Thalamocortical axons are influenced by chemorepellent and chemoattractant activities localized to decision points along their path

Thalamocortical axons (TCAs), which originate in dorsal thalamus, project ventrally in diencephalon and then dorsolaterally in ventral telencephalon to their target, the neocortex. To elucidate potentially key decision points in TCA pathfinding and hence the possible localization of guidance cues, we used DiI-tracing to describe the initial trajectory of TCAs in mice. DiI-labeled TCAs extend ventrally on the lateral surface of ventral thalamus. Rather than continuing this trajectory onto the lateral surface of the hypothalamus, TCAs make a sharp lateral turn into ventral telencephalon. This behavior suggests that the hypothalamus is repulsive and the ventral telencephalon attractive for TCAs. In support of this hypothesis, we find that axon outgrowth from explants of dorsal thalamus is biased away from hypothalamus and toward ventral telencephalon when cocultured at a distance in collagen gels. The in vivo DiI analysis also reveals a broad cluster of retrogradely labeled neurons in the medial part of ventral telencephalon positioned within or adjacent to the thalamocortical pathway prior to or at the time TCAs are extending through it. The axons of these neurons extend into or through dorsal thalamus and appear to be coincident with the oppositely extending TCAs. These findings suggest that multiple cues guide TCAs along their pathway from dorsal thalamus to neocortex: TCAs may fasciculate on the axons of ventral telencephalic neurons as they extend through ventral thalamus and the medial part of ventral telencephalon, and chemorepellent and chemoattractant activities expressed by hypothalamus and ventral telencephalon, respectively, may cooperate to promote the turning of TCAs away from hypothalamus and into ventral telencephalon.     

Early thalamocortical tract guidance and topographic sorting of thalamic projections requires LIM-homeodomain gene Lhx2

The thalamocortical tract is the primary source of sensory information to the cerebral cortex, but the mechanisms regulating its pathfinding are not completely understood. LIM-homeodomain (LIM-HD) gene Lhx2 has been proposed to participate in a combinatorial "code" to regulate dorsal thalamic patterning and also the topography of thalamocortical projections. Here, we report that Lhx2-/- embryos exhibit a gross disruption in the early development of the thalamocortical tract, such that thalamic axons are unable to enter the ventral telencephalon. A possible cause for this deficit is a severe reduction of "pioneer" cells in the mutant ventral telencephalon that constitutes a putative mechanism for guiding the entry of the thalamocortical tract into this structure in vivo. However, in vitro, the thalamocortical tract is able to enter the ventral telencephalon, and this permitted an examination of whether thalamocortical topography is normal in the Lhx2 mutant. Contrary to hypotheses that proposed a cell-autonomous role for Lhx2 in the thalamus, Lhx2-/- thalamic explants generate a normal topography of projections in control ventral telencephalic preparations. This is consistent with our findings of normal patterning of the Lhx2 mutant dorsal thalamus using a wide array of markers. In the reverse experiment, however, control thalamic explants display aberrant topography in Lhx2-/- telencephalic preparations. This perturbation is restricted to projections from caudal thalamic explants, while rostral and middle explants project normally. Thus Lhx2 is required for multiple steps in thalamocortical tract pathfinding, but these functions appear localized in the ventral telencephalon rather than in the dorsal thalamic neurons. Furthermore, the absence of Lhx2 in the ventral telencephalon selectively disrupts a subset of thalamic axon topography, indicating a specific rather than a general perturbation of cues in this structure.     

Tangential neuronal migration controls axon guidance: a role for neuregulin-1 in thalamocortical axon navigation

Neuronal migration and axon guidance constitute fundamental processes in brain development that are generally studied independently. Although both share common mechanisms of cell biology and biochemistry, little is known about their coordinated integration in the formation of neural circuits. Here we show that the development of the thalamocortical projection, one of the most prominent tracts in the mammalian brain, depends on the early tangential migration of a population of neurons derived from the ventral telencephalon. This tangential migration contributes to the establishment of a permissive corridor that is essential for thalamocortical axon pathfinding. Our results also demonstrate that in this process two different products of the Neuregulin-1 gene, CRD-NRG1 and Ig-NRG1, mediate the guidance of thalamocortical axons. These results show that neuronal tangential migration constitutes a novel mechanism to control the timely arrangement of guidance cues required for axonal tract formation in the mammalian brain.     

