Biomedical subjects
S Guthrie
Publications and source records attributed to S Guthrie.
Axon guidance: starting and stopping with slit.
As developing axons navigate, they exhibit various behaviours: extending and branching, pausing, changing direction, retracting. Now, the Slit protein has been discovered to have striking positive and negative effects on axon growth and guidance.
Rhombomere origin plays a role in the specificity of cranial motor axon projections in the chick.
Guidance of cranial motor axons to their targets conforms to a segmental plan in the chick embryo. Trigeminal motor neurons lie within rhombomeres 2 and 3 and project via an exit point in rhombomere 2 to innervate the first branchial arch. Facial motor neurons lie within rhombomeres 4 and 5 and grow out via an exit point in rhombomere 4 to innervate the second branchial arch. We have investigated the axial level-specific matching of motor neurons and branchial arches using donor to host transplantation in avian embryos. Previous work has shown that rostrocaudal reversal of a single hindbrain segment (rhombomere 3) leads to misprojection of a contingent of trigeminal axons via the facial nerve exit point. Using the same experimental manipulation in chick embryos and quail-chick chimaeras, we have analysed the pathways of these aberrant projections. We have found that in the majority of embryos analysed from stage 19 to 31, trigeminal axons from the transplanted rhombomere projected towards second branchial arch muscles, in addition to their normal first arch muscle targets. However, from stage 32 to 36, aberrant projections to second arch-derived muscles were detected only in a small minority of embryos. These experiments show that trigeminal motor neurons show a lack of specificity in their early projection into the periphery but that inappropriate projections may be later eliminated. This suggests that segmental mechanisms intrinsic to the hindbrain specify motor neurons with respect to their eventual innervation pattern.
Transplantation of avian neural tissue.
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Dorsal spinal cord neuroepithelium generates astrocytes but not oligodendrocytes.
There is evidence that oligodendrocytes in the spinal cord are derived from a restricted part of the ventricular zone near the floor plate. An alternative view is that oligodendrocytes are generated from all parts of the ventricular zone. We reinvestigated glial origins by constructing chick-quail chimeras in which dorsal or ventral segments of the embryonic chick neural tube were replaced with equivalent segments of quail neural tube. Ventral grafts gave rise to both oligodendrocytes and astrocytes. In contrast, dorsal grafts produced astrocytes but not oligodendrocytes. In mixed cultures of ventral and dorsal cells, only ventral cells generated oligodendrocytes, whereas both ventral and dorsal cells generated astrocytes. Therefore, oligodendrocytes are derived specifically from ventral neuroepithelium, and astrocytes from both dorsal and ventral.
Cost analysis of methylprednisolone treatment of multiple sclerosis patients.
BACKGROUND: Intravenous methylprednisolone (IVMP) is the treatment of choice for multiple sclerosis (MS) patients undergoing acute exacerbation of disease symptoms and yet its cost has not been accurately determined. Determination of this cost in different settings is also pertinent to consideration of cost-saving alternatives to in-patient treatment. METHODS: Cost analysis from the point of view of the health care system of IVMP treatment of MS patients receiving treatment in association with a selected Toronto teaching hospital in fiscal year 1994/95 was carried out. Costs of any concurrent treatments were excluded. RESULTS: Total cost for 92 patients, based on a 4 dose regime, was estimated to be $78,527. The the cost per patient was $1,1181.84 for in-patients (IP), $714.64 for out-patients of the MS Clinic (OP) and $774.21 for patients whose treatment was initiated in the Clinic, but completed in the home (HC). Sensitivity analyses indicated: 1) IP treatment was in all cases more expensive than that of OP or HC; 2) the cost savings of OP vs. HC was sensitive to assumptions made regarding Clinic overhead, Clinic nursing costs and Home Care Program overhead. CONCLUSION: Alternatives to in-patient care must be considered carefully. In this study, both out-patient and in-home treatment were cost-saving alternatives to in-patient treatment, but large differences in the cost of hospital out-patient vs. in-home care could not be demonstrated.
