Pathways of Retinotectal Projection in Developing Xenopus Tadpoles Revealed by Selectively Labeling of Retinal Axons with Horseradish Peroxidase (HRP)

Anatomical mapping was made of the retinal central pathways from the chiasm to the targets within the tectum in the developing Xenopus tadpoles, after labeling a specific regional population of retinal axons with horseradish peroxidase (HRP). In the tadpoles at stage 50, pathway sorting of retinal axons within the optic tract was clear for the dorsoventral axis of the retina, but not for the nasotemporal axis. Most nasal retinal axons and some dorsal and ventral retinal axons invaded the tectum directly at the diencephalotectal junction, and arrived at their correct sites of innervation after running through ectopic parts of the tectum. These findings indicate that the pathway orientation before targets is not a prerequisite factor for establishment of the orderly map of the retinotectal projection. Rather, a direct interaction between ingrowing retinal axons and tectal cells seems to be a predominant factor for specification of retinal central connections.     

Asymmetrical retinoic acid synthesis in the dorsoventral axis of the retina.

An aldehyde dehydrogenase present at high levels in the dorsal retina of the embryonic and adult mouse was identified as the isoform AHD-2 known to oxidize retinaldehyde to retinoic acid. Comparative estimates of retinoic acid levels with a reporter cell line placed the retinas among the richest tissues in the entire body of the early embryo; levels in ventral retina, however, exceeded dorsal levels. Retinoic acid synthesis from retinaldehyde in the dorsal pathway was less effective than the ventral pathway at low substrate levels and more effective at high levels. The dorsal pathway was preferentially inhibited by disulfiram, while ventral synthesis was preferentially inhibited by p-hydroxymercuribenzoate. When protein fractions separated by isoelectric focusing were analyzed for retinoic acid synthesizing capacity by a zymography-bioassay, most of the synthesis in dorsal retina was found to be mediated by AHD-2, and ventral synthesis was mediated by dehydrogenase activities distinct in charge from AHD-2. Postnatally, levels of highest retinoic acid synthesis shifted from ventral to dorsal retina. In the adult retina, the dorsal pathway persisted, but the preferential ventral pathway was no longer detectable. Our observations raise the possibility that retinoic acid plays a role in the determination and maintenance of the dorsoventral axis of the retina, and that the morphogenetically significant asymmetry here lies in the spatial arrangement of synthetic pathways.     

Retinal axon pathfinding in the optic chiasm: divergence of crossed and uncrossed fibers

In the developing mammalian visual system, retinal fibers grow through the optic chiasm, where one population crosses to the opposite side of the brain and the other does not. Evidence from labeling growing retinal axons with the carbocyanine dye Dil in mouse embryos indicates that the two subpopulations diverge at a zone along the midline of the optic chiasm. At the border of this zone, crossed fibers grow directly across, whereas uncrossed fibers turn back, developing highly complex terminations with bifurcating and wide-ranging growth cones. When one eye is removed at early stages, uncrossed fibers from the remaining eye stall at the chiasm midline. These results suggest that crossed and uncrossed retinal fibers respond differently to cues along the midline of the chiasm and that the uncrossed fibers from one eye grow along crossed fibers from the other eye, both guidance mechanisms contributing to the establishment of the bilateral pattern of visual projections in mammalian brain.     

Retinoic acid in the formation of the dorsoventral retina and its central projections

Dynamic expression patterns of four retinoid-metabolizing enzymes create rapidly changing retinoic acid (RA) patterns in the emerging eye anlage of the mouse. First, a RA-rich ventral zone is set up, then a RA-poor dorsal zone, and finally a tripartite organization consisting of dorsal and ventral RA-rich zones separated by a horizontal RA-poor stripe. This subdivision of the retina into three RA concentration zones is directly visible as beta-galactosidase labeling patterns in retinas of RA-reporter mice. Because the axons of retinal ganglion cells transport the reporter product anterogradely, the central projections from dorsal and ventral retina can be visualized as two heavily labeled axon bundles. Comparisons of the axonal labeling with physiologic recordings of visual topography in the adult mouse show that the labeled axons represent the upper and the lower visual fields. The RA-poor stripe develops into a broad horizontal zone of higher visual acuity. Comparisons of the retina labeling with eye-muscle insertions show that the axis of the RA pattern lines up with the dorsoventral axis of the oculomotor system. These observations indicate that the dorsoventral axis of the embryonic eye anlage determines the functional coordinates of both vision and eye movements in the adult.     

Absence of dorsal-ventral differences in energy metabolism in early embryos of Xenopus laevis.

