[Show abstract][Hide abstract] ABSTRACT: Axons of retinal ganglion cells (RGCs) make a divergent choice at the optic chiasm to cross or avoid the midline in order to project to ipsilateral and contralateral targets, thereby establishing the binocular visual pathway. The zinc-finger transcription factor Zic2 and a member of the Eph family of receptor tyrosine kinases, EphB1, are both essential for proper development of the ipsilateral projection at the mammalian optic chiasm midline. Here, we demonstrate in mouse by functional experiments in vivo that Zic2 is not only required but is also sufficient to change the trajectory of RGC axons from crossed to uncrossed. In addition, our results reveal that this transcription factor regulates the expression of EphB1 in RGCs and also suggest the existence of an additional EphB1-independent pathway controlled by Zic2 that contributes to retinal axon divergence at the midline.
Development 06/2008; 135(10):1833-41. · 6.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In animals with binocular vision, retinal ganglion cell (RGC) axons from each eye sort in the developing ventral diencephalon to project to ipsi- or contralateral targets, thereby forming the optic chiasm. Ipsilaterally projecting axons arise from the ventrotemporal (VT) retina and contralaterally projecting axons primarily from the other retinal quadrants. The winged helix transcription factor Foxd1 (previously known as BF-2, Brain Factor 2) is expressed in VT retina, as well as in the ventral diencephalon during the formation of the optic chiasm. We report here that in embryos lacking Foxd1, both retinal development and chiasm morphogenesis are disrupted. In the Foxd1 deficient retina, proteins designating the ipsilateral projection, such as Zic2 and EphB1, are missing, and the domain of Foxg1 (BF-1) expands from nasal retina into the VT crescent. In retina-chiasm co-cultures, VT RGCs from Foxd1 deficient retina are not repulsed by chiasm cells, and in vivo many VT RGCs aberrantly project contralaterally. However, even though the ipsilateral program is lost in the retina, a larger than normal uncrossed component develops in Foxd1 deficient embryos. Chiasm defects include axon stalling in the chiasm and a reduction in the total number of RGCs projecting to the optic tract. In addition, in the Foxd1 deficient ventral diencephalon, Foxg1 invades the Foxd1 domain, Zic2 and Islet1 expression are minimized, and Slit2 prematurely expands, changes that could contribute to axon projection errors. Thus, Foxd1 plays a dual role in the establishment of the binocular visual pathways: first, in specification of the VT retina, acting upstream of proteins directing the ipsilateral pathway; and second, in the patterning of the developing ventral diencephalon where the optic chiasm forms.
Development 12/2004; 131(22):5727-39. · 6.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The structurally related cell adhesion molecules L1 and Nr-CAM have overlapping expression patterns in cerebellar granule cells. Here we analyzed their involvement in granule cell development using mutant mice. Nr-CAM-deficient cerebellar granule cells failed to extend neurites in vitro on contactin, a known ligand for Nr-CAM expressed in the cerebellum, confirming that these mice are functionally null for Nr-CAM. In vivo, Nr-CAM-null cerebella did not exhibit obvious histological defects, although a mild size reduction of several lobes was observed, most notably lobes IV and V in the vermis. Mice deficient for both L1 and Nr-CAM exhibited severe cerebellar folial defects and a reduction in the thickness of the inner granule cell layer. Additionally, anti-L1 antibodies specifically disrupted survival and maintenance of Nr-CAM-deficient granule cells in cerebellar cultures treated with antibodies. The combined results indicate that Nr-CAM and L1 play a role in cerebellar granule cell development, and suggest that closely related molecules in the L1 family have overlapping functions.
