecular mechanisms that influence its expression. The zeIF4E gene spans about 14 kb and like its human counterpart is comprised of seven exons. Alternative splicing between the first and second exon generates two mRNA splice-forms called SF1 and SF2. Nuclease-S1-protection and primer-extension reveal two zeIF4E transcriptional start-sites. Transcripts initiating from the distal start-site during oogenesis are exclusively SF1, while initiation from the proximal start-site generates both splice-forms. Although translation in vitro of SF1 mRNA gives rise to a protein consistent in mass with affinity-purified zeIF4E, SF2 mRNA does not. Instead, SF2 mRNA inhibits in vitro protein synthesis in a concentration-dependent manner, suggesting it functions as a translational attenuator. Thus, specific transcriptional activation from the distal start-site may provide a unique mechanism for transcriptional regulation of the levels, as well as the function of zeIF4E mRNAs.
Alternative Splicing↗
Short inverted-repeat transposable elements in teleost fish and implications for a mechanism of their amplification.
Angel is the first miniature inverted-repeat transposable element (MITE) isolated from fish. Angel elements are imperfect palindromes with the potential to form stem-loop structures in vitro. Despite sequence divergence of elements of up to 55% within and between species, their inverted repeat structures have been maintained, implying functional importance. We estimate that there are about 10(3)-10(4) Angels scattered throughout the zebrafish genome, evidence that this family of transposable elements has been significantly amplified over the course of evolution. Angel elements and Xenopus MITEs carry common sequence motifs at their termini, indicating common origin and/or related mechanisms of transposition. We present a model in which MITEs take advantage of the basic cellular mechanism of DNA replication for their amplification, which is dependent on the characteristic inverted repeat structures of these elements. We propose that MITEs are genomic parasites that transpose via a DNA intermediate, which forms by a folding-back of a single strand of DNA, that borrow all of the necessary factors for their amplification from products encoded in the genomes in which they reside. DNA polymorphisms in different lines of zebrafish were detected by PCR using Angel-specific primers, indicating that such elements, combined with other transposons in vertebrate genomes, will be useful molecular tools for genome mapping and genetic analyses of mutations.
Animals↗
Genetic applications of transposons and other repetitive elements in zebrafish.
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Animals↗
Position-independent expression of transgenes in zebrafish.
The variability in expression patterns of transgenes, caused by the influence of neighboring chromatin, is called 'position effect'. Border elements are DNA sequences, which have the ability to alleviate position effects. The abilities of two types of border elements, scs/scs' from the D. melanogaster 87A7 heat shock locus and the A-element from the chicken lysozyme gene, to protect transgenes from position effects were quantified in developing zebrafish embryos. The transgenic construct used was FV3CAT, which consists of the carp beta-actin transcriptional regulatory region, the chloramphenicol acetyltransferase (CAT) gene and the 3'-untranslated region from the Chinook salmon growth hormone gene. FV3CAT constructs flanked by either scs/scs'-elements or A-elements were introduced into zebrafish chromosomes and the spatial and temporal expression patterns of the transgenes were quantified in multiple generations of transgenic zebrafish. Levels of transgene expression were uniform in the pre-differentiated and fully differentiated populations of cells present during embryonic development. Levels of transgene expression were proportional to the numbers of integrated transgenes. Expression of transgenes per cell varied less than two-fold in different transgenic lines. Both types of border elements were able to prevent the influences of neighboring chromatin on transgene expression through three generations of fish. The results are consistent with the ability of border elements to function with equal efficiencies in the many cell types found in vertebrates. Thus, inclusion of border elements in genetic constructs can provide reliable and reproducible levels of gene expression in multiple lines of fish.
Animals↗
Overexpression of thyroid hormone receptor alpha 1 during zebrafish embryogenesis disrupts hindbrain patterning and implicates retinoic acid receptors in the control of hox gene expression.