A distinct developmental programme for the cranial paraxial mesoderm in the chick embryo.
Cells of the cranial paraxial mesoderm give rise to parts of the skull and muscles of the head. Some mesoderm cells migrate from locations close to the hindbrain into the branchial arches where they undergo muscle differentiation. We have characterised these migratory pathways in chick embryos either by DiI-labelling cells before migration or by grafting quail cranial paraxial mesoderm orthotopically. These experiments demonstrate that depending on their initial rostrocaudal position, cranial paraxial mesoderm cells migrate to fill the core of specific branchial arches. A survey of the expression of myogenic genes showed that the myogenic markers Myf5, MyoD and myogenin were expressed in branchial arch muscle, but at comparatively late stages compared with their expression in the somites. Pax3 was not expressed by myogenic cells that migrate into the branchial arches despite its expression in migrating precursors of limb muscles. In order to test whether segmental plate or somitic mesoderm has the ability to migrate in a cranial location, we grafted quail trunk mesoderm into the cranial paraxial mesoderm region. While segmental plate mesoderm cells did not migrate into the branchial arches, somitic cells were capable of migrating and were incorporated into the branchial arch muscle mass. Grafted somitic cells in the vicinity of the neural tube maintained expression of the somitic markers Pax3, MyoD and Pax1. By contrast, ectopic somitic cells located distal to the neural tube and in the branchial arches did not express Pax3. These data imply that signals in the vicinity of the hindbrain and branchial arches act on migrating myogenic cells to influence their gene expression and developmental pathways.
Two regulatory genes, cNkx5-1 and cPax2, show different responses to local signals during otic placode and vesicle formation in the chick embryo.
The early stages of otic placode development depend on signals from neighbouring tissues including the hindbrain. The identity of these signals and of the responding placodal genes, however, is not known. We have identified a chick homeobox gene cNkx5-1, which is expressed in the otic placode beginning at stage 10 and exhibits a dynamic expression pattern during formation and further differentiation of the otic vesicle. In a series of heterotopic transplantation experiments, we demonstrate that cNkx5-1 can be activated in ectopic positions. However, significant differences in otic development and cNkx5-1 gene activity were observed when placodes were transplanted into the more rostral positions within the head mesenchyme or into the wing buds of older hosts. These results indicate that only the rostral tissues were able to induce and/or maintain ear development. Ectopically induced cNkx5-1 expression always reproduced the endogenous pattern within the lateral wall of the otocyst that is destined to form vestibular structures. In contrast, cPax2 which is expressed in the medial wall of the early otic vesicle later forming the cochlea never resumed its correct expression pattern after transplantation. Our experiments illustrate that only some aspects of gene expression and presumably pattern formation during inner ear development can be established and maintained ectopically. In particular, the dorsal vestibular structures seem to be programmed earlier and differently from the ventral cochlear part.
Molecules making waves in axon guidance.
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Axon guidance: netrin receptors are revealed.
Netrins are molecules that guide growing axons and that are strikingly similar in sequence and in function in flies, nematodes and vertebrates. Now, members of a family of netrin receptors have been identified in all three animal groups and shown to have crucial, conserved roles in axon navigation.
Motor axon subpopulations respond differentially to the chemorepellents netrin-1 and semaphorin D.
During development, growing motor axons are excluded from the ventral midline of the neural tube by diffusible chemorepellents emanating from this region. Molecular candidates for this chemorepellent activity include semaphorin D and netrin-1; the latter is known to repel trochlear motor axons. Qualitatively or quantitatively different responses to these molecules might underlie the initial deflection from the midline and subsequent segregation of motor axon trajectories. To test this idea, we have cocultured cell aggregates secreting netrin-1 or semaphorin D at a distance from tissue explants containing different motor neuron subpopulations, in collagen gels. Cranial motor axons that project dorsally in vivo such as those of the trigeminal, facial, and glossopharyngeal nuclei were repelled by both netrin-1 and semaphorin D. By contrast, ventrally projecting spinal motor axons and abducens axons were not affected by netrin-1. Spinal and abducens motor neurons also responded to semaphorin D. The ventrally projecting axons of oculomotor neurons were not repelled by netrin-1 or semaphorin D. Differential responsiveness to netrin-1 and semaphorin D could thus contribute to the generation of dorsal and ventral motor axon pathways during development.