The Journal of Cell Biology 10/2001; 154(6):1259-73. · 10.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nr-CAM is a member of the L1 subfamily of cell adhesion molecules (CAMs) that belong to the immunoglobulin superfamily. To explore the role of Nr-CAM in the developing nervous system, we prepared specific antibodies against both chick and mouse Nr-CAM using recombinant Fc fusion proteins of chick Nr-CAM and mouse Nr-CAM, respectively. First, we show the specificity of the new anti-chick Nr-CAM antibody compared with a previously employed antibody using the expression patterns of Nr-CAM in the chick spinal cord and floor plate and on commissural axons, where Nr-CAM has been implicated in axon guidance. Using the anti-mouse Nr-CAM antibody, we then studied the expression patterns of Nr-CAM in the developing mouse nervous system along with the patterns of two related CAMs, L1, which labels most growing axons, and TAG-1, which binds to Nr-CAM and has a more restricted distribution. Major sites that are positive for Nr-CAM are specialized glial formations in the ventral midline, including the floor plate in the spinal cord, the hindbrain and midbrain, the optic chiasm, and the median eminence in the forebrain. Similar to what is seen in the chick spinal cord, Nr-CAM is expressed on crossing fibers as they course through these areas. In addition, Nr-CAM is found in crossing fiber pathways, including the anterior commissure, corpus callosum, and posterior commissure, and in nondecussating pathways, such as the lateral olfactory tract and the habenulointerpeduncular tract. Nr-CAM, for the most part, is colocalized with TAG-1 in all of these systems. Based on in vitro studies indicating that the Nr-CAM-axonin-1/TAG-1 interaction is involved in peripheral axonal growth and guidance in the spinal cord [Lustig et al. (1999) Dev Biol 209:340-351; Fitzli et al. (2000) J Cell Biol 149:951-968], the expression patterns described herein implicate a role for this interaction in central nervous system axon growth and guidance, especially at points of decussation. Nr-CAM also is expressed in cortical regions, such as the olfactory bulb. In the hippocampus, however, TAG-1-positive areas are segregated from Nr-CAM-positive areas, suggesting that, in neuropilar regions, Nr-CAM interacts with molecules other than TAG-1.
The Journal of Comparative Neurology 06/2001; 434(1):13-28. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Studies in vitro have revealed a great deal about growth cone behaviors, especially responses to guidance molecules, both positive and negative, and the signaling systems mediating these responses. Little, however, is known about these events as they take place in vivo. With new imaging methods, growth cone behaviors can be chronicled in the complex settings of intact or semi-intact systems. With the retinal projection through the optic chiasm as a model, we examined the hypothesis previously drawn from static material that growth cone form is position-specific: growth cone form in fact reflects specific behaviors, including rate and tempo of extension, that are more or less prominent in different locales in which growth cones are situated. Other studies show that growth cones interact with cells along the pathway, both specialized nonneuronal cells and other neurons, some expressing known guidance molecules. The present challenge is to bridge dynamic imaging with electron microscopy and molecular localization, in order to link growth cone behaviors with cell and molecular interactions in the natural setting in which growth cones extend.
Journal of Neurobiology 09/2000; 44(2):260-70. · 3.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ventral midline of the nervous system is an important choice point at which growing axons decide whether to cross and project contralaterally or remain on the same side of the brain. In Drosophila, the decision to cross or avoid the CNS midline is controlled, at least in part, by the Roundabout (Robo) receptor on the axons and its ligand, Slit, an inhibitory extracellular matrix molecule secreted by the midline glia. Vertebrate homologs of these molecules have been cloned and have also been implicated in regulating axon guidance. Using in situ hybridization, we have determined the expression patterns of robo1,2 and slit1,2,3 in the mouse retina and in the region of the developing optic chiasm, a ventral midline structure in which retinal ganglion cell (RGC) axons diverge to either side of the brain. The receptors and ligands are expressed at the appropriate time and place, in both the retina and the ventral diencephalon, to be able to influence RGC axon guidance. In vitro, slit2 is inhibitory to RGC axons, with outgrowth of both ipsilaterally and contralaterally projecting axons being strongly affected. Overall, these results indicate that Robos and Slits alone do not directly control RGC axon divergence at the optic chiasm and may additionally function as a general inhibitory guidance system involved in determining the relative position of the optic chiasm at the ventral midline of the developing hypothalamus.
Journal of Neuroscience 08/2000; 20(13):4975-82. · 6.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In a new mouse mutant, circletail (Crc), failure of neural tube closure (embryonic day [E] 8-9) is associated with errors in retinal axon projection at the optic chiasm (E12-18), such that many axons normally projecting contralaterally instead grow to ipsilateral targets. Although the architecture of the chiasmatic region is altered, neurons and glia containing putative cues for axon guidance are present. The aberrant ipsilateral-projecting cells originate from a nonrandom expansion of the wild-type uncrossed retinal region. These axon pathway defects are found in two other mutants with cephalic neural tube defects (NTD), loop-tail (Lp) and Pax3 (splotch; Sp(2H)). Crc is phenotypically similar to Lp, exhibiting an open neural tube from midbrain to tail (craniorachischisis), while splotch has spina bifida with or without a cranial NTD. The retinal axon abnormalities occur only in the presence of NTD and not in homozygous mutants lacking cranial NTD. Thus, failure of neural tube closure is associated with failure of many retinal axons to cross the ventral midline. This study therefore reveals an unexpected connection between closure of the neural tube at the dorsal midline and development of ventral axon tracts. genesis 27:32-47, 2000.
