Combinatorial expression patterns of LIM-homeodomain and other regulatory genes parcellate developing thalamus.

The anatomical and functional organization of dorsal thalamus (dTh) and ventral thalamus (vTh), two major regions of the diencephalon, is characterized by their parcellation into distinct cell groups, or nuclei, that can be histologically defined in postnatal animals. However, because of the complexity of dTh and vTh and difficulties in histologically defining nuclei at early developmental stages, our understanding of the mechanisms that control the parcellation of dTh and vTh and the differentiation of nuclei is limited. We have defined a set of regulatory genes, which include five LIM-homeodomain transcription factors (Isl1, Lhx1, Lhx2, Lhx5, and Lhx9) and three other genes (Gbx2, Ngn2, and Pax6), that are differentially expressed in dTh and vTh of early postnatal mice in distinct but overlapping patterns that mark nuclei or subsets of nuclei. These genes exhibit differential expression patterns in dTh and vTh as early as embryonic day 10.5, when neurogenesis begins; the expression of most of them is detected as progenitor cells exit the cell cycle. Soon thereafter, their expression patterns are very similar to those that we observe postnatally, indicating that unique combinations of these genes mark specific cell groups from the time they are generated to their later differentiation into nuclei. Our findings suggest that these genes act in a combinatorial manner to control the specification of nuclei-specific properties of thalamic cells and the differentiation of nuclei within dTh and vTh. These genes may also influence the pathfinding and targeting of thalamocortical axons through both cell-autonomous and non-autonomous mechanisms.

Differential expression of COUP-TFI, CHL1, and two novel genes in developing neocortex identified by differential display PCR.

Genes that control the specification and differentiation of the functionally specialized areas of the mammalian neocortex are likely expressed across the developing neocortex in graded or restricted patterns. To search for such genes we have performed a PCR-based differential display screen using RNAs from rostral neocortex, which included the primary motor area, and caudal neocortex, which included the primary visual area, of embryonic day 16 rats. We identified 82 differentially expressed gene fragments. Secondary screening by in situ hybridization confirmed that five fragments, representing four genes, are differentially expressed across developing rat neocortex. Two of the genes, chick ovalbumin upstream transcription factor I (COUP-TFI) and close homolog of L1 (CHL1), have been cloned previously, but their differential expression in cortex has not been reported. Sequences from the other two fragments suggest that they represent novel genes. The expression patterns include graded, restricted, and discontinuous expression with abrupt borders that might correlate with those of areas. The differential expression patterns of all four genes are established before the arrival of thalamocortical afferents, suggesting that they are independent of thalamic influence, and could direct or reflect arealization. In addition, COUP-TFI and CHL1 exhibit dynamic expression patterns that undergo substantial changes after thalamocortical afferents invade the cortical plate, suggesting that thalamic axons may influence their later expression. Postnatally, COUP-TFI is most prominently expressed in layer 4, in both rats and mice, and CHL1 is expressed in layer 5. COUP-TFI expression in cortex, and in ventral telencephalon and dorsal thalamus, suggests several possible causes for the loss of layer 4 neurons and the reduced thalamocortical projection reported in COUP-TFI knock-out mice.

Regulation of area identity in the mammalian neocortex by Emx2 and Pax6.

The contribution of extrinsic and genetic mechanisms in determining areas of the mammalian neocortex has been a contested issue. This study analyzes the roles of the regulatory genes Emx2 and Pax6, which are expressed in opposing gradients in the neocortical ventricular zone, in specifying areas. Changes in the patterning of molecular markers and area-specific connections between the cortex and thalamus suggest that arealization of the neocortex is disproportionately altered in Emx2 and Pax6 mutant mice in opposing manners predicted from their countergradients of expression: rostral areas expand and caudal areas contract in Emx2 mutants, whereas the opposite effect is seen in Pax6 mutants. These findings suggest that Emx2 and Pax6 cooperate to regulate arealization of the neocortex and to confer area identity to cortical cells.

