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M Westerfield

Publications and source records attributed to M Westerfield.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1989

Early expression of acetylcholinesterase activity in functionally distinct neurons of the zebrafish.

The first expression and distribution of acetylcholinesterase (AChE) activity was studied among a distinct population of early neurons in embryonic zebrafish by using histochemical and retrograde labeling techniques. AChE first appeared in the nervous system in the primary motoneurons of the rostral spinal cord when the embryo had nine somites, approximately 14 hours postfertilization. Subsequent expression of AChE activity in the spinal cord proceeded in a rostral-to-caudal sequence. Cranial neurons expressed AChE activity shortly after it appeared in the rostral spinal cord. Several hours later, near the end of the first day, primary neurons in the hind-brain and spinal cord all contained AChE, including sensory neurons, reticulospinal interneurons, and primary motoneurons. AChE activity was also detected in the nucleus of the medial longitudinal fasciculus. Presumptive cranial ganglia transiently expressed AChE activity between 14 and 24 hours of development. These results, combined with previous observations that examined the time of origin and axogenesis of primary neurons, suggest that primary neurons in the embryonic zebrafish contain AChE before they sprout axons. The primary neurons appear to follow a common sequence of development consisting of a withdrawal from the cell division cycle, the expression of AChE, and axogenesis. Although this sequence is followed by all primary neurons, lack of a rostral-to-caudal sequence in the time of birth and variability in the time of axon outgrowth demonstrate that the relative timing of these three events is not rigidly programmed in individual neurons. Moreover, the very early expression of AChE in such diverse cell types suggests that it may have a developmental role in addition to its function in transmitter metabolism.

Acetylcholinesterase↗
PubMed1988

A neural degeneration mutation that spares primary neurons in the zebrafish.

We describe an embryonic lethal mutation in the zebrafish Brachydanio rerio that specifically affects the viability of most cells in the embryonic central nervous system (CNS). The mutation ned-1 (b39rl) was induced with gamma-irradiation and segregates as a single recessive allele closely linked to its centromere. It produces massive cell death in the CNS but a small set of specific neurons, including Rohon-Beard sensory neurons, large hindbrain interneurons, and primary motoneurons, survive embryogenesis and are functional. Synaptic connections between embryonic motoneurons and muscle cells appear physiologically normal, and the normally observed spontaneous flexions are present. Correlated with the presence of sensory neurons and interneurons, mutant embryos display reflexive movements in response to mechanical stimulation. Together, the surviving neurons, called primary neurons, form a class of cells that are prominent in size and arise early during development. Thus, this mutation may define a function that is differentially required by developmentally distinguishable sets of cells in the embryonic CNS.

Animals↗
PubMed1988

Function of identified motoneurones and co-ordination of primary and secondary motor systems during zebra fish swimming.

1. The activity of the two classes of motoneurones, primary and secondary, which innervate myotomal muscle fibres in the zebra fish, was monitored with electromyographic and intracellular techniques. 2. Simultaneous EMG and intracellular recordings from muscle fibres showed that the activity of the two motor systems and of individual primary motoneurones can be distinguished by recording EMG spikes during swimming. 3. Measurements of EMG spikes demonstrated that primary and secondary motoneurones are co-ordinately activated over a wide range of conditions during normal swimming. 4. During swimming the primary motoneurones within a given segment are usually co-activated although they sometimes fire independently. 5. When different primary motoneurones within a given segment are co-activated, they fire nearly synchronously. 6. We conclude that the primary motoneurones are used principally, although not exclusively, during fast swimming, struggling and the startle response, whereas secondary motoneurones function primarily during slower swimming.

Action Potentials↗
PubMed1988

Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo.

