Biomedical subjects

M Westerfield

Publications and source records attributed to M Westerfield.

At least 37 records · Page 2Linked to original sources

PubMed1996

Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation.

We have examined the development of specific muscle fiber types in zebrafish axial muscle by labeling myogenic precursor cells with vital fluorescent dyes and following their subsequent differentiation and fate. Two populations of muscle precursors, medial and lateral, can be distinguished in the segmental plate by position, morphology and gene expression. The medial cells, known as adaxial cells, are large, cuboidal cells adjacent to the notochord that express myoD. Surprisingly, after somite formation, they migrate radially away from the notochord, becoming a superficial layer of muscle cells. A subset of adaxial cells develop into engrailed-expressing muscle pioneers. Adaxial cells differentiate into slow muscle fibers of the adult fish. We have named the lateral population of cells in the segmental plate, lateral presomitic cells. They are smaller, more irregularly shaped and separated from the notochord by adaxial cells; they do not express myoD until after somite formation. Lateral presomitic cells remain deep in the myotome and they differentiate into fast muscle fibers. Thus, slow and fast muscle fiber types in zebrafish axial muscle arise from distinct populations of cells in the segmental plate that develop in different cellular environments and display distinct behaviors.

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PubMed1995

Differential induction of four msx homeobox genes during fin development and regeneration in zebrafish.

To study the genetic regulation of growth control and pattern formation during fin development and regeneration, we have analysed the expression of four homeobox genes, msxA, msxB, msxC and msxD in zebrafish fins. The median fin fold, which gives rise to the unpaired fins, expresses these four msx genes during development. Transcripts of the genes are also present in cells of the presumptive pectoral fin buds. The most distal cells, the apical ectodermal ridge of the paired fins and the cleft and flanking cells of the median fin fold express all these msx genes with the exception of msxC. Mesenchymal cells underlying the most distal cells express all four genes. Expression of the msx genes in the fin fold and fin buds is transient and, by 3 days after fertilization, msx expression in the median fin fold falls below levels detectable by in situ hybridization. Although the fins of adult zebrafish normally have levels of msx transcripts undetectable by in situ hybridization, expression of all four genes is strongly reinduced during regeneration of both paired and unpaired fins. Induction of msx gene expression in regenerating caudal fins occurs as early as 30 hours postamputation. As the blastema forms, the levels of expression increase and reach a maximum between the third and fifth days. Then, msx expression progressively declines and disappears by day 12 when the caudal fin has grown back to its normal size. In the regenerating fin, the blastema cells that develop at the tip of each fin ray express msxB and msxC. Cells of the overlying epithelium express msxA and msxD, but do not express msxB or msxC. Amputations at various levels along the proximodistal axis of the fin suggest that msxB expression depends upon the position of the blastema, with cells of the rapidly proliferating proximal blastema expressing higher levels than the cells of the less rapidly proliferating distal blastema. Expression of msxC and msxD is independent of the position of the blastema cell along this axis. Our results suggest distinct roles for each of the four msx genes during fin development and regeneration and differential regulation of their expression.

Amino Acid Sequence↗

PubMed1994

Autonomous expression of the nic1 acetylcholine receptor mutation in zebrafish muscle cells.

The nic1b107 (nic1) mutation blocks expression of both functional and clustered acetylcholine receptors (AChRs) in zebrafish muscle. Normally, signaling between motoneurons and muscles regulates AChR clustering. To learn if signaling is affected and to identify the primary cellular target of the nic1 mutation, we made mosaic embryos by transplanting motoneurons and muscle precursors from wild-type to mutant embryos. Genotypically mutant muscle cells fail to cluster AChRs even when contacted by wild-type motoneurons, whereas genotypically mutant motoneurons induce AChR clustering on wild-type muscle cells. Moreover, mutant muscle cells fail to cluster AChRs under culture conditions that induce AChR clustering on wild-type cells. We conclude that the nic1 mutation acts autonomously in muscle cells rather than by affecting signaling between motoneurons and muscle. The wild-type nic1 gene is necessary in muscle for expression and clustering of AChRs.