Graded and areal expression patterns of regulatory genes and cadherins in embryonic neocortex independent of thalamocortical input.

The differentiation of areas of the mammalian neocortex has been hypothesized to be controlled by intrinsic genetic programs and extrinsic influences such as those mediated by thalamocortical afferents (TCAs). To address the interplay between these intrinsic and extrinsic mechanisms in the process of arealization, we have analyzed the requirement of TCAs in establishing or maintaining graded or areal patterns of gene expression in the developing mouse neocortex. We describe the differential expression of Lhx2, SCIP, and Emx1, representatives of three different classes of transcription factors, and the type II classical cadherins Cad6, Cad8, and Cad11, which are expressed in graded or areal patterns, as well as layer-specific patterns, in the cortical plate. The differential expression of Lhx2, SCIP, Emx1, and Cad8 in the cortical plate is not evident until after TCAs reach the cortex, whereas Cad6 and Cad11 show subtle graded patterns of expression before the arrival of TCAs, which later become stronger. We find that these genes exhibit normal-appearing graded or areal expression patterns in Mash-1 mutant mice that fail to develop a TCA projection. These findings show that TCAs are not required for the establishment or maintenance of the graded and areal expression patterns of these genes and strongly suggest that their regulation is intrinsic to the developing neocortex.

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.

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.

Retinal axon guidance by region-specific cues in diencephalon.

Retinal axons show region-specific patterning along the dorsal-ventral axis of diencephalon: retinal axons grow in a compact bundle over hypothalamus, dramatically splay out over thalamus, and circumvent epithalamus as they continue toward the dorsal midbrain. In vitro, retinal axons are repulsed by substrate-bound and soluble activities in hypothalamus and epithalamus, but invade thalamus. The repulsion is mimicked by a soluble floor plate activity. Tenascin and neurocan, extracellular matrix molecules that inhibit retinal axon growth in vitro, are enriched in hypothalamus and epithalamus. Within thalamus, a stimulatory activity is specifically upregulated in target nuclei at the time that retinal axons invade them. These findings suggest that region-specific, axon repulsive and stimulatory activities control retinal axon patterning in the embryonic diencephalon.

Graded and lamina-specific distributions of ligands of EphB receptor tyrosine kinases in the developing retinotectal system.

Molecular gradients have been postulated to control the topographic mapping of retinal axons in their central targets. Based initially on their expression patterns, and more recently on functional studies, members of the EphA subfamily of receptor tyrosine kinases and their ephrin-A ligands have been implicated in the guidance of retinal axons along the anterior-posterior axis of the chick optic tectum. The report that a receptor of the EphB subfamily, EphB2/Cek5/Nuk/Sek3, is expressed in a high ventral to low dorsal gradient in the developing chick retina and is present on ganglion cell axons suggests that it may be involved in the mapping of retinal axons along the corresponding dorsal-ventral axis of the tectum. To address this issue, we have determined the expression and distribution of ephrin-B1/LERK-2/Cek5-L and ephrin-B2/LERK-5/Htk-L/ELF-2, ligands for EphB2, in the developing chick retinotectal system using riboprobes, immunocytochemistry, and receptor affinity probes. Both ephrin-B1 and ephrin-B2 transcripts are expressed in a high dorsal to low ventral gradient in the developing retina, complementary to the distribution of EphB2. Ephrin-B1 and ephrin-B2 proteins are predominantly found in the developing plexiform layers, suggesting a role in the development of intraretinal connections. Neither protein is detected on ganglion cell axons. In tectum, ephrin-B1 transcripts are expressed in a high dorsal to low ventral gradient in the neuroepithelium and the protein is present along the processes of radial glia and is concentrated at their endfeet in the stratum opticum, at the time retinal axons are growing through it. This distribution of ephrin-B1 suggests that it influences retinal axon mapping along the dorsal-ventral tectal axis and may also be involved in intratectal development. In contrast, ephrin-B2 transcripts and protein are localized to the deeper retinorecipient laminae in the tectum at the time retinal axons begin to arborize in them, suggesting that this ligand may influence the laminar patterning of retinal axon terminations.