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.     

Neogenin interacts with RGMa and netrin-1 to guide axons within the embryonic vertebrate forebrain

In the embryonic forebrain, pioneer axons establish a simple topography of dorsoventral and longitudinal tracts. The cues used by these axons during the initial formation of the axon scaffold remain largely unknown. We have investigated the axon guidance role of Neogenin, a member of the immunoglobulin (Ig) superfamily that binds to the chemoattractive ligand Netrin-1, as well as to the chemorepulsive ligand repulsive guidance molecule (RGMa). Here, we show strong expression of Neogenin and both of its putative ligands in the developing Xenopus forebrain. Neogenin loss-of-function mutants revealed that this receptor was essential for axon guidance in an early forming dorsoventral brain pathway. Similar mutant phenotypes were also observed following loss of either RGMa or Netrin-1. Simultaneous partial knock downs of these molecules revealed dosage-sensitive interactions and confirmed that these receptors and ligands were acting in the same pathway. The results provide the first evidence that Neogenin acts as an axon guidance molecule in vivo and support a model whereby Neogenin-expressing axons respond to a combination of attractive and repulsive cues as they navigate their ventral trajectory.     

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.     

Mechanisms of ventral patterning in the vertebrate nervous system

Dorsoventral patterning of the neural tube has a crucial role in shaping the functional organization of the CNS. It is well established that hedgehog signalling plays a key role in specifying ventral cell types throughout the neuroectoderm, and major progress has been made in elucidating how hedgehog signalling works in this ventral specification. In addition, other molecular pathways, including nodal, retinoic acid and fibroblast growth factor signalling, have been identified as important molecular cues for ventral patterning of the spinal cord, telencephalon and eye. Here, we discuss recent advances in this field, highlighting the emerging interplay of these signalling pathways in the molecular specification of ventral patterning at different rostrocaudal levels of the CNS.     

Region-specific requirement for cholesterol modification of sonic hedgehog in patterning the telencephalon and spinal cord

Sonic hedgehog (Shh) secreted from the axial signaling centers of the notochord and prechordal plate functions as a morphogen in dorsoventral patterning of the neural tube. Active Shh is uniquely cholesterol-modified and the hydrophobic nature of cholesterol suggests that it might regulate Shh spreading in the neural tube. Here, we examined the capacity of Shh lacking the cholesterol moiety (ShhN) to pattern different cell types in the telencephalon and spinal cord. In mice expressing ShhN, we detected low-level ShhN in the prechordal plate and notochord, consistent with the notion that ShhN can rapidly spread from its site of synthesis. Surprisingly, we found that low-level ShhN can elicit the generation of a full spectrum of ventral cell types in the spinal cord, whereas ventral neuronal specification and ganglionic eminence development in the Shh(N/-) telencephalon were severely impaired, suggesting that telencephalic patterning is more sensitive to alterations in local Shh concentration and spreading. In agreement, we observed induction of Shh pathway activity and expression of ventral markers at ectopic sites in the dorsal telencephalon indicative of long-range ShhN activity. Our findings indicate an essential role for the cholesterol moiety in restricting Shh dilution and deregulated spread for patterning the telencephalon. We propose that the differential effect of ShhN in patterning the spinal cord versus telencephalon may be attributed to regional differences in the maintenance of Shh expression in the ventral neuroepithelium and differences in dorsal tissue responsiveness to deregulated Shh spreading behavior.     

