Potential Role of Pax-2 in Retinal Axon Navigation through the Chick Optic Nerve Stalk and Optic Chiasm

ArticleinJournal of Neurobiology 59(1):8-23 · May 2004with 35 Reads
DOI: 10.1002/neu.20001 · Source: PubMed
Abstract
The degree of fiber decussation at the optic chiasm differs between species, ranging from complete crossing in lower vertebrates to highly complex patterns of intermingling of the fibers from the two eyes seen in mammals and birds. Understanding the genetic control of fiber guidance through the chiasm is therefore important to unravel the developmental mechanisms within the visual system. Here we first report on early stages of chiasm formation, with pioneering axons from the left eye consistently arriving earlier than their counterparts from the right eye. This initial left-right asymmetry is transient and no functional significance is assigned to it yet. Secondly, we examined formation of the chiasm in relation with the expression of the transcription factor Pax-2 along the ventral eye cup and optic nerve stalk. Finally, in order to examine causal involvement of Pax-2 in chiasm formation, the gene was overexpressed along the neuraxis and in the eye cup at embryonic stages preceding the exit of axons from the eye, and hence arrival of axons at the chiasm. When studied with neuroanatomical tracing, Pax-2 overexpression resulted in visibly anomalous decussation of axons at the chiasm. A likely consequence of this perturbation was erroneous arrival of axons at the tectum, as observed by anterograde staining from the retina. These data suggest that balanced expression of Pax-2 results in the correct formation of the chick chiasm at early stages by imposing accurate pathfinding within the optic stalk and the midline.
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    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.
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    We investigated whether regenerating mature axons recapitulate embryonic features essential to successful reconnectivity within the injured nervous system. Strips from embryonic and adult chick retinae were cultured, and outgrowing axons were examined morphometrically and immunohistochemically. In addition, the target-recognition properties of adult neurites were analyzed. Regenerating adult axons elongate on a poly-L-lysine/laminin substratum with a speed about one order of magnitude slower than that of embryonic axons. Morphologically, adult axonal tips differ dramatically from embryonic growth cones in that they possess only filopodial extensions whereas embryonic growth cones possess both lamellipodial and filopodial processes. Both embryonic and adult neurites express the growth-associated protein GAP-43. When cultured on alternating stripes of anterior and posterior embryonic tectal membranes, both adult and embryonic retinal axons distinguish between the two membrane preparations. Our results demonstrate that during axonal regeneration the mature neurons express embryonic properties that are involved in the recognition of tectal positional cues.
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    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.
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    Carbocyanine dyes, fluorescent lipophilic substances used for optical recordings of membrane voltage and for studies of membrane fluidity, have recently been shown to provide intense and long-lasting staining of neurones in vivo and in vitro (Schwartz & Agranoff, 1981; Honig & Hume, 1985, 1986; Catsicas, Thanos & Clarke, 1986; Landmesser & Honig, 1986; Thanos & Bonhoeffer, 1987). We report here that two of these dyes, diI (1,1',dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate) and diO (3,3'-dioctadecyloxacarbocyanine perchlorate), can also label neurones in embryonic mouse and chicken brain tissue that has been previously fixed in aldehyde fixatives. Neuronal processes and perikarya can be labelled along considerable distances in both anterograde and retrograde directions. The staining of processes and cells, including their finest extensions is smooth and clear, rivalling intracellular injections of HRP or Lucifer Yellow. The appearance and time course of progression of the staining along axons suggest that the staining in fixed tissue occurs due to a process of diffusion of dyes along the plasma membranes of cells. This technique has allowed us to study the first stages in the development of optic fibres in mouse embryos, especially at the optic chiasm. The early retinal projection (E13-E13 1/2) is mainly crossed, but some optic fibres grow to the ipsilateral side of the brain at the outset. Retrogradely labelled ganglion cells from the dorsocentral area of the retina participate in the formation of both the ipsilateral and the contralateral projection. Thus, at early stages, crossed and uncrossed projections arise from identical subregions of the retina and the partition of the retina with respect to the laterality of its projection arises later.