Nuclear receptors play key roles in anterior/posterior (A/P) axis formation during vertebrate embryogenesis. Within this gene family, retinoic acid receptors and retinoic acid itself have profound influences on the establishment of the A/P axis. Thyroid hormone receptors are expressed during early periods of development, long before the establishment of the thyroid gland, and are able to interact with retinoic acid receptors. Here we examined the ability of the thyroid hormone receptor alpha 1 to affect early embryonic development by mRNA injection of either repressor or activator forms of the thyroid hormone receptor. Overexpression of either the thyroid hormone receptor alpha 1 or a constitutive repressor form, v-erbA, caused a swelling in the rostral hindbrain. These defects were associated with disorganization and loss of rhombomere borders as well as an increase in the number of acetylcholine esterase positive cells. This phenotype correlated with a reduction in hoxa1 expression during gastrulation. Furthermore, injection of either thyroid hormone receptor alpha 1 or v-erbA mRNA repressed a reporter gene that contained a retinoic acid response element, demonstrating the ability of the thyroid hormone receptor alpha 1 to repress retinoic acid signaling during gastrulation. In contrast, embryos treated with retinoic acid alone or embryos injected with thyroid hormone receptor alpha 1 and treated with the thyroid hormone analog TRIAC displayed a similar set of defects, including loss of the midbrain-hindbrain border and severe disruption of the rostral hindbrain. These studies support the involvement of retinoic acid and its receptors in the direct control of Hox gene expression and the early patterning of the zebrafish central nervous system.
Animals↗
Dynamic and tissue-specific expression of eIF4E during zebrafish embryogenesis.
The regulation of protein synthesis is critical to diverse cellular processes and plays a pivotal role in regulating gene expression during embryogenesis. The cap-binding protein eIF4E is a translational factor whose activity appears to be both ubiquitous and central to the regulation of protein synthesis in all cell-types. As a cell-cycle regulator, mesoderm inducer and proto-oncogene, the amount and activity of the translational factor eIF4E must be under strict control, but the range of its expression and its concentration as a function of position and time in the developing embryo are unknown. Consequently, we have initiated studies to elucidate the expression of the eIF4E gene and its role in the regulating embryonic development. We have cloned a zebrafish gene encoding eIF4E, zeIF4E, and measured its developmental expression. Unexpectedly, we found that the zeIF4E gene produces two alternatively spliced transcripts that potentially encode different forms of the initiation factor. Molecular analyses and in situ hybridization reveal a potential role for eIF4E in regulating protein synthesis during vertebrate oogenesis, gastrulation, and erythropoiesis. The dynamic and asymmetric expression of eIF4E during zebrafish embryogenesis reveals that this ostensibly general translation factor may act as a tissue-specific translational enhancer.
Alternative Splicing↗
Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells.
Members of the Tc1/mariner superfamily of transposons isolated from fish appear to be transpositionally inactive due to the accumulation of mutations. Molecular phylogenetic data were used to construct a synthetic transposon, Sleeping Beauty, which could be identical or equivalent to an ancient element that dispersed in fish genomes in part by horizontal transmission between species. A consensus sequence of a transposase gene of the salmonid subfamily of elements was engineered by eliminating the inactivating mutations. Sleeping Beauty transposase binds to the inverted repeats of salmonid transposons in a substrate-specific manner, and it mediates precise cut-and-paste transposition in fish as well as in mouse and human cells. Sleeping Beauty is an active DNA-transposon system from vertebrates for genetic transformation and insertional mutagenesis.
Amino Acid Sequence↗
The zebrafish thyroid hormone receptor alpha 1 is expressed during early embryogenesis and can function in transcriptional repression.
Nuclear receptors are a large family of ligand dependent transcription factors which participate in many diverse processes during development. In this report, we describe the cloning of the zebrafish thyroid hormone receptor alpha 1 (TR alpha 1) gene, the cellular counterpart of the viral oncogene v-erbA. TR alpha 1 is expressed during oogenesis and maternally supplied to the embryo. TR alpha 1 is expressed again after the mid blastula transition. By examining the effects of increased expression of TR alpha 1 on expression of a reporter gene which responds to both TR alpha 1 and retinoic acid receptors (RARs), we show that the zebrafish TR alpha 1 can act as a repressor during early zebrafish development before thyroid hormone is present in the embryo. In addition, our data suggest that TR alpha 1 can repress retinoic acid (RA)-signaling during early development. We propose that TR alpha 1 functions during early development as a transcriptional repressor, similar to the constitutive repressor activity of its viral counterpart v-erbA, which regulates anterior-posterior (A/P) patterning by repressing RA-signaling.
Amino Acid Sequence↗
Repetitive elements and their genetic applications in zebrafish.