Motoneurons are essential for vascular pathfinding

The neural and vascular systems share common guidance cues that have direct and independent signaling effects on nerves and endothelial cells. Here, we show that zebrafish Netrin 1a directs Dcc-mediated axon guidance of motoneurons and that this neural guidance function is essential for lymphangiogenesis. Specifically, Netrin 1a secreted by the muscle pioneers at the horizontal myoseptum (HMS) is required for the sprouting of dcc-expressing rostral primary motoneuron (RoP) axons and neighboring axons along the HMS, adjacent to the future trajectory of the parachordal chain (PAC). These axons are required for the formation of the PAC and, subsequently, the thoracic duct. The failure to form the PAC in netrin 1a or dcc morphants is phenocopied by laser ablation of motoneurons and is rescued both by cellular transplants and overexpression of dcc mRNA. These results provide a definitive example of the requirement of axons in endothelial guidance leading to the parallel patterning of nerves and vessels in vivo.     

EphA4-dependent axon guidance is mediated by the RacGAP alpha2-chimaerin

Neuronal network formation in the developing nervous system is dependent on the accurate navigation of nerve cell axons and dendrites, which is controlled by attractive and repulsive guidance cues. Ephrins and their cognate Eph receptors mediate many repulsive axonal guidance decisions by intercellular interactions resulting in growth cone collapse and axon retraction of the Eph-presenting neuron. We show that the Rac-specific GTPase-activating protein alpha2-chimaerin binds activated EphA4 and mediates EphA4-triggered axonal growth cone collapse. alpha-Chimaerin mutant mice display a phenotype similar to that of EphA4 mutant mice, including aberrant midline axon guidance and defective spinal cord central pattern generator activity. Our results reveal an alpha-chimaerin-dependent signaling pathway downstream of EphA4, which is essential for axon guidance decisions and neuronal circuit formation in vivo.     

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.     

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum¹. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra². The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions^3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or βIII tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.     

Expression of extracellular matrix molecules in the embryonic rat olfactory pathway

Primary olfactory neurons arise from placodal neuroepithelium that is separate from the neuroepithelial plate that forms the neural tube and crest. The axons of these neurons course along a stereotypical pathway and invade the rostral telencephalic vesicle where they induce the formation of the olfactory bulb. In the present study we examined the expression of several extracellular matrix constituents during formation of the olfactory nerve pathway in order to identify putative developmentally significant molecules. Double-label immunofluorescence was used to simultaneously map the trajectory of growing primary olfactory axons by expression of growth associated protein 43 (GAP-43) and the distribution of either laminin, heparan sulfate proteoglycans (HSPG), or chondroitin sulfate proteoglycans (CSPG). At embryonic day 12.5 (E12.5) primary olfactory axons have exited the olfactory neuroepithelium of the nasal pit and formed a rudimentary olfactory nerve. These axons together with migrating neural cells form a large mass outside the rostral surface of the telencephalon. This nerve pathway is clearly defined by a punctate distribution of laminin and HSPG. CSPG is selectively present in the mesenchyme between the olfactory nerve pathway and the nasal pit and in the marginal zone of the telencephalon. At E14.5 primary olfactory axons pierce the telencephalon through gaps that have emerged in the basement membrane. At this age both laminin and HSPG are colocalized with the primary olfactory axons that have entered the marginal zone of the telencephalon. CSPG expression becomes downregulated in this same region while it remains highly expressed in the marginal zone adjacent to the presumptive olfactory bulb. By E16.5 most of the basement membrane separating the olfactory nerve from the telencephalon has degraded, and there is direct continuity between the olfactory nerve pathway and the central nervous system. This strict spatiotemporal regulation of extracellular matrix constituents in the olfactory nerve pathway supports an important role of these molecules in axon guidance. We propose that laminin and HSPG are expressed by migrating olfactory Schwann cells in the developing olfactory nerve pathway and that these molecules provide a conducive substrate for axon growth between the olfactory neuroepithelium and the brain. CSPG in the surrounding mesenchyme may act to restrict axon growth to within this pathway. The regional degradation of the basement membrane of the telencephalon and the downregulation of CSPG within the marginal zone probably facilitates the passage of primary olfactory axons into the brain to form the presumptive nerve fiber layer of the olfactory bulb. © 1996 John Wiley & Sons, Inc.     