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    When optic fibers first approach the chiasmatic region of the diencephalon in the chick embryo on days 3 and 4 (E3-4), they rarely grow rostrally into the olfactory region of the telencephalon. Conversely, olfactory tract axons grow as far as, but never cross the diencephalic/telencephalic (D/T) boundary to enter the optic chiasm. In this study, a region of specialized neuroepithelium, originally named the "knot" in mouse by Silver (1984), has been identified at the D/T border of chick embryos. At pre-axonal stages, the presumptive knot region undergoes a cataclysmic cell death, with concomitant phagocytosis of necrotic debris by the remaining cells. When fibers subsequently appear in the chiasm and olfactory tracts, the knot consists of a very dense, interwoven cluster of non-neuronal cells that lack marginal radial processes, and whose cell bodies directly abut the glial limiting membrane. Thus, the morphology of the knot is in sharp contrast to the cell body-free marginal zone and endfoot regions along which axons tend to grow. In addition, we found that the neural cell adhesion molecule (N-CAM), which is expressed on neuroepithelial cell processes within the central optic and olfactory pathways, is not present on cells in the knot region during periods of axon growth. These results suggest that the knot, through its elimination of the marginal zone processes, absence of large extracellular spaces, and relative absence of adhesion molecules, functions as an axon-refractory barrier that effectively separates the optic and olfactory projections.
  • Article
    The events that occur during the early development of the optic chiasm of the chick embryo have been studied by light and scanning electron microscopy. In developmental stages previous to the arrival of the first optic fibres in the floor of the diencephalon, as well as during the arrival of the leading fibres, extracellular spaces can be seen in the diencephalon ventral wall. These spaces are defined by external cell prolongations which end in a foot-shaped formation. During stages 25 and 26 a prechiasmic degenerative centre appears in the area immediately rostral to the early chiasm, leading to a notable degree of disorganization in the diencephalon wall. This centre appears to be related to the reorganization of the system of external cell processes and extracellular spaces which become progressively more irregularly distributed, coinciding with the arrival of the first optic fibre fascicles to the midline of the floor of the diencephalon. The optic fibre fascicles change their latero-medial directionality in the medial-most regions of the ventral diencephalon, where their course becomes rostrocaudal. This reorientation of the optic fibres seems to be mediated by primitive glial cells which first appear in the ventrorostral region of the early chiasm (previously occupied by the system of external cell processes and extracellular spaces) in stage 26, increasing in number from this stage on. The morphology of the primitive glial cells is laminar in nature and the cells are seen to be densely packed together with no large extracellular spaces between them.
  • Article
    The formation of the very orderly neuronal projection from the retina to the optic tectum is not yet understood, but several mechanisms are thought to be involved in a coordinated fashion. These mechanisms may include mechanical or chemical guidance in channels, guidance by spatial gradients of positional markers, gradients of temporal (maturation) markers or specific inter-axon interactions (see ref. 1 for review). The last-mentioned mechanism could explain the fibre order found in optic nerve and tract. It requires that some or all growing retinal axons can distinguish between retinal axons of various origins and grow preferentially along retinal axons originating from the same area as themselves. The in vitro experiments described here show that growth cones from the temporal half of the chick retina grow preferentially along temporal axons, whereas growth cones from nasal retina do not distinguish between nasal and temporal axons.
  • Article
    Antibodies against the neural cell adhesion molecule (NCAM) were used in vivo both to localize NCAM antigenic determinants in developing tissues of the chicken visual system and to perturb cell-cell adhesion during growth of optic fibers to the tectum. The immunohistochemical studies revealed a staining pattern on neuroepithelial cells which coincided with certain regions of the presumptive route for optic axons, not only with respect to the overall pathway from the eye to the tectum, but also in the preferential distribution of the antigen on the marginal endfeet which are contacted by optic axon growth cones. The antibody-perturbation studies, which involved intraocular injection of anti-NCAM Fab at embryonic Day 3.5, demonstrated that inhibition of NCAM-mediated adhesion results in a dramatic distortion of growth cone-neuroepithelial cell relationships and consequently of the optic pathway. Together, these studies suggest that guidance of optic axons along the margin of the brain is at least in part influenced by a preformed adhesive pathway on neuroepithelial cells associated with NCAM antigens.
  • Article
    Following discrete lesions of the chick retina, the distribution of degenerating retinal ganglion cell axons in the optic chiasm was examined. A feature of particular interest was the dorsal aspect of the optic chiasm which was found to contain fibres originating from the superior temporal retina. The significance of this finding is discussed in relation to possible damage to the optic chiasm resulting as a consequence of sectioning the supraoptic decussation.