Determination of neuroepithelial cell fate: induction of the oligodendrocyte lineage by ventral midline cells and sonic hedgehog.
Near the floor plate of the embryonic neural tube there is a group of neuroepithelial precursor cells that are specialized for production of the oligodendrocyte lineage. We performed experiments to test whether specification of these neuroepithelial oligodendrocyte precursors, like other ventral neural cell types, depends on signals from the notochord and/or floor plate. We analyzed heterozygous Danforth's short tail (Sd/+) mutant mice, which lack a notochord and floor plate in caudal regions of the neural tube, and found that oligodendrocyte precursors did not appear at the ventricular surface where there was no floor plate. Moreover, oligodendrocytes did not develop in explant cultures of Sd/+ spinal cord in the absence of a floor plate. When a second notochord was grafted into an ectopic position dorsolateral to the endogenous notochord of a chicken embryo, an additional floor plate was induced along with an ectopic focus of oligodendrocyte precursors at the ventricular surface. Oligodendrocytes developed in explants of intermediate neural tube only when they were cocultured with fragments of notochord or in the presence of purified Sonic hedgehog (Shh) protein. Thus, signals from the notochord/floor plate, possibly involving Shh, are necessary and sufficient to induce the development of ventrally derived oligodendroglia. These signals appear to act by specifying the future fate(s) of neuroepithelial cells at the ventricular surface rather than by influencing the proliferation or differentiation of prespecified progenitor cells in the parenchyma of the cord.
Differential expression of LIM homeobox genes among motor neuron subpopulations in the developing chick brain stem.
During development of the chick brain stem, cranial motor neuron subpopulations differentiate at distinct axial levels and extend their axons along specific pathways into the periphery. Differences in phenotype and axonal trajectory of these neuronal populations might be governed by the expression of different repertoires of transcription factors. In 2- to 7-day chick embryos, we find that genes of the LIM homeobox family are expressed differentially among cranial motor nuclei. Whereas Islet-1 is expressed by motor neurons of all cranial nerves, Islet-2 is expressed only in nuclei that contain somatic motor neurons and transiently in a specialized population of contralateral vestibuloacoustic efferent neurons. Lim-3 is expressed in the hypoglossal and accessory abducens nuclei only, and Lim-1 and Lim-2 are not expressed by cranial motor neurons. Our findings are consistent with a role of these transcription factors in determining neuronal phenotype and axonal pathfinding.
Patterning the hindbrain.
Of paramount importance for hindbrain patterning is positional information that is laid down along the rostrocaudal axis. Recent findings suggest that retinoic acid may establish rostrocaudal domains of gene expression that confer on rhombomeres their specific identities; these domains display different responses to dorsoventral signals that further refine the repertoire of cellular fates therein. After rhombomere boundaries form, a high degree of segmental autonomy is balanced by a continuing capacity for interaction along the rostrocaudal axis, exemplified by the generation of the neural crest.
Cranial motor axons respond differently to the floor plate and sensory ganglia in collagen gel co-cultures.
