Sonic hedgehog contributes to oligodendrocyte specification in the mammalian forebrain

This study addresses the role of Sonic hedgehog (Shh) in promoting the generation of oligodendrocytes in the mouse telencephalon. We show that in the forebrain, expression of the early oligodendrocyte markers Olig2, plp/dm20 and PDGFR(alpha) corresponds to regions of Shh expression. To directly test if Shh can induce the development of oligodendrocytes within the telencephalon, we use retroviral vectors to ectopically express Shh within the mouse embryonic telencephalon. We find that infections with Shh-expressing retrovirus at embryonic day 9.5, result in ectopic Olig2 and PDGFR(alpha) expression by mid-embryogenesis. By postnatal day 21, cells expressing ectopic Shh overwhelmingly adopt an oligodendrocyte identity. To determine if the loss of telencephalic Shh correspondingly results in the loss of oligodendrocyte production, we studied Nkx2.1 mutant mice in which telencephalic expression of Shh is selectively lost. In accordance with Shh playing a role in oligodendrogenesis, within the medial ganglionic eminence of Nkx2.1 mutants, the early expression of PDGFR(alpha) is absent and the level of Olig2 expression is diminished in this region. In addition, in these same mutants, expression of both Shh and plp/dm20 is lost in the hypothalamus. Notably, in the prospective amygdala region where Shh expression persists in the Nkx2.1 mutant, the presence of plp/dm20 is unperturbed. Further supporting the idea that Shh is required for the in vivo establishment of early oligodendrocyte populations, expression of PDGFR(alpha) can be partially rescued by virally mediated expression of Shh in the Nkx2.1 mutant telencephalon. Interestingly, despite the apparent requirement for Shh for oligodendrocyte specification in vivo, all regions of either wild-type or Nkx2.1 mutant telencephalon are competent to produce oligodendrocytes in vitro. Furthermore, analysis of CNS tissue from Shh null animals definitively shows that, in vitro, Shh is not required for the generation of oligodendrocytes. We propose that oligodendrocyte specification is negatively regulated in vivo and that Shh generates oligodendrocytes by overcoming this inhibition. Furthermore, it appears that a Shh-independent pathway for generating oligodendrocytes exists.     

Slits contribute to the guidance of retinal ganglion cell axons in the mammalian optic tract

RGC axons extend in the optic tracts in a manner that correlates with the expression in the hypothalamus and epithalamus of a soluble factor inhibitory to RGC axon outgrowth. Additionally, although the RGC axons extend adjacent to the telencephalon, they do not normally grow into this tissue. Here, we show that slit1 and slit2, known chemorepellents for RGC axons expressed in specific regions of the diencephalon and telencephalon, help regulate optic tract development. In mice lacking slit1 and slit2, a subset of RGC axons extend into the telencephalon and grow along the pial surface but not more deeply into this tissue. Surprisingly, distinct guidance errors occur in the telencephalon of slit1 -/-; slit2 +/- and slit1/2 -/- embryos, suggesting that the precise level of Slits is critical for determining the path followed by individual axons. In mice lacking both slit1 and slit2, a subset of RGC axons also project aberrantly into the epithalamus, pineal and across the dorsal midline. However, many axons reach their primary target, the superior colliculus. This demonstrates that Slits play an important role in directing the guidance of post-crossing RGC axons within the optic tracts but are not required for target innervation.     

Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum.

The telencephalon is organized into distinct longitudinal domains: the cerebral cortex and the basal ganglia. The basal ganglia primarily consists of a dorsal region (striatum) and a ventral region (pallidum). Within the telencephalon, the anlage of the pallidum expresses the Nkx2.1 homeobox gene. A mouse deficient in Nkx2.1 function does not form pallidal structures, lacks basal forebrain TrkA-positive neurons (probable cholinergic neurons) and has reduced numbers of cortical cells expressing GABA, DLX2 and calbindin that migrate from the pallidum through the striatum and into the cortex. We present evidence that these phenotypes result from a ventral-to-dorsal transformation of the pallidal primordium into a striatal-like anlage.     