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  • Article
    What are the forces residing at the presumptive chiasm of embryonic mice that control the directionality (i.e., side specificity) of the optic axons? In an attempt to answer this question, the overall trajectories of individual fascicles of early growing axons and the various environments that they encounter along their pathway have been charted from the eye through the nerve and into the base of the diencephalon. Serial sections and reconstructive computer graphic techniques were used for the analysis. The early optic axons (embryonic (E) day 13.5) arrive at the chiasm in a stereotyped topographic arrangement. However, the fiber array at the primitive chiasm is not retinotopically organized nor is it maintained with the same level of spatial precision as it is at the disc. Thus, the annular, inverted retinotopic contingent of “pioneering” axons that exists in the primitive nerve becomes reorganized at the chiasm into a crescent-shaped configuration, with fascicles from ventrotemporal and ventronasal retina at either side of the crescent and with fascicles from dorsal retina interposed. Because of their gross locations in the crescent, particular clusters of fibers, each largely originating from different retinal sectors, but “contaminated” with fibers from other regions, come in contact with different types of nonneuronal structures at the chiasm. One, a dense, knotlike glial formation that lies along the margin of the diencephalic-telencephalic junction, directs all adjacent (ventronasal) fibers contralaterally. The other, a discrete pathway of lengthy marginal glial processes, separated by an anastomotic system of large extracellular spaces, guides all nearby fibers from ventrotemporal retina ipsilaterally. The results suggest that fiber topography as well as local environmental factors may play important roles in guiding axons at the chiasm.
  • Article
    Recent evidence indicates that the retinotectal projection has a field-to-field rather than a point-to-point precision. Therefore, individual fibres can vary their relative position in the fibre pathway to some extent. In addition it is not necessary that retinotopy is maintained throughout the whole pathway; transformations may also occur. In fact, in the chick embryo outgrowing retinal fibres maintain not an absolute but only a high degree of order. Transformations occur at the entrance to the optic nerve and probably also right behind the chiasm. The origin of the map is determined by the fact that central retinal fibres which are formed first connect to tectal neurons near the centre of the optic tectum where neurons mature first. There they immediately invade the cellular tectal layers and form functional synapses soon after. Supernumerary fibres degenerate.
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    The retinofugal pathway is a useful model for axon guidance because fibers from each eye project to targets on both sides of the brain. Studies using static and real time analyses in mice at E15-17 demonstrated that uncrossed axons from ventrotemporal retina diverge from crossed axons in the optic chiasm, where specialized resident cells may direct divergence. Other studies, however, suggest that pioneering uncrossed retinal axons derive from a different retinal region, take a different course, and enter the ipsilateral optic tract independent of fiber-fiber interactions. We examine these differences by dye-labeling the earliest optic axons and immunocytochemically identifying cells in their path. The first optic axons arising from dorsocentral retina, enter the diencephalon at E12.5. All axons initially grow caudally, lateral to a radial glial palisade. In contrast to later growing axons, early uncrossed axons enter the ipsilateral optic tract directly. Crossed axons enter the glial palisade and course medially, then anteriorly, in a pathway corresponding to the border of an early neuronal population that expresses SSEA-1, CD44, and beta-tubulin. Axon patterning occurs independent of fiber-fiber interactions from both eyes, as the first uncrossed axons enter the optic tract before crossed ones from opposite eye. These analysis, in conjunction with our previous studies during the principal period of retinal axon growth in the diencephalon, suggest that the adult visual projection arises from age-dependent variations in the types and relative contribution of cues along the path through the emerging optic chiasm.
  • Article
    We report a newly identified syndrome in which nasal retinal fibres fail to decussate due to the inborn absence of an optic chiasm. Visual evoked potential (VEP) assessment and neuro-opththalmic evaluation in two unrelated, non-albino children revealed the unusual visual pathway anomaly in the form of misrouted retinal-fugal projections. Monocular VEP responses across the occiput, regardless of stimulus mode (full- or partial-field pattern onset, pattern reversal, luminance flash or high temporal frequency luminance flicker) showed unequivocal evidence of pathological VEP ipsilateral asymmetry. Marked attenuation of primary visual evoked responses from the occiput contralateral to the eye of stimulation, indicative of aberrant contralateral retinal-fugal projections, was confirmed by MRI which depicted the remarkable achiasmatic condition. MRIs and neurological evaluation also confirmed the absence of accompanying congenital or acquired brain malformations or anomalies. Ophthalmic evaluation revealed that both achiasmatic children had reduced distance acuity for age, alternating esotropia, torticollis, head tremor and ocular motor instability; visual fields were normal. Eye movements were also monitored and indicated congenital nystagmus waveforms in the horizontal plane; see-saw nystagmus was observed in the the horizontal plane; see-saw nystagmus was observed in the vertical and torsional planes. The age range of the two children during evaluation and follow-up, over a 6-year period, was about 4-15 years. Comparisons of VEP responses from age-matched normal, albino and idiopathic congenital nystagmus controls, recorded under the same VEP test conditions, were also performed. In contrast to the achiasmatic ipsilateral inter-ocular asymmetry, the albinos showed the expected monocular VEP topography pattern of contralateral asymmetry. Also as expected, VEP profiles from the normal controls and those with congenital nystagmus, evinced no aberrant asymmetry patterns. In general, the results indicate that the VEP misrouting protocol is indispensable for the non-invasive electrophysiological detection and differential diagnosis of optic pathway mutations and may well identify individuals with purported idiopathic congenital nystagmus or albinism that are, in fact, achiasmatic.