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PubMed1994

Neural selective activation and temporal regulation of a mammalian GAP-43 promoter in zebrafish.

Neurons throughout the vertebrate nervous system selectively activate the gene for a growth cone component, GAP-43, during embryonic development, and then decrease its expression abruptly as they form synapses. Distal interruption of mature axons in the central nervous system (CNS) of fish and amphibians, but not in the mammalian CNS reverses the developmental down-regulation of GAP-43 expression. To explore functional conservation and divergence of cis-acting elements that regulate expression of the GAP-43 gene, we studied activation, in transgenic zebrafish embryos, of mammalian GAP-43 genomic sequences fused to a marker gene. The DNA fragments containing the GAP-43 promoter, including a short fragment of 386 base pairs, were preferentially activated in the embryonic fish nervous system at times when extensive neuronal differentiation and neurite outgrowth take place. After 2 days of development, expression of the mammalian transgenes was specifically downregulated in the fish spinal cord but increased in more rostral regions of the CNS. This expression pattern was well correlated with the regulation of the endogenous fish GAP-43 gene revealed by in situ hybridization. Elements of the mammalian gene located a substantial distance upstream of the minimal promoter directed additional expression of the marker gene in a specific set of non-neural cells in zebrafish embryos. Our results indicate that cis-acting elements of the GAP-43 gene, and signaling pathways controlling these elements during embryonic development, have been functionally conserved in vertebrate evolution.

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PubMed1994

Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head.

We describe analysis of zebrafish distal-less-related homeobox genes that may serve as specifiers of positional information in anterior regions of the CNS and in peripheral structures. We isolated three zebrafish genes, dlx2, dlx3, and dlx4, by screening embryonic cDNA libraries. Comparisons of the predicted sequences of the Dlx2, Dlx3, and Dlx4 proteins with distal-less proteins from other species suggest that vertebrate distal-less genes can be divided into four orthologous groups. We observed similarities but also unique features of the expression patterns of the zebrafish dlx genes. Among the three genes, dlx3 alone is expressed during gastrulation. Shortly after gastrulation, cells in the ventral forebrain rudiment express dlx2 and dlx4, but not dlx3, and hindbrain neural crest cells express only dlx2. Presumptive precursor cells of the olfactory placodes express dlx3 and dlx4 but not dlx2. Transcripts of dlx3 and dlx4 are present in overlapping subsets of cells in the auditory vesicle and in cells of the median fin fold, whereas dlx2 is never expressed in the auditory vesicle and only at low levels in localized regions of the median fin fold. Cells of the visceral arches and their primordia express all three dlx genes, but with different developmental time courses. We suggest that combinatorial expression of the dlx genes is part of a homeobox gene code specifying pattern formation or cell fate determination in the forebrain, in peripheral structures of the head, and in the fins.

Amino Acid Sequence↗

PubMed1993

Monkey P3 in an "oddball" paradigm: pharmacological support for multiple neural sources.

Two experiments examined the effects of the alpha-2 antagonist L657,743 on monkey electroencephalographic (EEG) and event-related potential (ERP) activity following auditory and visual stimuli. In the first experiment, EEG was recorded before and after the administration of a saline placebo or L657,743 (0.01, 0.05 mg/kg IM). No drug effects occurred on EEG spectral frequencies. In the second experiment, ERPs were recorded during an 80-10-10 passive oddball paradigm. The paradigm consisted of a repetitive tone as background, a different-pitch tone as the auditory oddball, and a yellow rectangle as an infrequent visual stimulus. Waveforms were collected before and after the administration of placebo or drug (0.01, 0.03, 0.05 mg/kg IM). Both auditory oddball and visual stimuli elicited large P3-like potentials with different distributions, suggesting different sources. However, their magnitudes were similarly reduced by drug administration. These effects were significant at parietal but unchanged at other sites. The data support the hypothesis that norepinephrine is a modulatory neurotransmitter common to auditory and visual P3s in parietal areas. On the other hand, midline and temporal P3s are unaffected by manipulations of noradrenergic activity, supporting the existence of two independent P3 sources.