Pioneers in the Ventral Telencephalon

OL-protocadherin is a member of the non-clustered-type protocadherin family. A recent study of ours showed that it is essential not only for growth of striatal axons but also for higher ordered neural circuit formation in the ventral telencephalon. The phenotype of OL-protocadherin-deficient mice is striking: several major neural pathways such as thalamocortical pathway, corticothalamic pathway, corticospinal pathway and strionigral pathway were misrouted and/or congested in the ventral telencephalon. Moreover, we detected abnormal patterning of putative guidance cues for thalamocortical axons such as the Nkx2.1+ cell domain and permissive "corridor" in the ventral telencephalon. Analyses of the expression pattern and phenotypes suggested that deficiency of striatal axons is the primary cause of these phenotypes. With these observations in mind, we proposed a novel hypothesis that proper growth of striatal axons is essential in patterning guidance cues and subsequent formation of neural circuits in the ventral telencephalon. This hypothesis will open a new possibility to reveal the unknown mechanism of neural circuit formation in the ventral telencephalon.     

Defects in thalamocortical axon pathfinding correlate with altered cell domains in Mash-1-deficient mice

We have analyzed the pathfinding of thalamocortical axons (TCAs) from dorsal thalamus to neocortex in relation to specific cell domains in the forebrain of wild-type and Mash-1-deficient mice. In wild-type mice, we identified four cell domains that constitute the proximal part of the TCA pathway. These domains are distinguished by patterns of gene expression and by the presence of neurons retrogradely labeled from dorsal thalamus. Since the cells that form these domains are generated in forebrain proliferative zones that express high levels of Mash-1, we studied Mash-1 mutant mice to assess the potential roles of these domains in TCA pathfinding. In null mutants, each of the domains is altered: the two Pax-6 domains, one in ventral thalamus and one in hypothalamus, are expanded in size; a complementary RPTP(delta) domain in ventral thalamus is correspondingly reduced and the normally graded expression of RPTP(delta) in that domain is no longer apparent. In ventral telencephalon, a domain characterized in the wild type by Netrin-1 and Nkx-2.1 expression and by retrogradely labeled neurons is absent in the mutant. Defects in TCA pathfinding are localized to the borders of each of these altered domains. Many TCAs fail to enter the expanded, ventral thalamic Pax-6 domain that constitutes the most proximal part of the TCA pathway, and form a dense whorl at the border between dorsal and ventral thalamus. A proportion of TCAs do extend further distally into ventral thalamus, but many of these stall at an aberrant, abrupt border of high RPTP(delta) expression. A small proportion of TCAs extend around the RPTP(delta) domain and reach the ventral thalamic-hypothalamic border, but few of these axons turn at that border to extend into the ventral telencephalon. These findings demonstrate that Mash-1 is required for the normal development of cell domains that in turn are required for normal TCA pathfinding. In addition, these findings support the hypothesis that ventral telencephalic neurons and their axons guide TCAs through ventral thalamus and into ventral telencephalon.     

Area specificity and topography of thalamocortical projections are controlled by ephrin/Eph genes

The mechanisms generating precise connections between specific thalamic nuclei and cortical areas remain poorly understood. Using axon tracing analysis of ephrin/Eph mutant mice, we provide in vivo evidence that Eph receptors in the thalamus and ephrins in the cortex control intra-areal topographic mapping of thalamocortical (TC) axons. In addition, we show that the same ephrin/Eph genes unexpectedly control the inter-areal specificity of TC projections through the early topographic sorting of TC axons in an intermediate target, the ventral telencephalon. Our results constitute the first identification of guidance cues involved in inter-areal specificity of TC projections and demonstrate that the same set of mapping labels is used differentially for the generation of topographic specificity of TC projections between and within individual cortical areas.     

Olfactory bulb axonal outgrowth is inhibited by draxin

Olfactory bulb (OB) projection neurons receive sensory input from olfactory receptor neurons and precisely relay it through their axons to the olfactory cortex. Thus, olfactory bulb axonal tracts play an important role in relaying information to the higher order of olfactory structures in the brain. Several classes of axon guidance molecules influence the pathfinding of the olfactory bulb axons. Draxin, a recently identified novel class of repulsive axon guidance protein, is essential for the formation of forebrain commissures and can mediate repulsion of diverse classes of neurons from chickens and mice. In this study, we have investigated the draxin expression pattern in the mouse telencephalon and its guidance functions for OB axonal projection to the telencephalon. We have found that draxin is expressed in the neocortex and septum at E13 and E17.5 when OB projection neurons form the lateral olfactory tract (LOT) rostrocaudally along the ventrolateral side of the telencephalon. Draxin inhibits axonal outgrowth from olfactory bulb explants in vitro and draxin-binding activity in the LOT axons in vivo is detected. The LOT develops normally in draxin−/− mice despite subtle defasciculation in the tract of these mutants. These results suggest that draxin functions as an inhibitory guidance cue for OB axons and indicate its contribution to the formation of the LOT.     