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    The developmental, regulatory gene Pax-2 is activated during early kidney morphogenesis and repressed in mature renal epithelium. Persistent Pax-2 expression is also observed in a variety of kidney tumors. Yet, little is known about the signals regulating this transient expression pattern in the developing kidney. We have examined the spatial and temporal expression patterns of Pax-2 and the Wilm's tumor suppresser protein WT1 with specific antibodies in developing mouse kidneys. A marked increase in WT1 protein levels coincided precisely with down-regulation of the Pax-2 gene in the individual precursor cells of the visceral glomerular epithelium, suggesting a direct effect of the WT1 repressor protein on Pax-2 regulatory elements. To examine whether WT1 could directly repress Pax-2 transcription, binding of WT1 to three high affinity sites in the 5' untranslated Pax-2 leader sequence was demonstrated by DNAseI footprinting analysis. Furthermore, co-transfection assays using CAT reporter constructs under the control of Pax-2 regulatory sequences demonstrated WT1-dependent transcriptional repression. These three WT1 binding sites were also able to repress transcription, in a WT1-dependent manner, when inserted between a heterologous promoter and the reporter gene. The data indicate that Pax-2 is a likely target gene for WT1 and suggest a direct link, at the level of transcriptional regulation, between a developmental control gene, active in undifferentiated and proliferating cells, and a known tumor suppressor gene.
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  • Article
    Previous investigations have identified proteoglycans in the central nervous system during development and have implicated some proteoglycans as axon guidance molecules that act by inhibiting axon extension. The present study investigated the pattern of immunoreactivity for several glycosaminoglycans common to certain proteoglycans relative to growing retinal axons in the developing chick visual system and in retinal explant cultures. Immunostaining for chondroitin-6-sulfate, chondroitin-4-sulfate, and keratan sulfate was observed to colocalize with retinal axons throughout the retinofugal pathway during the entire period of retinal axon growth. The proteoglycan form of collagen IX, however, was only observed in the retina, primarily peripheral to the areas with actively growing axons. The pattern of immunostaining for chondroitin sulfate in tissue sections suggested that the retinal axons might be a source for some of the chondroitin sulfate immunostaining in the developing visual pathway. This was confirmed in that chondroitin sulfate immunostaining was also observed on neurites emanating from cultured retinal explants. These findings indicate that retinal axons grow in the presence of chondroitin sulfate and keratan sulfate proteoglycans and that these proteoglycans in the developing chick visual pathway have functions other than to inhibit axon growth.
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    The retinotectal projection of the chick is established between Embryonic Days 3 and 13 (E3 to E13). Fate mappings of the eye anlage by local injections of the fluorescent dyes DiI and DiA revealed that the anteroposterior axis of the optic vesicle corresponds to the nasotemporal axis of the retina. To investigate possible alterations in retinotopic specificity after ablating parts of the early eye anlage, we resected either most of the presumptive temporal or a large part of the presumptive nasal half of the eye anlage around stage 11 of the Hamburger-Hamilton scale (40-45 hr). After such treatment, the axes are restored in the healed optic vesicle. In the healing process the wound is closed by cells moving in from surrounding areas. After early posterior (i.e., temporal) ablation, the projection from the restored temporal half-retina onto the optic tectum was examined in embryos (E13 to E17) and juvenile chicken (P16) by retrograde and anterograde labeling of ganglion cells and their axons with DiI and DiASP. Normally, only a small fraction of ganglion cells from the temporal retina (between 6.4% on E13 and 0.08% on P16) projects onto the caudal part of the tectum. In experimental embryos and juvenile chicken this fraction is significantly increased (up to 80%). Retrograde double-labeling from the rostral and the caudal tectum reveals that temporal cells project onto either the rostral or the caudal tectum, but not via collaterals upon both areas. The ganglion cells with "displaced nasal" identity within the temporal retina that were backlabeled from the caudal tectum were to a large extent segregated into distinct clusters, indicating their derivation from few or possibly even single progenitor cells. Likewise, ablation of the anterior half of the optic vesicle led to clusters of rostrally projecting cells of "displaced temporal" identity within the restored nasal retina. In these experiments the dorsal-ventral retinotectal relationship remained intact. The simplest, though not exclusive interpretation of the findings is that positional information is imposed as a temporal-to-nasal step function already on the multipotent progenitors of the ganglion cells in the eye anlage by stage 11 or earlier. The positional values remain stable when during the healing of the optic vesicle cells of one specification become surrounded by cells of another specification.