Acoustic Stimulation↗

PubMed1992

Identification of two families of satellite-like repetitive DNA sequences from the zebrafish (Brachydanio rerio).

To further our understanding of the structure and organization of the zebrafish genome, we have undertaken the analysis of highly and middle-repetitive DNA sequences. We have cloned and sequenced two families of tandemly repeated DNA fragments. The monomer units of the Type I satellite-like sequence are 186 bp long, A+T-rich (65%), and exhibit a high degree of sequence conservation. The Type I satellite-like sequence constitutes 8% of the zebrafish genome, or approximately 8 x 10(5) copies per haploid genome. Southern analysis of genomic DNA, digested with several restriction endonucleases, shows a ladder of hybridizing bands, consistent with a tandem array, and suggests longer range periodic variations in the sequence of the tandem repeats. The Type II satellite has a monomer length of 165 bp, is also A+T-rich (68%), and constitutes 0.2% of the zebrafish genome (22,000 copies per haploid genome). Southern analysis reveals a complex pattern rather than a ladder of regularly spaced hybridizing bands.

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PubMed1992

Regional expression of three homeobox transcripts in the inner ear of zebrafish embryos.

The inner ear of all jawed vertebrates arises from the epithelium of the otic vesicle and contains three semicircular canals, otoliths, and sets of sensory neurons, all positioned precisely within the cranium to detect head orientation and movement. The msh-C gene and two new homebox genes, msh-D and a gene related to distal-less, dlx-3, are each expressed in distinct regions of the otic vesicle during its early development in zebrafish embryos. Cells in the ectoderm express dlx-3 before induction of the otic vesicle, suggesting that dlx-3 has an early function in this process. Later, cells aligned with the future axes of the semicircular canals specifically express either dlx-3 or msh-D. Even later, sensory hair cells express msh-C and msh-D, while other cells of the epithelium express dlx-3. The early expression of these genes could specify the orientation and morphogenesis of the inner ear, whereas their later expression could specify the fates of particular cell types.

Amino Acid Sequence↗

PubMed1992

Comparative analysis of Pax-2 protein distributions during neurulation in mice and zebrafish.

Members of different vertebrate species share a number of developmental mechanisms and control genes, suggesting that they have similar genetic programs of development. We compared the expression patterns of the Pax-2 protein in Mus musculus and Brachydanio rerio to gain a better understanding of the evolution of developmental control genes. We found that the tissue specificity and the time course of Pax-2 expression relative to specific developmental processes are remarkably similar during the early development of the two organisms. The brain, the optic stalk, the auditory vesicle, the pronephros, and single cells in the spinal cord and the hindbrain express Pax-2 in both species. The Pax-2 expression domain in the prospective brain of E8 mouse embryos has not been described previously. Expression appears first during early neurulation at the junction between the midbrain and hindbrain. However, there are some differences in Pax-2 expression between the two species. Most notable, expression at the midbrain/hindbrain boundary is no longer detectable after E11 in the mouse. Using monoclonal antibodies, we could exclude that primary neurons express Pax-2 in the zebrafish spinal cord. Our results confirm that Pax genes are highly conserved both in sequences and in expression patterns, indicating that they may have a function during early development that has been conserved during vertebrate evolution.

Animals↗

PubMed1992

Motor axon pathfinding.

Recent studies have provided a significant advance in our understanding of motor axon pathfinding. In particular, sources of guidance cues and some of the mechanisms used by motor growth cones during pathfinding have been identified.

Animals↗

PubMed1992

Identification of polymorphic simple sequence repeats in the genome of the zebrafish.