Positional cues that are strictly localized in the telencephalon induce preferential growth of mitral cell axons

In mice, mitral cells are the major efferent neurons of the main olfactory bulb and elongate axons into a very narrow part of the telencephalon to form a fiber bundle referred to as the lateral olfactory tract (LOT). To clarify the mechanisms responsible for guidance of mitral cell axons along this particular pathway, we co-cultured mouse embryo main olfactory bulbs with the telencephalons, and analyzed the pathways taken by mitral cell axons. Ingrowth of mitral cell axons into the telencephalon was observed in those co-cultures in which the olfactory bulbs had been exactly combined to their normal pathway (the LOT position) of the telencephalon. The axons grew preferentially along the LOT position, and formed a LOT-like fiber bundle. When the olfactory bulbs were grafted at positions apart from their normal pathway, however, no mitral cell axons grew into the telencephalon. Neocortical fragments combined with the telencephalon projected fibers into the telencephalon in random directions. These results suggest that the LOT position of the telencephalon offers a guiding pathway for mitral cell axons and that guiding cues for mitral cell axons are extremely localized. © 1996 John Wiley & Sons, Inc.     

Emergent growth cone responses to combinations of Slit1 and Netrin 1 in thalamocortical axon topography

How guidance cues are integrated during the formation of complex axonal tracts remains largely unknown. Thalamocortical axons (TCAs), which convey sensory and motor information to the neocortex, have a rostrocaudal topographic organization initially established within the ventral telencephalon [1-3]. Here, we show that this topography is set in a small hub, the corridor, which contains matching rostrocaudal gradients of Slit1 and Netrin 1. Using in vitro and in vivo experiments, we show that Slit1 is a rostral repellent that positions intermediate axons. For rostral axons, although Slit1 is also repulsive and Netrin 1 has no chemotactic activity, the two factors combined generate attraction. These results show that Slit1 has a dual context-dependent role in TCA pathfinding and furthermore reveal that a combination of cues produces an emergent activity that neither of them has alone. Our study thus provides a novel framework to explain how a limited set of guidance cues can generate a vast diversity of axonal responses necessary for proper wiring of the nervous system.     

Mechanisms and Molecules of Neuronal Wiring: A Primer

The complex patterns of neuronal wiring in the adult nervous system depend on a series of guidance events during neural development that establish a framework on which functional circuits can be built. In this subject collection, the cellular and molecular mechanisms that underlie neuronal guidance are considered from several perspectives, ranging from how cytoskeletal dynamics within extending neuronal growth cones steer axons, to how guidance cues influence synaptogenesis. We introduce here some basic topics to frame the more detailed reviews in following articles, including the cellular strategies that define basic themes governing neuronal wiring throughout life, an enumeration of the molecular cues and receptors known to play key guidance roles during neural development, and an overview of the signaling mechanisms that transduce guidance information into growth-cone steering.Nerve processes extend toward their immediate and final targets with remarkable precision. At the tip of an extending axon is a flattened, fan-shaped structure called a growth cone, with many long, thin spikes that radiate outward much like fingers on a glove. Classical observations of neuronal growth cones and the formation of axonal and dendritic trajectories during neural development led to the conclusion that extrinsic cues must exist that have the capacity to steer extending neuronal processes. For over 100 years, neuroscientists have searched for these cues, their cell surface receptors, and an understanding of how the cues signal spatial information to the extending neuronal processes to direct neural circuit formation.A wealth of cellular observations indicate that growth cones are actively directed along their prescribed pathways. In this collection, Raper and Mason review the extensive body of experiments that support this view (Raper and Mason 2010). These studies reveal that neural wiring occurs through a combination of initial neuronal activity-independent guidance events, and that these early formed connections are subsequently refined through electrical signaling among neurons. The cues that initially guide axons and dendrites can function at both long and short ranges, and they are capable of influencing the bundling of axons together into nerves or fascicles (termed “fasciculation”) and also of mediating interactions between nerves and the substrates on which they extend (Fig. 1). Guidance cues associated with particular intermediate or final targets can be chemoattractive or chemorepulsive, and provide the information essential for selective guidance of distinct neuronal populations. Sequential responses to guidance cues as axons extend over very long distances toward their targets allow for complex pathways to develop, but this often requires that neurons extinguish their responses to certain cues and acquire responsiveness to others at key choice points. Much work over the past several decades has been devoted to identifying these guidance cues and their receptors, and to understanding how cellular responses to these cues change to allow for guidance of extending neuronal processes along discrete segments of their journey to their final targets.Open in a separate windowFigure 1.The diversity of neuronal guidance mechanisms. Neuronal processes are guided by cues that can function at long and short distances to mediate either attractive or repulsive guidance.     