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    A novel homeobox gene, SOHo-1, was isolated from embryonic chicken retina. On embryonic day 2 (E2), SOHo-1 is expressed in the retina, posterolateral otic pit, and neural tube anterior to the spinal cord. On E4, SOHo-1 is expressed at high levels in anterior retina and low levels in posterior retina, suggestive of a role in patterning the anterior-posterior axis. It is also expressed on E4 in the otocyst, the dorsal root ganglia, some cranial ganglia, and the second branchial arch. SOHo-1 expression in the otic pit and otocyst is restricted to regions that will give rise to the nonsensory tissues of the inner ear. SOHo-1 is not closely related to any identified vertebrate or Drosophila homeobox-containing genes. Since it is expressed in sensory-related structures and does not fit into existing classes of homeobox genes, we propose the name SOHo-1, for sensory organ homeobox-1.
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    To study how retinal ganglion cell axons diverge in the optic chiasm, the behavior of dye-labeled fibers was monitored in real time with video microscopy in an isolated preparation of embryonic mouse brain, with a focus on embryonic day 15-16. These real-time studies have revealed the dynamics of the growth of individual retinal axons, especially the tempo of extension and growth cone behaviors during divergence in the chiasm, a model for "decision" regions in developing pathways. Within the chiasm, retinal growth cones extend by saltatory growth, consisting of bursts of rapid advance alternating with pauses in extension. During pauses, growth cone appendages remain motile, and develop asymmetries prior to a change in the axis of growth. In a zone straddling the midline, retinal fibers, irrespective of destination, display long pauses for up to several hours, making small advances and retractions with no net extension. While crossed fibers ultimately progress through the midline, uncrossed fibers from inferior temporal retina develop wide-ranging branched growth cones, and then turn back to the ipsilateral side. Turns are effected by the selective retraction or micropruning of asymmetric foci of motile activity, and by the transformation of a backward-directed filopodium into a new growth cone. The behavior of retinal axons at the midline supports the hypothesis that this locus contains cues important for retinal axon divergence. Moreover, the observations of growth cone kinetics in the chiasm elucidate which growth cone forms seen in static preparations mediate growth cone turning, and suggest a model of axon navigation in decision regions in the intact nervous system.
  • Article
    Neurogenesis, migration and maturation of ganglion cells in the posterior pole of chick retina have been studied using embryonic incorporation of [3H]thymidine, immunocytochemistry and retrograde labeling. Unlike previous studies, we have examined the neurogenesis of independently identified ganglion cells that have survived the period of naturally occurring cell death (embryonic days 11-16). Embryos were labeled with [3H]thymidine at different embryonic ages (embryonic days 3, 5 and 7). After the chicks hatched, ganglion cells were retrogradely labeled with rhodamine microspheres and the retinas were processed for autoradiography and fluorescent microscopy. The results indicate that 40% of the ganglion cells in the posterior pole undergo a final mitosis by embryonic day 3 and that more than 25% of the ganglion cells are born on or after embryonic day 7. These results also suggest that naturally occurring cell death does not preferentially affect ganglion cells born on specific embryonic days. Using immunocytochemistry with an antibody against neuron-specific beta-tubulin and retrograde labeling with the carbocyanine dye DiI we show that ganglion cells begin to differentiate before the completion of their migration to the presumptive ganglion cell layer. These results suggest the following developmental sequence. (1) Ganglion cells of the posterior pole undergo their final mitosis near the ventricular margin between embryonic days 2 and 8. (2) They maintain contacts with both retinal surfaces and their nuclei move toward the ganglion cell layer. At this time they start to differentiate, expressing a form of neuron-specific tubulin and growing axons that can reach the optic chiasm. (3) Once migration is completed dendritic development commences.