The zebrafish has drawn a great deal of attention as a developmental system because it offers the ability to combine excellent embryology and genetics. Here, we report that simple sequence repeats are abundant in the zebrafish genome and are highly polymorphic between two outbred lines, making them useful markers for the construction of a genetic map of this organism.

Animals↗

PubMed1992

Specific activation of mammalian Hox promoters in mosaic transgenic zebrafish.

Homeo box-containing genes (Hox) are expressed in restricted regions of vertebrate embryos and may specify positional information. The organization and expression patterns of these genes are highly conserved among different species, suggesting that their regulation may also have been conserved. We developed a transient expression system, using mosaically transgenic zebrafish, which allows rapid analysis of transgene expression, and examined the activities of two mammalian Hox genes, mouse Hox-1.1 and human HOX-3.3. We found that these Hox promoters are activated in specific regions and tissues of developing zebrafish embryos and that this specificity depends upon the same regulatory elements within the promoters that specify the spatial expression of these genes in mice. Our results suggest that the promoter activities have been remarkably conserved from fish to mammals. To study the regulation of Hox expression in the developing nervous system, we analyzed the promoter activities in spt-1 mutants that have a mesodermal deficiency. Our results suggest that interactions, probably with the paraxial mesoderm, differentially regulate the activities of Hox promoters in the developing nervous system.

Animals↗

PubMed1992

Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice.

Despite obvious differences in the patterns of early embryonic development, vertebrates share a number of developmental mechanisms and control genes, suggesting that they use similar genetic programs at some stages of development. To examine this idea, we isolated and characterized one such gene, pax-6, a member of the pax gene family, from the zebrafish Brachydanio rerio and determined the evolutionary conservation in the structure and expression of this gene by comparison to its homolog in mice. We found two alternatively spliced forms of the zebrafish pax-6 message. Sequence and expression pattern of the zebrafish pax-6 gene are remarkably similar to its murine homolog. pax-6 expression begins during early neurulation. A stripe of cells in the neuroectoderm, including the prospective diencephalon and a part of the telencephalon, expresses pax-6 as well as the hindbrain and the ventral spinal cord extending from the level of the first rhombomere to the posterior end of the CNS. During later development more limited regions of the brain including the eye, the olfactory bulb and the pituitary gland express pax-6. Cells at the midbrain-hindbrain junction express eng genes and are separated from the neighboring pax-6 regions by several cells that express neither gene, indicating a complex subdivision of this region. pax-6 expression appears during processes when cell-to-cell signalling is thought to be important, for example during induction of the eye and regionalization of the spinal cord and brain, suggesting that it may be one component mediating the response to inductive interactions.

Amino Acid Sequence↗

PubMed1992

Coordinate embryonic expression of three zebrafish engrailed genes.

We have identified three genes, expressed in zebrafish embryos, that are members of the engrailed gene family. On the basis of sequence comparisons and analyses of their expression patterns, we suggest that two of these genes, eng2 and eng3, are closely related to the En-2 gene of other vertebrates. The third gene, eng1, is probably the zebrafish homolog of En-1. Subsets of cells at the developing junction between the midbrain and hindbrain express three different combinations of these genes, revealing a previously unknown complexity of this region of the CNS. Other cells, for example, jaw and myotomal muscle precursors, express two of the three genes in combinations which, in the myotomal muscles, change during development. Cells in the developing hindbrain and fins express only a single engrailed gene. We propose that the fates and patterning of these cells may be regulated by the coordinate expression of particular combinations of these closely related homeoproteins.

Amino Acid Sequence↗

PubMed1992

Clustering of muscle acetylcholine receptors requires motoneurons in live embryos, but not in cell culture.