In the ventral hindbrain and spinal cord of zebrafish embryos, the first neurones that can be identified appear as single cells or small clusters of cells, distributed periodically at intervals equal to the length of a somite. In the hindbrain, a series of neuromeres of corresponding length is present, and the earliest neurones are located in the centres of each neuromere. Young neurones within both the hindbrain and spinal cord were identified in live embryos using Nomarski optics, and histochemically by labelling for acetylcholinesterase activity and expression of an antigen recognized by the monoclonal antibody zn-1. Among them are individually identified hindbrain reticulospinal neurones and spinal motoneurones. These observations suggest that early development in these regions of the CNS reflects a common segmental pattern. Subsequently, as more neurones differentiate, the initially similar patterning of the cells in these two regions diverges. A continuous longitudinal column of developing neurones appears in the spinal cord, whereas an alternating series of large and small clusters of neurones is present in the hindbrain.

Acetylcholinesterase↗
PubMed1988

Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos.

To generate stable lines of transgenic fish, early zebrafish embryos were injected with high concentrations of a linear bacterial plasmid. After injection, the foreign DNA was converted into a high molecular weight form and then amplified approximately tenfold during the initial rapid cleavages characteristic of the early embryo prior to gastrulation. While most of this DNA was subsequently degraded during gastrulation, some of the foreign sequences survived the gastrula stage and could be found in most of the injected fish at 3 weeks of age. Only about 5% of fish analysed 4 months after the injection retained foreign DNA in their fins, usually at less than one copy per cell. One of these fish was also found to contain about 100 copies per cell of foreign DNA in a fraction of its germ cells. Approximately 20% of the F1 offspring from this germ-line-positive parent inherited the foreign DNA, whereas 50% of F2 progeny obtained from an identified F1 individual inherited these sequences. The 50% transmission rate in F2 progeny was as expected for a single, heterozygous genomic insert. These observations indicate that injected DNA can be integrated into the fish genome, that the resulting transgenic fish are mosaic and that some of these mosaic individuals give rise to stable lines of transgenic fish.

Animals↗
PubMed1987

Substrate interactions affecting motor growth cone guidance during development and regeneration.

Most serious injuries of spinal nerves or roots in man and other higher vertebrates lead to permanent loss of control of skeletal muscles. In some cases this may be due to a failure of motor axons to regenerate, although even when functional neuromuscular connections are re-established, coordinated use of body and limb muscles may be absent. In both mammals and lower vertebrates, damaged motor axons usually regrow and reform functional connections with muscles, although these connections are often inappropriate. The selectivity of reinnervation is improved by maintaining alignment of the severed ends of the nerve. Thus, factors operating near the lesion site may direct regenerating motor axons into fascicles in the distal nerve stump that lead to inappropriate muscles. The identity of some of these factors is suggested by recent studies of developing systems which have shown that motor axons are directed in their growth. (a) The filopodia of their growth cones sample a limited region of the periphery. If motor growth cones extend too far from their normal pathways they establish connections with inappropriate muscles. (b) Motor growth cones normally extend into regions of embryos rich in the extracellular matrix molecule laminin, and avoid regions containing fibronectin. Moreover, motor growth cones extend on laminin but not on fibronectin substrates in vitro. In peripheral nerves, these two molecules are differentially distributed; laminin is expressed by Schwann cells in the endoneurium whereas fibronectin is expressed by fibroblasts primarily in the perineurium. These studies suggest that regenerating motor growth cones may be directed to appropriate muscles if their original fascicles within the distal nerve stump are within filopodial reach but may not be able to escape the fibronectin-rich perineurial sheath once directed into an inappropriate fascicle.

Animals↗
PubMed1986

Identified motoneurons and their innervation of axial muscles in the zebrafish.

The organization of spinal cord motoneurons and their innervation of axial (white) muscles in the zebrafish were studied. Motoneurons can be divided into 2 classes, primary and secondary, on the basis of their cell-body sizes and positions. Each side of each spinal segment contains 3 primary motoneurons that are uniquely identifiable as individuals by their stereotyped cell-body positions and peripheral branching patterns. Moreover, these motoneurons precisely innervate cell-specific subsets of contiguous muscle fibers in mutually exclusive regions of their own body segment. Individual muscle fibers receive inputs from a single primary motoneuron and, in addition, from up to 3 secondary motoneurons. The results demonstrate that the precision of innervation previously described in invertebrates is also present in some vertebrates.

Animals↗
PubMed1986

Development and axonal outgrowth of identified motoneurons in the zebrafish.