Sonic hedgehog maintains the identity of cortical interneuron progenitors in the ventral telencephalon

Fate determination in the mammalian forebrain, where mature phenotypes are often not achieved until postnatal stages of development, has been an elusive topic of study despite its relevance to neuropsychiatric disease. In the ventral telencephalon, major subgroups of cerebral cortical interneurons originate in the medial ganglionic eminence (MGE), where the signaling molecule sonic hedgehog (Shh) continues to be expressed during the period of neuronogenesis. To examine whether Shh regulates cortical interneuron specification, we studied mice harboring conditional mutations in Shh within the neural tube. At embryonic day 12.5, NestinCre:Shh(Fl/Fl) mutants have a relatively normal index of S-phase cells in the MGE, but many of these cells do not co-express the interneuron fate-determining gene Nkx2.1. This effect is reproduced by inhibiting Shh signaling in slice cultures, and the effect can be rescued in NestinCre:Shh(Fl/Fl) slices by the addition of exogenous Shh. By culturing MGE progenitors on a cortical feeder layer, cell fate analyses suggest that Shh signaling maintains Nkx2.1 expression and cortical interneuron fate determination by MGE progenitors. These results are corroborated by the examination of NestinCre:Shh(Fl/Fl) cortex at postnatal day 12, in which there is a dramatic reduction in cell profiles that express somatostatin or parvalbumin. By contrast, analyses of Dlx5/6Cre:Smoothened(Fl/Fl) mutant mice suggest that cell-autonomous hedgehog signaling is not crucial to the migration or differentiation of most cortical interneurons. These results combine in vitro and ex vivo analyses to link embryonic abnormalities in Shh signaling to postnatal alterations in cortical interneuron composition.     

Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons

GABAergic interneurons have major roles in hippocampal function and dysfunction. Here we provide evidence that, in mice, virtually all of these cells originate from progenitors in the basal telencephalon. Immature interneurons tangentially migrate from the basal telencephalon through the neocortex to take up their final positions in the hippocampus. Disrupting differentiation in the embryonic basal telencephalon (lateral and medial ganglionic eminences) through loss of Dlx1/2 homeobox function blocks the migration of virtually all GABAergic interneurons to the hippocampus. On the other hand, disrupting specification of the medial ganglionic eminence through loss of Nkx2.1 homeobox function depletes the hippocampus of a distinct subset of hippocampal interneurons. Loss of hippocampal interneurons does not appear to have major effects on the early development of hippocampal projection neurons nor on the pathfinding of afferrent tracts.     

The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio

We have examined neuronal differentiation and the formation of axon tracts in the embryonic forebrain and midbrain of the zebrafish, between 1 and 2 days postfertilisation. Axons were visualised with three techniques; immunocytochemistry (using HNK-1 and antiacetylated tubulin antibodies) and horseradish peroxidase (HRP) labelling in whole-mounted brains, and transmission electron microscopy. Differentiation was monitored by histochemical staining for acetylcholinesterase (AChE). These independent methods demonstrated that a simple grid of tracts and commissures forms the initial axon scaffold of the brain. At 1 day, the olfactory nerve, four commissures, their associated tracts and three other non-commissural tracts are present. By 2 days, these tracts and commissures have all greatly enlarged and, in addition, the optic nerve and tract, and three new commissures and their associated tracts have been added. Small applications of HRP at various sites revealed the origins and projections of some of these earliest axons. Retrogradely labelled cell bodies originated from regions that were also positive for AChE activity. At 1 day, HRP-labelled axons were traced: (1) from the olfactory placode through the olfactory nerve to the dorsal telencephalon; (2) from the telencephalon into the tract of the anterior commissure and also to the postoptic region of the diencephalon; (3) from the hindbrain through the ventral midbrain and diencephalon to the postoptic commissure; (4) from the dorsal diencephalon (in or near the epiphysis) to the tract of the postoptic commissure; (5) from ventral and rostral midbrain through the posterior commissure. Three new projections were demonstrated at 2 days: (1) from the retina through the tract of the postoptic commissure to the tectum; (2) from the telencephalon to the contralateral diencephalon; and (3) from the telencephalon to the ventral flexure. These results show that at 1 day, the zebrafish brain is impressively simple, with a few small, well-separated tracts but by 2 days the brain is already considerably more complex. Most of the additional axons added onto pre-existent tracts rather than pioneered new ones supporting the notion that other axons play a crucial role in the guidance of early central nervous system (CNS) axons.     