[Show abstract][Hide abstract] ABSTRACT: In mammals, some axons from each retina cross at the optic chiasm, whereas others do not. Although several loci have been identified within the chiasmatic region that appear to provide guidance cues to the retinal axons, the underlying molecular mechanisms that regulate this process are poorly understood. Here we investigate whether the earliest retinal axon trajectories and a cellular population (CD44 and stage-specific embryonic antigen 1 [SSEA] neurons), previously implicated in directing axon growth in the developing chiasm (reviewed in Mason and Sretavan  Curr. Op. Neurobiol. 7:647-653), correlate with the expression patterns of several regulatory genes (BF-1, BF-2, Dlx-2, Nkx-2.1, Nkx-2.2, and Shh). These studies demonstrate that gene expression patterns in the chiasmatic region reflect the longitudinal subdivisions of the forebrain in that axon tracts in this region generally are aligned parallel to these subdivisions. Moreover, zones defined by overlapping domains of regulatory gene expression coincide with sites implicated in providing guidance information for retinal axon growth in the developing optic chiasm. Together, these data support the hypothesis that molecularly distinct, longitudinally aligned domains in the forebrain regulate the pattern of retinal axon projections in the developing hypothalamus.
The Journal of Comparative Neurology 02/1999; 403(3):346-58. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neurotrophins cooperate with neural activity to modulate CNS neuronal survival and dendritic differentiation. In a previous study, we demonstrated that a critical balance of neurotrophin and neural activity is required for Purkinje cell survival in cocultures of purified granule and Purkinje cells (Morrison and Mason, 1998). Here we investigate whether TrkB signaling regulates dendrite and spine development of Purkinje cells. BDNF treatment of purified Purkinje cells cultured alone did not elicit formation of mature dendrites or spines. In cocultures of granule and Purkinje cells, however, continuous treatment with BDNF over a 2 week postnatal culture period increased the density of Purkinje cell dendritic spines relative to controls without causing a shift in the proportions of headed and filopodia-like spines. The increase in spine number was blocked by adding TrkB-IgG to the medium together with BDNF. Although BDNF alone did not consistently modify the morphology of dendritic spines, treatment with TrkB-IgG alone yielded spines with longer necks than those in control cultures. None of these treatments altered Purkinje cell dendritic complexity. These analyses reveal a role for TrkB signaling in modulating spine development, consistent with recently reported effects of neurotrophins on synaptic function. Moreover, spine development can be uncoupled from dendrite outgrowth in this reductionist system of purified presynaptic and postsynaptic neurons.
Journal of Neuroscience 12/1998; 18(21):8559-70. · 6.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Granule neurons, presynaptic afferents of Purkinje cells, are potent regulators of Purkinje cell development. Purified Purkinje cells survive and differentiate poorly, whereas coculture with granule neurons enhances their survival and dendritic development. Here we investigate the role of neurotrophins in granule-Purkinje cell interactions. BDNF or NT-4 improves, but NT-3 or CNTF reduces, survival of isolated Purkinje cells. When granule neurons are present, however, BDNF or NT-4 treatment leads to Purkinje cell loss. This decrease is overcome by anti-BDNF or TrkB-IgG-blocking reagents or by CNQX, a non-NMDA glutamate receptor antagonist. Furthermore, BDNF increases the spine density on the surviving Purkinje cells. These results suggest that Purkinje cell survival and differentiation are context-dependent and require a balance between neurotrophin- and activity-dependent signaling.
Journal of Neuroscience 06/1998; 18(10):3563-73. · 6.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In an in vitro model system based on purified target cerebellar granule neurons and explants of afferents, pontine mossy fiber afferents stop growing through contact-mediated mechanisms when they encounter granule neurons. Here we studied the developmental regulation of the stop signal posed by granule cells and the response of mossy fibers to the stop signal in two culture systems. Granule neurons presented in slices or as dissociated cells from postnatal day (P) 4 and P7 cerebellum were more potent in the arrest of P0 pontine neurites than younger (P0-P2) or older (up to P14) granule neurons. In contrast, pontine neurites at embryonic day (E) 18, during their period of normal growth toward the cerebellum, grew extensively on both cerebellar slices of all ages from P0 to P10 and dissociated P4 granule neurons. When E18 explants were maintained for 2 days before plating in medium conditioned by neonatal cerebellar cells, E18 pontine explants were rendered more responsive to the stop signal from P4 granule cells. These results indicate that the stop signal, and the response of afferents to it, are developmentally regulated. Moreover, factors within the target region may initiate these interactions.