The establishment of dorsoventral polarity in amphibian development begins prior to the first cleavage with the formation of the gray crescent. It has been suggested that quantitative differences exist in the metabolic activity of dorsal and ventral regions at these stages and that this metabolic activity gradient is involved in the maintenance of dorsoventral polarity. In this study regional metabolism has been examined by the microinjection of various radioactive compounds into dorsal or ventral cells of the four-celled Xenopus laevis embryo. The activities of the Embden-Meyerhof pathway, the pentose shunt, and the Krebs cycle and phosphate metabolism have been measured. No difference was detected in the rate or pattern of metabolism between dorsal and ventral regions, suggesting that metabolic activity gradients do not exist in these pathways during early Xenopus embryogenesis.     

Abnormal anteroposterior and dorsoventral patterning of the limb bud in the absence of retinoids.

We describe here how the early limb bud of the quail embryo develops in the absence of retinoids, including retinoic acid. Retinoid-deficient embryos develop to about stage 20/21, thus allowing patterns of early gene activity in the limb bud to be readily examined. Genes representing different aspects of limb polarity were analysed. Concerning the anteroposterior axis, Hoxb-8 was up-regulated and its border was shifted anteriorly whereas shh and the mesodermal expression of bmp-2 were down-regulated in the absence of retinoids. Concerning the apical ectodermal genes, fgf-4 was down-regulated whereas fgf-8 and the ectodermal domain of bmp-2 were unaffected. Genes involved in dorsoventral polarity were all disrupted. Wnt-7a, normally confined to the dorsal ectoderm, was ectopically expressed in the ventral ectoderm and the corresponding dorsal mesodermal gene Lmx-1 spread into the ventral mesoderm. En-1 was partially or completely absent from the ventral ectoderm. These dorsoventral patterns of expression resemble those seen in En-1 knockout mouse limb buds. Overall, the patterns of gene expression are also similar to the Japanese limbless mutant. These experiments demonstrate that the retinoid-deficient embryo is a valuable tool for dissecting pathways of gene activity in the limb bud and reveal for the first time a role for retinoic acid in the organisation of the dorsoventral axis.     

Topography of retinal axons in the diencephalon of goldfish

Summary The projections of horseradish peroxidase-filled axons from each quadrant of the retina were studied to determine whether retinal projections of goldfish are topographically organized in diencephalic target nuclei. A distinct topography of the dorsal, nasal, ventral and temporal retina exists in the lateral geniculate nucleus and in the dorsolateral optic nucleus of the thalamus. The projections of retinal quadrants show minimal spatial overlap in each of these nuclei. The suprachiasmatic nucleus of the hypothalamus is extensively innervated by ventral retinal fibers, whereas the nucleus is sparsely innervated by fibers from the other three retinal quadrants. A rudimentary topography also exists in the pretectum where the dorsal pretectal area receives projections primarily from the ventral retina and the ventral pretectal area receives projections mostly from the dorsal retina. These data show that retinal projections to some diencephalic nuclei are topographically organized.This work was supported by Research Grant EY-01426 to S.C.S.     

The early development of retinal ganglion cells with uncrossed axons in the mouse: retinal position and axonal course

The carbocyanine dye, DiI, has been used to study the retinal origin of the uncrossed retinofugal component of the mouse and to show the course taken by these fibres through the optic nerve and chiasm during development. Optic axons first arrive at the chiasm at embryonic day 13 (E13) but do not cross the midline until E14. After this stage, fibres taking an uncrossed course can be selectively labelled by unilateral tract implants of DiI. The earliest ipsilaterally projecting ganglion cells are located in the dorsal central retina. The first sign of the adult pattern of distribution of ganglion cells with uncrossed axons located mainly in the ventrotemporal retina is seen on embryonic day 16.5, thus showing that the adult line of decussation forms early in development. A small number of labelled cells continue to be found in nasal and dorsal retina at all later stages. At early stages (E14-15), retrogradely labelled uncrossed fibres are found in virtually all fascicles of the developing nerve, intermingling with crossed axons throughout the length of the nerve. At later stages of development (E16-17), although uncrossed fibres pass predominantly within the temporal part of the stalk, they remain intermingled with crossed axons. A significant number of uncrossed axons also lie within the nasal part of the optic stalk. The position of uncrossed fibres throughout the nerve in the later developmental stages is comparable to that seen in the adult rodent (Baker and Jeffery, 1989). The distribution of uncrossed axons thus indicates that positional cues are not sufficient to account for the choice made by axons when they reach the optic chiasm.     

