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M Westerfield

Publications and source records attributed to M Westerfield.

At least 19 recordsLinked to original sources
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.

Animals↗
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.

Animals↗
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.

Animals↗
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.

Animals↗
PubMed1990

Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression.

To study the frequency of germ-line transformation and to examine the reproducibility of tissue-specific transgene expression, we produced several lines of transgenic zebrafish expressing a recombinant chloramphenicol acetyltransferase (CAT) gene. Supercoiled plasmids containing both Rous sarcoma virus and SV-40 promoter sequences upstream of the CAT coding region were injected into zebrafish embryos prior to first cleavage. CAT activity could be detected in batches of injected embryos as early as 8 h and up to at least 12 days post-fertilization. Approximately 18% of injected fish raised to maturity exhibited CAT activity in their fins, and approximately 5% of injected fish became stable germ-line transformants. Breeding studies indicated that although transgenic founder fish were frequently germ-line mosaics, transgenic individuals of subsequent generations were fully hemizygous for the transgene marker. The transgenes present in the F1 progeny of four independent lines were relatively well expressed in fin and skin, while lower levels of expression were observed in heart, gill and muscle. Little or no CAT expression was observed in the brain, liver and gonad. A monoclonal antibody directed against the CAT gene product consistently revealed variegated patterns of CAT expression in ectodermally derived fin epidermal cells in three of these lines. These results show that it is possible to efficiently produce stable germ-line transformants of the zebrafish and to observe reproducible tissue-specific patterns of transgene expression in this organism. Possible mechanisms for the variegated expression observed within tissues are also considered.

Animals↗
PubMed1990

The formation of terminal fields in the absence of competitive interactions among primary motoneurons in the zebrafish.

To make specific synaptic connections, projection neurons extend neurites to regions containing appropriate targets, then form synapses with the correct type and number of target cells. To investigate the mechanisms controlling this process, we have studied the formation of motoneuronal terminal fields in live zebrafish embryos. The primary motoneurons of the zebrafish are identifiable as individuals and innervate neighboring but mutually exclusive territories. To study the first week of their development, which includes embryonic and early larval stages, we labeled identified motoneurons with fluorescent dyes and made sequential observations of the axonal branches of individual neurons. We assessed the roles of competitive interactions and synapse elimination in the formation of specific synapses by identified neurons that innervate neighboring territories. Our results demonstrate that primary motoneurons establish their cell-specific terminal fields primarily by directed outgrowth of branches and formation of neuromuscular junctions almost exclusively on appropriate muscle fibers, rather than by overproduction and selective elimination of inappropriate branches. Retraction of the few branches that are inappropriately placed, though correlated in time with the ingrowth of branches from appropriate motoneurons, occurs independently of the influences of these other cells and when neuromuscular transmission is blocked. We suggest that, similar to the way in which they pioneer peripheral nerve pathways, primary motoneurons establish their cell-specific terminal fields using mechanisms that operate independently of activity and competition. The target or substrate interactions that are likely to instruct directed growth-cone navigation may be similar to the interactions that determine the locations of territorial borders and that instruct the retraction of misplaced branches.

Animals↗
PubMed1978

A numerical method to model excitable cells.

We have extended a fast, stable, and accurate method for the numerical solution of cable equations to include changes in geometry and membrane properties in order to model a single excitable cell realistically. In addition, by including the provision that the radius may be a function of distance along an axis, we have achieved a general and powerful method for simulating a cell with any number of branched processes, any or all of which may be nonuniform in diameter, and with no restriction on the branching pattern.

Action Potentials↗
PubMed1978

Temperature-sensitive conduction failure at axon branch points.

1. The propagation of action potentials through the branching regions of squid axons was examined experimentally and with computer simulations over a temperature range of 5-25 degrees C. 2. Above a critical ratio of postbranch to prebranch diameters, propagation of an action potential failed. The value of this critical ratio is very sensitive to temperature and is smaller at high temperatures. The experimentally measured Q10 of the critical ratio is 0.37 +/- 0.04. 3. Evaluation of a number of parameters of action-potential propagation showed that this effect is closely related to the change in the width of the action potential with temperature (Q10 = 0.29 +/- 0.01).

Action Potentials↗
PubMed1977

How Gymnodinium breve red tide toxin(s) produces repetitive firing in squid axons.

Partially purified toxin(s), GbTX, extracted from Gymnodinium breve red tide organisms elicits a spontaneous train of action potentials in the squid giant axon. The spikes have a shape similar to that in the normal seawater control except for an increase in the rate of recovery from the afterhyperpolarization. With this more rapid recovery, the membrane potential overshoots the resting potential and threshold, triggers another spike, and thus produces repetitive firing. Voltage-clamp studies revealed that the toxin has no effect on the normal sodium or potassium conductance changes produced by step depolarization. However, consistent with the faster recovery after an action potential, GbTX speeds recovery of the "shut-off" currents to their steady-state values after a depolarization. The most likely mechanism by which the toxin accelerates recovery after an action potential (leading to repetitive firing) is the induction of a small additional inward current which was found to be reduced by prehyperpolarization. This toxin-induced current which speeds recovery is blocked by tetrodotoxin and hence presumably flows through the sodium channel.

Action Potentials↗