[Show abstract][Hide abstract] ABSTRACT: The importance of vision in the behavior of animals, from invertebrates to primates, has led to a good deal of interest in how projection neurons in the retina make specific connections with targets in the brain. Recent research has focused on the cellular interactions occurring between retinal ganglion cell (RGC) axons and specific glial and neuronal populations in the embryonic brain during formation of the mouse optic chiasm. These interactions appear to be involved both in determining the position of the optic chiasm on the ventral diencephalon (presumptive hypothalamus) and in ipsilateral and contralateral RGC axon pathfinding, development events fundamental to binocular vision in the adult animal.
Current Opinion in Neurobiology 11/1997; 7(5):647-53. · 7.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Video time-lapse microscopy has made it possible to document growth cone motility during axon navigation in the intact brain. This approach prompted us to reanalyze the hypothesis, originally derived from observations of fixed tissue, that growth cone form is position-specific. The behaviors of Dil-labeled retinal axon growth cones were tracked from retina through the optic tract in mouse brain at embryonic day (E) 15-17, and these behaviors were matched with different growth cone forms. Patterns of behavior were then analyzed in the different locales from the retina through the optic tract. Throughout the pathway, episodes of advance were punctuated by pauses in extension. Irrespective of locale, elongated streamlined growth cones mediated advance and complex forms developed during pauses. The rate of advance and the duration of pauses were surprisingly similar in different parts of the pathway. In contrast, the duration of periods of advance was more brief in the chiasm compared to those in the optic nerve and tract. Consequently, in the chiasm, growth cones spent relatively more time pausing and less time advancing than in the optic nerve or tract. Thus, because growth cone form is behavior-specific and certain behaviors predominate in particular loci, growth cone form appears to be position-specific in static preparations, due to the fraction of time spent in a given state in different locales.
Journal of Neuroscience 03/1997; 17(3):1086-100. · 6.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: After entering target regions, afferent growth cones grow among putative target cells, stop extending upon meeting target cells, and transit into a synaptic ending. During these events, signals are transmitted to and from target cells to stimulate programs of differentiation. Here we describe three approaches to unraveling mechanisms of these phases of synaptogenesis. First, dye-labeling in the intact cerebellum has revealed the orchestration of afferent ingrowth and contacts with target cells. Second, an in vitro model based on purified granule neurons has shed light on the role of target cells in the arrest of afferent extension. Third, coculture of purified granule neurons (parallel fiber afferents) with purified Purkinje cells has demonstrated facets of afferent regulation of target cell differentiation. These analyses have suggested molecular mechanisms that mediate maturation of afferents and their targets.
Perspectives on developmental neurobiology 01/1997; 5(1):69-82.
[Show abstract][Hide abstract] ABSTRACT: The Eph family of receptor tyrosine kinases and their ligands can be divided into two specificity subclasses: the Eck-related receptors and their GPI-anchored ligands, and the Elk-related receptors and their transmembrane ligands. Previous reports demonstrated that Eck- and Elk-related receptors in the retina distribute in high temporal-low nasal and high ventral-low dorsal gradients, respectively. While others have focused on complementary ligand gradients in the retinal axon target, the tectum, we report that ligands from each subclass also distribute in gradients opposing those of their corresponding receptors within the retina itself. Moreover, ligand gradients in the retina precede ganglion cell genesis. These results support an intraretinal role for Eph family members in addition to their previously proposed role in the development of retinotectal topography. The distinct distributions of Eph family members suggest that each subclass specifies positional information along independent retinal axes.
[Show abstract][Hide abstract] ABSTRACT: To determine whether diffusible guidance cues direct retinal axon growth and divergence at the optic chiasm, we cocultured mouse retinal and chiasm explants in collagen gels. The chiasm reduced retinal neurite lengths and numbers, but did not affect commissural or pontine neurite growth. This reduction in growth was equal for all retinal quadrants and occurred without reorienting the direction of neurite extension. The floor plate, another midline guidance locus, also suppressed retinal neurite outgrowth, whereas cortex or cerebellum explants did not. Growth suppression was not mediated by netrin-1, which instead enhanced retinal neurite extension. We propose that chemosuppression may be a general guidance mechanism that acts in intermediate targets to prime growth cones to perceive other, more specific cues.