We have observed the development of live, fluorescently labeled motoneurons in the spinal cord of embryonic and larval zebrafish. There are 2 classes of motoneurons: primary and secondary. On each side of each spinal segment there are 3 individually identifiable primary motoneurons, named CaP, MiP, and RoP. The motoneurons of the embryo and larva are similar in morphology and projection pattern to those of the adult. During initial development, axons of primary motoneurons make cell-specific, divergent pathway choices and grow without error to targets appropriate for their adult functions. We observed no period of cell death, and except for one consistently observed case, there was no remodeling of peripheral arbors. We have observed a consistent temporal sequence of axonal outgrowth within each spinal segment. The CaP motor axon is the first to leave the spinal cord, followed by the axons of the other primary motoneurons. The Mauthner growth cone enters the spinal cord after all the primary motoneurons of the trunk spinal cord have begun axonal outgrowth. Secondary motor growth cones appear only after the Mauthner growth cone has passed by. Our results suggest that this stereotyped temporal sequence of axonal outgrowth may play a role in defining the contacts between the Mauthner axon and the motoneurons; the behavior of growth cones in the periphery suggests that interactions with the environment, not timing, may determine path-finding and peripheral connectivity of the motoneurons.

Animals↗
PubMed1985

The growth of motor axons in the spinal cord of Xenopus embryos.

The innervation of the myotomal muscles in the trunk region of Xenopus embryos has been examined to see how the path taken by motoneurons within the spinal cord is formed. The growth of motor axons has been studied by retrograde labeling with horseradish peroxidase and the growth of the spinal cord and myotomes has been studied by labeling with fluorescent beads. Results show that motoneurons initially innervate the nearest muscles. Then through a process of differential growth whereby the muscles elongate more than the spinal cord, the axonal terminals in the muscles become displaced caudally relative to their cell bodies. In this manner the central pathway taken by the motor axons develops after initial innervation of their peripheral targets.

Animals↗
PubMed1984

The absence of specific dye-coupling among frog spinal neurons.

A double fluorescence labeling technique was developed to study the specificity of dye-coupling among frog spinal neurons. A pool of motoneurons known to be electrically coupled was prelabeled with a large molecule (rhodamine conjugated to horseradish peroxidase) that was not expected to pass through gap junctions. Then a single sensory or motor neuron within or outside this pool was injected with lucifer yellow to see if the dye spread specifically among neurons that are electrically coupled. We observed almost no examples of specific dye-coupling.

Animals↗
PubMed1983

Selective reinnervation of limb muscles by regenerating frog motor axons.

The selectivity of reinnervation of limb muscles by the regenerating axons of adult, frog spinal cord motoneurons was determined. Reinnervation patterns were compared following axotomy when the ventral root was crushed or cut. Appropriate peripheral connections were reformed only after a nerve crush. The results suggest that selective reinnervation occurs if alignment of the nerve sheath is maintained.

Animals↗
PubMed1983

Action potential propagation and threshold parameters in inhomogeneous regions of squid axons.

The squid giant axon was used as a model system in which to determine the independent contributions of membrane excitability and diameter changes to threshold parameters and propagation of action potentials in inhomogeneous regions. The membrane excitability of a segment of an axon was altered by changes in the bathing solution, while its effective electrical diameter was increased by the insertion of a low-resistance axial wire. In computer simulations of these experiments, similar alterations were made in the membrane's conductance and axon's diameter. The inflexions in the shapes of action potentials propagating into a region with abrupt decreases in axial resistance become more pronounced when the interval between impulses was shortened. At short intervals, propagation of the second impulse failed. In contrast, reduction of membrane excitability produced inflexion-free changes in action potential shape and allowed a close-following second impulse to pass through the inhomogeneity. A combined decrease in membrane excitability and increase in diameter of the same region exaggerated the changes in action potential shape characteristic of the diameter increase alone. Threshold parameters were obtained from 'strength-duration' excitability relationships measured by injection of current at different points along the axon. When only the membrane excitability was reduced, threshold characteristics changed smoothly from one region of the nerve to another. In contrast, lowering the internal resistance or increasing the diameter in one region of a nerve lowered the time constant of excitation and the threshold for brief (relative to rheobasic) current stimuli in the small-diameter region near the transition while raising them in the larger-diameter region.