Previous culture studies have demonstrated that muscle cells autonomously express and cluster ACh receptors (AChRs) and that contact by neurites induces a reorganization of these clusters. We studied these phenomena in zebrafish embryos where the same cells could be examined in vivo and in vitro, and where contacts between cells could be viewed repeatedly. Receptor clusters first appeared when the pioneer growth cones emerged from the spinal cord, were always associated with labeled branches, and developed normally in the presence of neuromuscular transmission blockers. When motoneurons were removed, the muscles failed to cluster receptors. In contrast, muscle cells grown in cell culture uncontacted by nerves clustered AChRs. Our results suggest that clustering of AChRs in living embryos is induced by the presence of neurites and does not occur in the absence of neuronal contact. We suggest that conditions in cell culture, which differ from those in the intact embryo, induce clusters on isolated muscle cells. Moreover, our results demonstrate that receptors cluster without binding transmitter and in the absence of neuronal activity.

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PubMed1991

Diversity of expression of engrailed-like antigens in zebrafish.

We have studied developmental expression of zebrafish engrailed-like (Eng) antigens. Many cell types are reproducibly labeled by two antibodies that recognize the Eng homeodomain, but other cells are labeled by only one or the other, suggesting a hitherto unrecognized complexity of Eng proteins. Expression patterns vary remarkably according to cell type and location. In the undifferentiated primordia of the brain and of each myotome, expression by a stripe of cells spatially subdivides the primordium at a location where a morphological boundary forms later, suggesting expression may be required for development of the boundaries. Supporting this hypothesis, trunk myotomal cells that express Eng are absent in spt-1 mutant embryos, just where the myotomal boundaries fail to form. Another pattern is present in rhombomeres, pharyngeal arches, and the pectoral girdle. In each of these cases, cells (neuron, muscle, cartilage) generating a subset of a series of repeated elements selectively express Eng. These subsets then form specialized derivatives, suggesting Eng homeoproteins are involved in determining the specializations. Epidermal expression is present in the ventral half of the pectoral fin rudiment, precisely 'compartmentalizing' the fin. Neuronal cells at a certain dorsoventral level in each hindbrain and spinal cord segment selectively express Eng, suggesting segmental control of neuronal identity. Specific expression patterns are observed in taste buds, otic vesicles and teeth. Thus we propose that eng genes function in diverse cell types in zebrafish, but play selector roles that can be classified into a few basic types.

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PubMed1990

Pathfinding and synapse formation in a zebrafish mutant lacking functional acetylcholine receptors.

We induced and characterized a recessive lethal mutation, nic-1, in zebrafish that blocks the function of muscle acetylcholine (ACh) receptors. Homozygous nic-1 embryos are nonmotile and fail to respond to exogenous application of cholinergic agonists, although their muscles contract in response to direct electrical stimulation. Moreover, we do not detect cell surface labeling by alpha-bungarotoxin or monoclonal antibodies that recognize the other three subunits of ACh receptors. Motoneurons, however, establish morphologically normal patterns of innervation and normal neuromuscular junctions. We suggest that neither transmitter-mediated nerve signaling nor any other aspect of ACh receptor function is required for the formation of appropriate nerve connections in this system.

Animals↗

PubMed1990

Mutations affecting skeletal muscle myofibril structure in the zebrafish.

We describe embryonic lethal mutations in the zebrafish, Brachydanio rerio, which affect organization of skeletal muscle myofibrils. The mutations, fub-1(b45) and fub-1(b126), were independently isolated from progeny of gamma-irradiated females. Each segregates as a single recessive gene: b45 is located about 23 map units from its centromere. The b126 mutation has a similar but slightly larger apparent gene-centromere distance and a less severe phenotype. The two mutations fail to complement, suggesting that they are allelic. Homozygous b45 mutant embryos are paralyzed, and their axial skeletal muscle cells are unstriated, containing severely disorganized myofibrillar components. Gel-electrophoretic comparisons of b45 mutant and wild-type muscle proteins failed to reveal absent or altered major myofibrillar proteins. Embryos genetically mosaic for b45 were also phenotypically mosaic, suggesting that the defect is cell-autonomous. We suggest that these mutations identify a gene required for proper organization of skeletal muscle myofibrils, and that the more severe mutation may represent a null allele.

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