Progressive determination of cell fates along the dorsoventral axis in the sea urchin Heliocidaris erythrogramma

In the direct-developing sea urchin Heliocidaris erythrogramma the first cleavage division bisects the dorsoventral axis of the developing embryo along a frontal plane. In the two-celled embryo one of the blastomeres, the ventral cell (V), gives rise to all pigmented mesenchyme, as well as to the vestibule of the echinus rudiment. Upon isolation, however, the dorsal blastomere (D) displays some regulation, and is able to form a small number of pigmented mesenchyme cells and even a vestibule. We have examined the spatial and temporal determination of cell fates along the dorsoventral axis during subsequent development. We demonstrate that the dorsoventral axis is resident within both cells of the two-celled embryo, but only the ventral pole of this axis has a rigidly fixed identity this early in development. The polarity of this axis remains the same in half-embryos developing from isolated ventral (V) blastomeres, but it can flip 180° in half-embryos developing from isolated dorsal (D) blastomeres. We find that cell fates are progressively determined along the dorsoventral axis up to the time of gastrulation. The ability of dorsal half-embryos to differentiate ventral cell fates diminishes as they are isolated at progressively later stages of development. These results suggest that the determination of cell fates along the dorsoventral axis in H. erythrogramma is regulated via inductive interactions organized by cells within the ventral half of the embryo.     

Commissural axon pathfinding on the contralateral side of the floor plate: a role for B-class ephrins in specifying the dorsoventral position of longitudinally projecting commissural axons.

In both invertebrate and lower vertebrate species, decussated commissural axons travel away from the midline and assume positions within distinct longitudinal tracts. We demonstrate that in the developing chick and mouse spinal cord, most dorsally situated commissural neuron populations extend axons across the ventral midline and through the ventral white matter along an arcuate trajectory on the contralateral side of the floor plate. Within the dorsal (chick) and intermediate (mouse) marginal zone, commissural axons turn at a conserved boundary of transmembrane ephrin expression, adjacent to which they form a discrete ascending fiber tract. In vitro perturbation of endogenous EphB-ephrinB interactions results in the failure of commissural axons to turn at the appropriate dorsoventral position on the contralateral side of the spinal cord; consequently, axons inappropriately invade more dorsal regions of B-class ephrin expression in the dorsal spinal cord. Taken together, these observations suggest that B-class ephrins act locally during a late phase of commissural axon pathfinding to specify the dorsoventral position at which decussated commissural axons turn into the longitudinal axis.     

Differential responses of temporal and nasal retinal neurites to regional-specific cues in the mouse retinofugal pathway

Retinal explants of mouse embryos were cultured together with explants of different regions in the retinofugal pathway in order to investigate whether ventral temporal (VT) and dorsal nasal (DN) retinal neurites showed differential responses to regional-specific cues in the pathway. In the presence of the chiasm, biased outgrowth of retinal neurites was found in explants of both retinal regions, which was accompanied by a reduction in total neurite growth in the VT but not the DN retina. Such differential responses to the diffusible negative influence were also observed when explants of two retinal origins were cocultured with the ventral diencephalon, but were not found with the dorsal diencephalon that contains targets of the optic axons. Indeed, extensive neurite invasion was found in the dorsal diencephalic explants and this ingrowth was more prominent for VT than DN neurites, showing a difference in axons from a distinct position in the retina to contact-mediated stimulatory activity within the target nuclei. We conclude that neurites from different regions of the retina show differential responses to the regional-specific cues in the diencephalon. These cues exist in both diffusible and contact-mediated forms that may shape the characteristic course and organization of retinal axons in decision regions of the optic pathway and the visual targets.     

The central morphology of the giant interneurons and their spatial relationship with the thoracic motorneurons in the cockroach, Periplaneta americana (Insecta)

Three groups of giant fibers are found in the cockroach ventral nerve cord. A latero-dorsal group (dorsal GIs), a latero-ventral group (ventral GIs) and a medio-ventral group. The morphology of all three groups of fibers within the thoracic ganglia is described. The morphology of the dorsal and ventral GI pathways in the abdominal and suboesophageal ganglia is also described. The projection patterns of the neurons in each ganglion are remarkably similar which suggests a common function. When motorneurons 5rl (depressor) and 6Br4 (levator) are stained simultaneously with the dorsal and ventral GI groups, some branches from both motor and giant neurons converge. The branching of the remaining medio-ventral group of fibers and their proximity to areas receiving motorneuronal input suggests that these are the small diameter axons described by Dagan and Parnas (1970).     