[Show abstract][Hide abstract] ABSTRACT: Weaver is a spontaneous mutation in mice characterized by the postnatal loss of external granule cells in the cerebellum and dopaminergic neurons of the midbrain, especially in the substantia nigra. We have shown previously that natural cell death with the morphology of apoptosis occurs in the substantia nigra of normal rodents during postnatal development. We therefore sought to determine whether the loss of dopaminergic neurons in homozygous weaver mice occurs during the period of natural cell death in the substantia nigra and whether it has the morphology of apoptosis. We have found, using a silver stain technique, that although apoptotic cell death does occur early postnatally in homozygous weaver substantia nigra, it also does so with equal magnitude in wild-type and heterozygous weaver littermates. Unique to homozygous weavers is the occurrence of degenerating neurons in the nigra that are not apoptotic. These degenerating neurons are observed at postnatal day 7, and they are most abundant on postnatal days 24-25. The nonapoptotic nature of this cell death is confirmed by negative in situ end labeling of nuclear DNA fragmentation and by ultrastructural analysis. Ultrastructural studies reveal irregular chromatin aggregates in the nucleus, as well as marked cytoplasmic changes, including the formation of vacuoles and distinctive stacks of dilated cisternae of endoplasmic reticulum. We interpret these changes as indicative of either a variant morphology of programmed cell death or a pathological degenerative process mediated by an as yet unknown mechanism related to the recently described mutation in the GIRK2 potassium channel.
Journal of Neuroscience 11/1996; 16(19):6134-45. · 6.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cerebellar granule neurons in vitro specifically arrest the extension of their appropriate presynaptic axons, mossy fibers. This "stop-growing signal" may be an essential step in the formation and specificity of synapses. Here, we have tested whether ionotropic glutamate receptors are involved in the stop-growing signal. When explants of basilar pontine nuclei, a mossy fiber source, were cultured on granule neurons, most pontine neurites terminated <200 microm from their explant of origin, a criterion for the stop-growing signal. In contrast, treatment with the NMDA antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-AP5) greatly increased the number of pontine neurites extending beyond 300 microm, whereas treatment with NMDA reduced the number of pontine neurites extending beyond 200 microm. A non-NMDA agonist (AMPA) and antagonist (6-cyano-7-nitroquinoxaline-2,3-dione) did not alter pontine neurite lengths. None of these agents affected neurite outgrowth from pontine explants in the absence of granule neurons, nor did any agent affect the survival of granule neurons. These results indicate that NMDA and D-AP5 specifically perturb an interaction between axons and target cells necessary for the stop-growing signal, and that NMDA receptors are critical for the development of a major cerebellar afferent system. These findings also suggest that NMDA-sensitive refinement of axon arbors during later development may involve the direct regulation of axon extension by target neurons.
Journal of Neuroscience 04/1996; 16(8):2642-8. · 6.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The visual pathway in albino animals is abnormal in that there is a smaller number of ipsilaterally projecting retinal ganglion cells. There are two possible sites of gene action that could result in such a defect. The first site is the retina where the amount of pigmentation in the retinal pigment epithelium is correlated with the degree of ipsilateral innervation (La Vail et al. (1978) J. Comp. Neurol. 182, 399-422). The second site is the optic chiasm, the site of retinal axon divergence. We investigated these two possibilities through a combination of in vivo and in vitro techniques. Our results demonstrate that the growth patterns of retinal axons and the cellular composition of the optic chiasm in albino mice are similar to those of normally pigmented mice, consistent with the albino mutation exerting its effects in the retina, and not on the cells from the chiasmatic midline. We directly tested whether the albino mutation affects the chiasm by studying 'chimeric' cultures of retinal explants and chiasm cells isolated from pigmented and albino mice. Crossed and uncrossed axons from pigmented or albino retinal explants display the same amount of differential growth when grown on either pigmented or albino chiasm cells, demonstrating that the albino mutation does not disrupt the signals for retinal axon divergence associated with the albino optic chiasm. Furthermore, in vitro, a greater proportion of albino retinal ganglion cells from ventrotemporal retina, origin of uncrossed axons, behave like crossed cells, suggesting that the albino mutation acts by respecifying the numbers of retinal ganglion cells that cross the chiasmatic midline.
Development 04/1996; 122(3):859-68. · 6.21 Impact Factor