Within the developing chick hindbrain, motor neurons differentiate in columns on either side of the ventral midline floor plate. Along the rostrocaudal axis, populations of motor neurons are organized segmentally with the trigeminal (V) and facial (VII) nuclei occupying successive pairs of rhombomeres. To reach their targets, motor axons follow stereotyped pathways. Branchiomotor and visceral motor axons of the Vth and VIIth nerves first project in a dorsal (lateral) direction away from the floor plate and towards the nerve exit point located in the alar plate of the even-numbered rhombomere of the pair. Having exited the hindbrain, axons grow in association with the cranial sensory ganglia before branchiomotor axons enter the branchial arches. We have investigated some of the factors that might guide cranial motor axons using a three-dimensional collagen gel culture system. When explants of hindbrain basal plate containing trigeminal or facial motor neurons were co-cultured with floor plate explants, axon outgrowth from the side facing the floor plate was inhibited in a manner consistent with chemorepulsion. When basal plate explants that contained an exit point were cultured alone, motor axons grew to the exit point and then stopped. When basal plate explants were co-cultured with trigeminal ganglia, motor outgrowth was increased in comparison with that in control cultures, suggesting a trophic influence. The findings presented here indicate that motor pathways are elaborated due to a progression of signals to which the growth cones respond in sequence.
Isoflavonoid compounds extracted from Pueraria lobata suppress alcohol preference in a pharmacogenetic rat model of alcoholism.
The extract from an edible vine, Pueraria lobata, has long been used in China to lessen alcohol intoxication. We have previously shown that daidzin, one of the major components from this plant extract, is efficacious in lowering blood alcohol levels and shortens sleep time induced by alcohol ingestion. This study was conducted to test the antidipsotropic effect of daidzin and two other major isoflavonoids, daidzein and puerarin, from Pueraria lobata administered by the oral route. An alcohol-preferring rat model, the selectively-bred P line of rats, was used for the study. All three isoflavonoid compounds were effective in suppressing voluntary alcohol consumption by the P rats. When given orally to P rats at a dose of 100 mg/kg/day, daidzein, daidzin, and puerarin decreased ethanol intake by 75%, 50%, and 40%, respectively. The decrease in alcohol consumption was accompanied by an increase in water intake, so that the total fluid volume consumed daily remained unchanged. The effects of these isoflavonoid compounds on alcohol and water intake were reversible. Suppression of alcohol consumption was evident after 1 day of administration and became maximal after 2 days. Similarly, alcohol preference returned to baseline levels 2 days after discontinuation of the isoflavonoids. Rats receiving the herbal extracts ate the same amounts of food as control animals, and they gained weight normally during the experiments. When administered orally, none of these compounds affected the activities of liver alcohol dehydrogenase and aldehyde dehydrogenase. Therefore, the reversal of alcohol preference produced by these compounds may be mediated via the CNS. Data demonstrate that isoflavonoid compounds extracted from Pueraria lobata is effective in suppressing the appetite for alcohol when taken orally, raising the possibility that other constituents of edible plants may exert similar and more potent actions.
The status of the neural segment.
A crucial phase of development in the vertebrate rhombencephalon involves transient organization into segments. Recent studies on the diencephalon and telencephalon pose the question of whether segmentation might also play a role in the development of more rostral brain regions. Criteria for segmentation formulated for the hindbrain might be met by the diencephalon, although there is disagreement as to the number and arrangement of segmental units. In contrast to the hindbrain, these segments appear when neurogenesis has begun, and might represent definitive functional units. Regarding the telencephalon, it is at present unclear whether domains of gene expression are associated with other features that are characteristic of segmental development, or whether other mechanisms control specification of this region.
Chemorepulsion of developing motor axons by the floor plate.
In the developing nervous system, motor axons grow away from the ventral midline floor plate, suggesting that the latter might be a source of repulsive axonal guidance cues. In donor to host transplantation experiments, ectopic pieces of floor plate were positioned between chick hindbrain motor neurons and their exit points. Immunohistochemistry and retrograde axonal labeling techniques demonstrated that motor axons diverted from their normal pathways to avoid grafted floor plate, often traversing abnormally long circuitous trajectories to reach exit points. When ventral explants of rat hindbrain and spinal cord were cocultured at a distance from floor plate explants within collagen gel matrices, the outgrowth of motor axons was dramatically reduced from explant borders that faced the floor plate. Thus, the floor plate secretes diffusible repulsive cues in vitro that may exclude motor axons from the midline during development.