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    The zone of polarizing activity (ZPA) is a region at the posterior margin of the limb bud that induces mirror-image duplications when grafted to the anterior of a second limb. We have isolated a vertebrate gene, Sonic hedgehog, related to the Drosophila segment polarity gene hedgehog, which is expressed specifically in the ZPA and in other regions of the embryo, that is capable of polarizing limbs in grafting experiments. Retinoic acid, which can convert anterior limb bud tissue into tissue with polarizing activity, concomitantly induces Sonic hedgehog expression in the anterior limb bud. Implanting cells that express Sonic hedgehog into anterior limb buds is sufficient to cause ZPA-like limb duplications. Like the ZPA, Sonic hedgehog expression leads to the activation of Hox genes. Sonic hedgehog thus appears to function as the signal for antero-posterior patterning in the limb.
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    In the rat, a small subpopulation of retinal ganglion cell axons forms a persistent projection to the ipsilateral half of the brain. These fibres originate almost exclusively from the ventrotemporal margin of the retina. In contrast to all other retinal axons they seem to be deflected from the midline of the optic chiasm and thereby led into the ipsilateral optic tract. In order to analyse the interactions between growing fibres and chiasm midline, we have developed the following in vitro model. Axons of the embryonic rat retina are grown on a carpet of tectal cell membranes used as a general growth-permissive substratum. At a certain distance from the explant (200-450 microns), the advancing fibres are confronted with two stripes of cell membranes prepared from the chiasm midline. Such chiasm membranes are shown to act as a barrier for the presumptive non-crossing axons, while they do not influence growth of fibres originating from any other regions of the retina, including the dorsotemporal part. The repulsion of non-crossing fibres by chiasm membranes is observed in vitro only when retinal explants from embryonic day (E) 17/18 and chiasm preparations from E14/15 are used. Fibres and tissue from different regions of the brain as well as from different developmental ages, and even from different species, can be combined in this assay system. In a first attempt to characterize the molecular basis of the repulsive effect of chiasm membranes on ventrotemporal fibres, similar assays were performed with membranes derived from other regions of the central nervous system midline, some of which are known to have repulsive properties against certain axon populations. Since these cell membranes did not act as a barrier for the ventrotemporal retinal axons, we suggest that the guidance cues at the chiasm are very specific. Our results are consistent with the hypothesis that certain cells at the chiasm midline (very likely radial glial cells) express 'repulsive or inhibitory' molecules, which act in a specific way on ipsilaterally projecting axons.
  • Article
    The specific routing of retinal ganglion cell axons at the mammalian optic chiasm into the ipsilateral or contralateral optic tracts results from axon pathfinding. Using time-lapse microscopy, we show that encounters between axons from opposite eyes at the chiasm induce axon turning, but do not always aim retinal axons into the optic tracts. Following removal of one eye before retinal axons have invaded the chiasm, axons from the remaining eye are still routed into the correct optic tracts. Ipsilaterally projecting axons make turning decisions without pausing over 10-20 min, whereas contralaterally projecting axons occasionally pause before crossing the midline. Thus, initial pathfinding at the chiasm does not depend on binocular axon interactions, but on local cues that trigger differential growth cone responses.
  • Article
    The development of retinal ganglion cells (RGC) was studied in the chick from stage 18 to adulthood. Our main objectives were to identify the retinal site where the first RGCs differentiate, to locate this site relative to the optically defined central retinal area, and to map the spatial arrangement of the RGC field at different stages in development. The eyes of the experimental animals were fixed and serially sectioned. The borders of RGC fields were determined from the presence of either ganglion cell perikarya or ganglion cell axons. In seven cases between stages 21 and 26, the borders of the RGC fields were confirmed electron microscopically. The serial sections together with the RGC fields were then reconstructed in three dimensions. The reconstructed retinae were projected onto a plane by using the radially equidistant polar azimuthal projection. First, RGCs appear dorsal to the apex of the optic fissure. Ganglion cell development then initially spreads out symmetrically with respect to the optic fissure. However, from stage 29 on, the nasal half of the retina expands much more than the temporal half. This asymmetrical growth entails that the optic fissure is eventually located in the temporal half of the retina in the mature animal. The RGC fields of the embryonic stages were superimposed on the retina of a visually active animal according to their real size and position. It turned out that the central retinal area was at least 2 mm away from the site where the first RGCs were generated. It is not before stage 28 that the prospective central retinal area is included into the expanding ganglion cell field. The fact that RGCs at the central retinal area are generated 2.5 days later than first RGCs near the apex of the optic fissure has important implications for the formation of the retinotectal projection.
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