Repetitive elements provide important clues about chromosome dynamics, evolutionary forces, and mechanisms for exchange of genetic information between organisms. Repetitive sequences, especially the mobile elements, have many potential applications in genetic research. DNA transposons and retroposons are routinely used for insertional mutagenesis, gene mapping, gene tagging, and gene transfer in several model systems. Once they are developed for the zebrafish, they will greatly facilitate the identification, mapping, and isolation of genes involved in development as well as the investigation of the evolutionary processes that have been shaping eukaryotic genomes. In this review repetitive elements are characterized in terms of their lengths and other physical properties, copy numbers, modes of amplification, and mobilities within a single genome and between genomes. Examples of how they can be used to screen genomes for species and individual strain differences are presented. This review does not cover repetitive gene families that encode well-studied products such as rRNAs, tRNAs, and the like.
Animals↗
Expression of zebrafish connexin43.4 in the notochord and tail bud of wild-type and mutant no tail embryos.
Communication via gap junctions provides a mechanism for the cell-cell transfer and coordination of developmental signals. The spatial restriction of gap junctions may also serve to organize cells into domains of coordinated behavior. To investigate the role of gap junctions during embryogenesis, we have characterized the expression of a member of the gap junction gene family, zebrafish connexin43.4, a homolog of connexin45 in chicken and mammals. Expression of connexin43.4 was induced in the early gastrula, coincident with the first definitive assignments of axial cell fate and the onset of the cell movements comprising convergence and extension in zebrafish. In situ hybridization and immunohistochemistry revealed that during gastrulation connexin43.4 mRNA and protein were progressively enriched in the germ ring and in the notochord primordia on the dorsal side of the embryo. Later in development connexin43.4 expression was detected in the notochord, the paraxial mesoderm, and the tail bud but was not observed after the differentiation of these tissues. In no tail mutant embryos which are defective in tail formation and proper morphogenesis of the notochord, connexin43.4 expression was absent during gastrulation from the caudal embryonic shield and notochord primordia. During somite stages in no tail embryos, connexin43.4 expression remained absent in the notochordal precursor cells and was lost in the tail bud. Thus, the no tail gene product, a transcription factor, was required for the expression of connexin43.4 in both the notochord and tail bud during morphogenesis. By microinjection of mRNA coding for a connexin43.4/green fluorescent protein fusion in the 1-cell zebrafish embryo, we showed that connexin43.4 is capable of assembling into structures reminiscent of gap junctions. The progressively restricted, developmental expression of the zebrafish connexin43.4 gene suggests that this gap junctional protein participates in the coordination of gastrulation and the formation of the notochord and tail.
Animals↗
Identification of functional domains and evolution of Tc1-like transposable elements.
Tc1-like transposable elements from teleost fish have been phylogenetically examined to determine the mechanisms involved in their evolution and conserved domains of function. We identified two new functional domains in these elements. The first is a bipartite nuclear localization signal, indicating that transposons can take advantage of the transport machinery of host cells for nuclear uptake of their transposases. The second is a novel combination of a paired domain-related protein motif juxtaposed to a leucine zipper-like domain located in the putative DNA-binding regions of the transposases. This domain coexists with a special inverted repeat structure in certain transposons in such phylogenetically distant hosts as fish and insects. Our data indicate that reassortment of functional domains and horizontal transmission between species are involved in the formation and spread of new types of transposable elements.
Amino Acid Sequence↗
DANA elements: a family of composite, tRNA-derived short interspersed DNA elements associated with mutational activities in zebrafish (Danio rerio).
DNA is the first SINE isolated from zebrafish (Danio rerio) exhibiting all the hallmarks of these tRNA-derived elements. DANA is unique in its clearly defined substructure of distinct cassettes. In contrast to generic SINE elements, DANA appears to have been assembled by insertions of short sequences into a progenitor, tRNA-derived element. Once associated with each other, these subunits were amplified as a new transposable element with such a remarkable success that DANA-related sequences comprise approximately 10% of the modern zebrafish genome. At least some of the sequences comprised by the full-length element were capable of movement, forming a new group of mobile, composite transposons, one of which caused an insertional mutation in the zebrafish no tail gene. Being present only in the genus Danio, and estimated to be as old as the genus itself, DANA may have played a role in Danio speciation by massive amplification and genome-wide dispersion. There are extensive DNA polymorphisms between zebrafish populations and strains detected by PCR amplification using primers specific to DANA, suggesting that the DANA element will be useful as a molecular tool for genetic and phylogenetic analyses.
Animals↗
Genetic engineering of Minnesota superfish.
There is a chronic need to develop growth-enhanced fish for aquaculture. To meet this need we have developed techniques for genetically engineering fish to grow larger and faster. We found that the major difficulty in genetically engineering fish is the extremely high rate of mosaicism due to the late integration of transgenes into the genome. This delay also reduces the chances of passage of the transgene through the germ line. Consequently, we have engineered new vectors and mechanisms for accelerating the rate of integration of exogenous DNA into fish chromosomes.