Neurochemical and Immunocytochemical Studies on the Distribution of N-Acetyl-Aspartylglutamate and N-Acetyl-Aspartate in Rat Spinal Cord and Some Peripheral Nervous Tissues

HPLC analysis of rat spinal cord revealed a uniform distribution of N-acetyl-aspartate (NAA) across both longitudinal and dorsoventral axes. In contrast, ventral cord N-acetyl-aspartylglutamate (NAAG) levels were significantly higher than those measured in dorsal halves of cervical, thoracic, and lumbar segments. Immunocytochemical studies using an affinity-purified antiserum raised against NAAG-bovine serum albumin revealed an intense staining of motoneurons within rat spinal cord. Along with the considerable NAAG content in ventral roots, these results suggest that NAAG may be concentrated in motoneurons and play a role in motor pathways. NAAG was also present in other peripheral neural tissues, including dorsal roots, dorsal root ganglia, superior cervical ganglia, and sciatic nerve. It is interesting that NAA levels in peripheral nervous tissues were lower than those in CNS structures and that NAA levels in ventral roots and sciatic nerve were lower than NAAG levels. These findings further document a lack of correlation between NAAG and NAA levels in both central and peripheral nervous tissues. Taken together, these data demonstrate the presence of NAAG in nonglutamatergic neuronal systems and suggest a more complex role of NAAG in neuronal physiology than previously postulated.     

Flexibility in the patterning and control of axial locomotor networks in lamprey

In lower vertebrates, locomotor burst generators for axial muscles generally produce unitary bursts that alternate between the two sides of the body. In lamprey, a lower vertebrate, locomotor activity in the axial ventral roots of the isolated spinal cord can exhibit flexibility in the timings of bursts to dorsally-located myotomal muscle fibers versus ventrally-located myotomal muscle fibers. These episodes of decreased synchrony can occur spontaneously, especially in the rostral spinal cord where the propagating body waves of swimming originate. Application of serotonin, an endogenous spinal neurotransmitter known to presynaptically inhibit excitatory synapses in lamprey, can promote decreased synchrony of dorsal-ventral bursting. These observations suggest the possible existence of dorsal and ventral locomotor networks with modifiable coupling strength between them. Intracellular recordings of motoneurons during locomotor activity provide some support for this model. Pairs of motoneurons innervating myotomal muscle fibers of similar ipsilateral dorsoventral location tend to have higher correlations of fast synaptic activity during fictive locomotion than do pairs of motoneurons innervating myotomes of different ipsilateral dorsoventral locations, suggesting their control by different populations of premotor interneurons. Further, these different motoneuron pools receive different patterns of excitatory and inhibitory inputs from individual reticulospinal neurons, conveyed in part by different sets of premotor interneurons. Perhaps, then, the locomotor network of the lamprey is not simply a unitary burst generator on each side of the spinal cord that activates all ipsilateral body muscles simultaneously. Instead, the burst generator on each side may comprise at least two coupled burst generators, one controlling motoneurons innervating dorsal body muscles and one controlling motoneurons innervating ventral body muscles. The coupling strength between these two ipsilateral burst generators may be modifiable and weakening when greater swimming maneuverability is required. Variable coupling of intrasegmental burst generators in the lamprey may be a precursor to the variable coupling of burst generators observed in the control of locomotion in the joints of limbed vertebrates.     

Dolphin peripheral visual pathway in chronic unilateral ocular atrophy: Complete decussation apparent

Components of the peripheral visual pathway were examined in two bottlenose dolphins, Tursiops truncatus, each with unilateral ocular degeneration and scarring of 3 or more years' duration. In both animals, the optic nerve associated with the blind eye right eye in Tg419 and left eye in Tt038 had a translucent, gel-like appearance upon gross examination. This translucency was also evident in the optic tract contralateral to the affected eye. In Tg419, myelinated axons of varying diameters were apparent in the left optic nerve, whereas the right optic nerve, serving the blind eye, appeared to be devoid of axons. In Tt038, myelinated axons were associated with the right optic nerve (serving the functional eye) and left optic tract but were essentially absent in the left optic nerve and right optic tract. Examined by light microscopy in serial horizontal sections, the optic chiasm of Tt038 was arranged along its central plane in segregated, alternating pathways for the decussation of right and left optic nerve fibers. Ventral to this plane, the chiasm was comprised of fibers from the left optic nerve, whereas dorsal to the central plane, fibers derived from the right optic nerve. Because of this architectural arrangement, the right and left optic nerves grossly appeared to overlap as they crossed the optic chiasm with the right optic nerve coursing dorsally to the left optic nerve. At the light and electron microscopic levels, the optic nerves and tracts lacking axons were well vascularized and dominated by glial cell bodies and glial processes, an expression of the marked glial scarring associated with postinjury axonal degeneration. The apparent absence of axons in one of the optic tract pairs (right in Tt038 and left in Tg419) supports the concept of complete decussation of right and left optic nerve fibers at the optic chiasm in the bottlenose dolphin. © 1994 Wiley-Liss, Inc.     