Acetylcholine influences growth cone motility and morphology of developing thalamic axons

The neurotransmitter acetylcholine (ACh) is expressed in the developing telencephalon at the time when thalamic axons project to the cortex, long before synapses are being formed. Since previous studies demonstrated an influence of ACh on neurite extension we used different in vitro assays to examine possible effects of ACh on the growth of thalamic axons. In explant cultures, application of ACh reduced the length of thalamic axons in a dose dependent manner, an effect that could also be evoked by selective muscarinic and nicotinic agonists. Time-lapse imaging of thalamic axons exposed to microscopic gradients of ACh revealed that growth cones no longer advanced, but maintained high filopodial activity. This growth cone pausing was not accompanied by axon retraction or growth cone collapse. It could at least partially be blocked by muscarinic and nicotinic antagonists, indicating that both types of ACh receptors contribute to mediate these effects on thalamic axons. Finally, we also found that ACh changed the morphology of growth cones; they became larger and extended more filopodia. Since such changes in the structure and motility of growth cones are observed at decision regions along the path of many fiber populations including thalamic axons, we suggest that ACh plays a role during the elaboration of thalamocortical projections.Key words: cortical development, thalamocortical projections, neurotransmitter, acetylcholine, growth cone, axonal guidance, wiring molecules     

Orientation of commissural axons in vitro in response to a floor plate-derived chemoattractant

Developing axons are guided to their targets by molecular cues in their local environment. Some cues are short-range, deriving from cells along axonal pathways. There is also increasing evidence for longer-range guidance cues, in the form of gradients of diffusible chemoattractant molecules, which originate from restricted populations of target cells. The guidance of developing commissural axons within the spinal cord depends on one of their intermediate cellular targets, the floor plate. We have shown previously that floor plate cells secrete a diffusible factor(s) that can alter the direction of commissural axon growth in vitro. Here we show that the factor is an effective chemoattractant for commissural axons. It can diffuse considerable distances through a collagen gel matrix and through dorsal and ventral neural epithelium in vitro to reorient the growth of virtually all commissural axons. The orientation of axons occurs in the absence of detectable effects on the survival of commissural neurons or on the rate of commissural axon extension. The regionally restricted expression of the factor suggests that it is present in the embryonic spinal cord in a gradient with its high point at the floor plate. These observations support the idea that the guidance of commissural axons to the ventral midline of the spinal cord results in part from the secretion of a chemoattractant by the floor plate.     

The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity

The Nodal and Hedgehog signaling pathways influence dorsoventral patterning at all axial levels of the CNS, but it remains largely unclear how these pathways interact to mediate patterning. Here we show that, in zebrafish, Nodal signaling is required for induction of the homeobox genes nk2.1a in the ventral diencephalon and nk2.1b in the ventral telencephalon. Hedgehog signaling is also required for telencephalic nk2.1b expression but may not be essential to establish diencephalic nk2.1a expression. Furthermore, Shh does not restore ventral diencephalic development in embryos lacking Nodal activity. In contrast, Shh does restore telencephalic nk2.1b expression in the absence of Nodal activity, suggesting that Hedgehog signaling acts downstream of Nodal activity to pattern the ventral telencephalon. Thus, the Nodal pathway regulates ventral forebrain patterning through both Hedgehog signaling-dependent and -independent mechanisms.