Animals↗
Autogenous regulation of RNA translation and packaging by Rous sarcoma virus Pr76gag.
Unspliced cytoplasmic retroviral RNA in chronically infected cells either is encapsidated by Gag proteins in the manufacture of virus or is used to direct synthesis of Gag proteins. Several models have been suggested to explain the sorting of viral RNA for these two purposes. Here we present evidence supporting a simple biochemical mechanism that accounts for the routing of retroviral RNA. Our results indicate that ribosomes compete with the Gag proteins to determine the fate of nascent retroviral RNA. Although the integrity of the entire Rous sarcoma virus leader sequence is important for retroviral packaging and translation, the RNA structure around the third small open reading frame, which neighbors the psi site required for packaging of the RNA, is particularly critical for maintenance of the balance between translation and packaging. These results support the hypothesis that Gag proteins autogenously regulate their synthesis and encapsidation of retroviral RNA and that an equilibrium exists between RNA destined for translation and packaging that is based on the intracellular levels of Gag proteins and ribosomes. To test the model, mRNAs with natural or mutated 5' leader sequences from Rous sarcoma virus were expressed in avian cells in the presence and absence of Pr76gag. We demonstrate that Pr76gag acts as a translational repressor of these mRNAs in a dose-dependent manner, supporting the hypothesis that Pr76gag can sort retroviral RNA for translation and encapsidation.
Animals↗
Development of cellular resistance to pp60v-src kinase-induced cell death.
The v-src gene of Rous sarcoma virus (RSV) encodes pp60v-src, a tyrosine kinase that can initiate cellular transformation. High levels of v-src gene expression can either be cytotoxic or the cause of altered expression of cellular genes. Examination of cytotoxic thresholds is difficult because cells expressing high levels of a cytotoxic oncogene will die. To evaluate quantitatively the cytotoxicity of pp60v-src on growth, we amplified two different v-src genes, under the control of the human hsp70B heat shock promoter to establish cell clones with varying copy numbers of the heat-inducible v-src gene. The viability of cell lines over a prolonged period of time depended on the particular src gene, the expression of v-src mRNA, synthesis of the pp60v-src protein and, most importantly, the tyrosine kinase activity of the pp60v-src protein. We found a relatively sharp threshold in v-src-encoded tyrosine kinase activity above which cell viability rapidly declines. However, over time, tyrosine kinase activity was exponentially suppressed at about a 10-fold higher rate than pp60v-src protein during passage. Our results indicate that homeostasis of tyrosine phosphorylation is important for cell viability, that perturbation of this balance results in cell mortality, and that cells can evolve to accommodate overexpression of oncogene by downregulating the level of tyrosine kinase activity.
Animals↗
Characterization of a Tc1-like transposable element in zebrafish (Danio rerio).
We have characterized Tdr1, a family of Tc1-like transposable elements found in the genome of zebrafish (Danio rerio). The copy number and distribution of the sequence in the zebrafish genome have been determined, and by these criteria Tdr1 can be classified as a moderately repetitive, interspersed element. Examination of the sequences and structures of several copies of Tdr1 revealed that a particular deletion derivative, 1250 bp long, of the transposon has been amplified to become the dominant form of Tdr1. The deletion in these elements encompasses sequences encoding the N-terminal portion of the putative Tdr1 transposase. Sequences corresponding to the deleted region were also detected, and thus allowed prediction of the nucleotide sequence of a hypothetical full-length element. Well conserved segments of Tc1-like transposons were found in the flanking regions of known fish genes, suggesting that these elements have a long evolutionary history in piscine genomes. Tdr1 elements have long, 208 bp inverted repeats, with a short DNA motif repeated four times at the termini of the inverted repeats. Although different from that of the prototype C. elegans transposon Tc1, this inverted repeat structure is shared by transposable elements from salmonid fish species and two Drosophila species. We propose that these transposons form a subgroup within the Tc1-like family. Comparison of Tc1-like transposons supports the hypothesis that the transposase genes and their flanking sequences have been shaped by independent evolutionary constraints. Although Tc1-like sequences are present in the genomes of several strains of zebrafish and in salmonid fishes, these sequences are not conserved in the genus Danio, thus raising the possibility that these elements can be exploited for gene tagging and genome mapping.
Amino Acid Sequence↗