Action Potentials↗
PubMed1983

On the site of impulse initiation in a neurone.

In the preceding paper (Moore & Westerfield, 1983) the effects of changes in membrane properties and non-uniform geometry on impulse propagation and threshold parameters were investigated. In this paper the contributions of these and other parameters to the site of initiation of an impulse were determined by computer simulations using the Hodgkin-Huxley membrane description, the cable equations, and geometry appropriate for a simplified motoneurone with a non-myelinated axon. Antidromic invasion of action potentials into the soma was found to depend upon (a) the ionic channel rate constants (determined by the temperature), (b) the abruptness of the transition from the small-diameter axon to the larger diameter (and increased load) of the soma-dendrite, (c) extensions of active properties into the dendrite, and (d) density of ion channels. The location of the apparent site of initiation of impulses was not necessarily at the site of synaptic input nor the nearest active membrane. Its position depended upon (a) the fraction of the dendritic tree with excitable membrane, and secondarily on (b) the stimulus strength. Even with uniform excitability in the active membrane, the apparent site of initiation could be moved a considerable distance from the soma and the site of stimulation by appropriate choice of the various parameters noted above.

Action Potentials↗
PubMed1983

Development of sensory-motor synapses in the spinal cord of the frog.

The development and specificity of monosynaptic sensory-motor synapses were studied in the brachial spinal cord of bullfrog tadpoles. Intracellular and extracellular recordings were made from motoneurones innervating several different muscles of the forelimb. Excitatory synaptic potentials (e.p.s.p.s) were elicited by stimulation of various peripheral muscle nerves. Sensory and motor axons in the triceps brachii muscle nerves were electrically excitable at stage XIII, the earliest stage studied. Their conduction velocities were 0.2-0.4 m/s. These velocities increased during subsequent development so that by stage XXII they were approximately 5 m/s. Before stage XVII, synaptic potentials evoked in motoneurones by stimulation of the triceps sensory fibres had a long central latency and fatigued easily. These potentials were probably mediated polysynaptically. At stage XVII, the first short-latency triceps synaptic potentials appeared. They had central latencies of less than 3 ms and represented the direct, monosynaptic input from muscle sensory cells on to motoneurones. During subsequent development the percentage of triceps motoneurones innervated by triceps sensory fibres increased, while the number of long-latency polysynaptic inputs decreased. Both the electrical and chemical components, characteristic of these monosynaptic e.p.s.p.s in adult frogs, were prominent from the time the e.p.s.p.s first appeared. The pattern of innervation of brachial motoneurones by triceps sensory afferents was specific from the beginning. Triceps sensory fibres innervated most triceps motoneurones but very few subscapular or pectoralis motoneurones, just as in adult frogs. At no time were there appreciable numbers of 'aberrant' connexions. The developmental time course of several different classes of sensory-motor connexions was similar. Thus the synaptic specificity of this system cannot be explained by a differential timing of synaptogenesis.

Animals↗
PubMed1982

Calcium-activated potassium conductance noise in snail neurons.

Current fluctuations were measured in small, 3-6 micrometers-diameter patches of soma membrane in bursting neurons of the snail, Helix pomatia. The fluctuations dramatically increased in magnitude with depolarization of the membrane potential under voltage clamp conditions. Two components of conductance noise were identified in the power spectra calculated from the membrane currents. One component had a corner frequency which increased with depolarization. This component was blocked by intracellular injection of TEA and was relatively insensitive to extracellular calcium levels (as long as the total number of effective divalent cations remained constant). It was identified as fluctuations of the voltage-dependent component of delayed outward current. The second component of conductance noise had a corner frequency which decreased with depolarization. It was relatively unaffected by TEA injection and was reversibly blocked by substitution of extracellular calcium with magnesium, cobalt, or nickel. This second component of noise was identified as fluctuations of the calcium-dependent potassium current. The results suggest that the two components of delayed outward current are conducted through physically distinct channels.

Animals↗