Aberrant development of hippocampal circuits and altered neural activity in netrin 1-deficient mice

Diffusible factors, including netrins and semaphorins, are believed to be important cues for the formation of neural circuits in the forebrain. Here we have examined the role of netrin 1 in the development of hippocampal connections. We show that netrin 1 and its receptor, Dcc, are expressed in the developing fimbria and in projection neurons, respectively, and that netrin 1 promotes the outgrowth of hippocampal axons in vitro via DCC receptors. We also show that the hippocampus of netrin 1-deficient mice shows a misorientation of fiber tracts and pathfinding errors, as detected with antibodies against the surface proteins TAG-1, L1 and DCC. DiI injections show that hippocampal commissural axons do not cross the midline in these mutants. Instead, when axons approach the midline, they turn ventrally and form a massive aberrant projection to the ipsilateral septum. In addition, both the ipsilateral entorhino-hippocampal and the CA3-to-CA1 associational projections show an altered pattern of layer-specific termination in netrin 1-deficient mice. Finally, optical recordings with the Ca(2+) indicator Fura 2-AM show that spontaneous neuronal activity is reduced in the septum of netrin 1-mutant mice. We conclude that netrin 1 is required not only for the formation of crossed connections in the forebrain, but also for the appropriate layer-specific targeting of ipsilateral projections and for the control of normal levels of spontaneous neural activity.     

Dorsoventral patterning is established in the telencephalon of mutants lacking both Gli3 and Hedgehog signaling

Considerable data suggest that sonic hedgehog (Shh) is both necessary and sufficient for the specification of ventral pattern throughout the nervous system, including the telencephalon. We show that the regional markers induced by Shh in the E9.0 telencephalon are dependent on the dorsoventral and anteroposterior position of ectopic Shh expression. This suggests that by this point in development regional character in the telencephalon is established. To determine whether this prepattern is dependent on earlier Shh signaling, we examined the telencephalon in mice carrying either Shh- or Gli3-null mutant alleles. This analysis revealed that the expression of a subset of ventral telencephalic markers, including Dlx2 and Gsh2, although greatly diminished, persist in Shh(-/-) mutants, and that these same markers were expanded in Gli3(-/-) mutants. To understand further the genetic interaction between Shh and Gli3, we examined Shh/Gli3 and Smoothened/Gli3 double homozygous mutants. Notably, in animals carrying either of these genetic backgrounds, genes such as Gsh2 and Dlx2, which are expressed pan-ventrally, as well as Nkx2.1, which demarcates the ventral most aspect of the telencephalon, appear to be largely restored to their wild-type patterns of expression. These results suggest that normal patterning in the telencephalon depends on the ventral repression of Gli3 function by Shh and, conversely, on the dorsal repression of Shh signaling by Gli3. In addition these results support the idea that, in addition to hedgehog signaling, a Shh-independent pathways must act during development to pattern the telencephalon.     

Specificity and Plasticity of Thalamocortical Connections in Sema6A Mutant Mice

The establishment of connectivity between specific thalamic nuclei and cortical areas involves a dynamic interplay between the guidance of thalamocortical axons and the elaboration of cortical areas in response to appropriate innervation. We show here that Sema6A mutants provide a unique model to test current ideas on the interactions between subcortical and cortical guidance mechanisms and cortical regionalization. In these mutants, axons from the dorsal lateral geniculate nucleus (dLGN) are misrouted in the ventral telencephalon. This leads to invasion of presumptive visual cortex by somatosensory thalamic axons at embryonic stages. Remarkably, the misrouted dLGN axons are able to find their way to the visual cortex via alternate routes at postnatal stages and reestablish a normal pattern of thalamocortical connectivity. These findings emphasize the importance and specificity of cortical cues in establishing thalamocortical connectivity and the spectacular capacity of the early postnatal cortex for remapping initial sensory representations.