Organization of the dorsoventral musculature in the wings of the pteropod mollusc Clione limacina

The wings of the pteropod mollusc Clione limacina provide forward propulsive force through flapping movements in which the wings bend throughout their length in both dorsal and ventral directions. The musculature of the wings includes oblique, striated muscle bundles that generate the swimming movements of the wings, longitudinal and transverse (smooth) muscle bundles that collapse the wings and pull them into the body during a wing withdrawal response, and dorsoventral muscles that control the thickness of the wings. All muscles act against a hydrostatic skeleton that forms a central hemocoelic space within the wings. Of these muscle types, all have been thoroughly described and studied except the dorsoventral muscles. The fortuitous discovery that the dorsoventral musculature can be intensely labeled with an antibody against the vertebrate hyperpolarization‐activated cation channel (HCN2) provided the opportunity to describe the organization of the dorsoventral musculature in detail. In addition, electrical recordings and microelectrode dye injections supported the immunohistochemical data, and provided preliminary data on the activity of the muscle fibers. The organization and activity of the dorsoventral musculature suggests it may be involved in regulation of wing stiffness during the change from slow to fast swimming.     

Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear

Organization of the vertebrate inner ear is mainly dependent on localized signals from surrounding tissues. Previous studies demonstrated that sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of ventral (auditory) and dorsal (vestibular) inner ear structures, yet it was not clear how this signaling activity is propagated. To elucidate the molecular mechanisms by which Shh regulates inner ear development, we examined embryos with various combinations of mutant alleles for Shh, Gli2 and Gli3. Our study shows that Gli3 repressor (R) is required for patterning dorsal inner ear structures, whereas Gli activator (A) proteins are essential for ventral inner ear structures. A proper balance of Gli3R and Gli2/3A is required along the length of the dorsoventral axis of the inner ear to mediate graded levels of Shh signaling, emanating from ventral midline tissues. Formation of the ventral-most otic region, the distal cochlear duct, requires robust Gli2/3A function. By contrast, the formation of the proximal cochlear duct and saccule, which requires less Shh signaling, is achieved by antagonizing Gli3R. The dorsal vestibular region requires the least amount of Shh signaling in order to generate the correct dose of Gli3R required for the development of this otic region. Taken together, our data suggest that reciprocal gradients of GliA and GliR mediate the responses to Shh signaling along the dorsoventral axis of the inner ear.     

Neuronal control of swimming in the medicinal leech

Summary The cell bodies and function of twelve neurons whose impulse pattern is clearly related to that of the swimming rhythm were identified in the segmental ganglion of the leech. These include excitatory and inhibitory motor neurons of the dorsal and ventral longitudinal muscles and the excitatory flattener motor neuron of the dorsoventral muscles. During swimming the membrane potential of these cells oscillates between a depolarized and a hyperpolarized phase. The activity of this ensemble of cells is sufficient to account for the contractile rhythm of the swimming animal. The following connections were found between these motor neurons. Electrotonic junctions link: (1) bilaterally homologous cells; (2) excitors of the dorsal longitudinal muscles; (3) excitors of the ventral longitudinal muscles; (4) inhibitors of both dorsal and ventral longitudinal muscles. The dorsal inhibitors project via an inhibitory pathway to the dorsal excitors, and the ventral inhibitor projects via an inhibitory pathway to the ventral excitors. The membrane potential oscillation of the excitors is at least partly attributable to the phasic inhibitory synaptic input which they receive from the inhibitors. The excitatory shortener motor neuron of the entire longitudinal musculature is maintained in an inactive state during swimming. This control is achieved by rectifying electrotonic junctions linking this neuron to the dorsal and ventral excitors. These junctions allow passage of only depolarizing current from the shortener to the dorsal and ventral excitors and of only hyperpolarizing current in the reverse direction. Furthermore, both dorsal and ventral inhibitors project via inhibitory pathways to the shortener neuron.We are greatly indebted to Ann Stuart for advice and help in this study, and for communicating to us some unpublished findings. We thank Elizabeth Mullenbach for excellent technical assistance.This research was supported by grant GB 31933 X from the National Science Foundation, and by Public Health Service Research grant GM 17866 and Training Grant GM 01389 from the Institute for General Medical Sciences.