Glutamate receptor blockade at cortical synapses disrupts development of thalamocortical and columnar organization in somatosensory cortex.

The segregation of thalamocortical inputs into eye-specific stripes in the developing cat or monkey visual cortex is prevented by manipulations that perturb or abolish neural activity in the visual pathway. Such findings show that proper development of the functional organization of visual cortex is dependent on normal patterns of neural activity. The generalisation of this conclusion to other sensory cortices has been questioned by findings that the segregation of thalamocortical afferents into a somatotopic barrel pattern in developing rodent primary somatosensory cortex (S1) is not prevented by activity blockade. We show that a temporary block of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat S1 during the critical period for barrel development disrupts the topographic refinement of thalamocortical connectivity and columnar organization. These effects are evident well after the blockade is ineffective and thus may be permanent. Our findings show that neural activity and specifically the activation of postsynaptic cortical neurons has a prominent role in establishing the primary sensory map in S1, as well as the topographic organization of higher order synaptic connections.

Labeling neural cells using adenoviral gene transfer of membrane-targeted GFP.

We describe an experimental system to visualize the soma and processes of mammalian neurons and glia in living and fixed preparations by using a recombinant adenovirus vector to transfer the jellyfish green fluorescent protein (GFP) into postmitotic neural cells both in vitro and in vivo. We have introduced several modifications of GFP that enhance its fluorescence intensity in mammalian axons and dendrites. This method should be useful for studying the dynamic processes of cell migration and the development of neuronal connections, as well as for analyzing the function of exogenous genes introduced into cells using the adenovirus vector.

Maturation-dependent upregulation of growth-promoting molecules in developing cortical plate controls thalamic and cortical neurite growth.

We have tested the hypothesis that maturation-dependent changes in the cortical plate affect the spatiotemporal growth patterns of developing thalamocortical and corticocortical axonal projections. Given a choice between alternating lanes of embryonic (E18-19) and neonatal (P0-1) rat cortical plate membranes, embryonic (E18-19) thalamic and cortical neurites prefer to extend on neonatal membranes. Thalamic and cortical explants do extend neurites on uniform carpets of E19 cortical plate membranes, but the outgrowth is consistently greater on uniform carpets of P1 cortical plate membranes. These experiments demonstrate a maturation-dependent enhancement in the ability of cortical plate to support neurite growth from thalamic and cortical explants. In contrast, retinal and cerebellar neurites, which do not grow into cortex in vivo, generally grew poorly on these membranes, suggesting a degree of specificity to the neurite growth response. Immunohistochemical analysis of developing cortex suggests that several extracellular matrix (ECM) and cell adhesion molecules are upregulated in cortical plate. However, immunocharacterization of membrane carpets for these same ECM and cell adhesion molecules suggests that the growth preferences of thalamic and cortical neurites in vitro are predominantly influenced by membrane-anchored, rather than ECM, molecules. Western analysis of E19 and P1 cortical plate membranes supports this conclusion, and indicates that the membrane-anchored cell adhesion molecules L1 and N-CAM are more abundant in the P1 cortical plate membrane preparation. Experiments in which cortical plate membranes were treated to remove molecules sensitive to phosphatidylinositol (PI)-specific phospholipase C demonstrate that neurite growth promoters present in E19 cortical plate membranes are predominantly PI linked, whereas those present in P1 membranes are predominantly non-PI linked. These findings indicate that the neurite growth preferences are mediated, at least in part, by an upregulation of neurite growth-promoting molecules in developing cortical plate that are not PI linked. Taken together, these findings suggest that a maturation-dependent upregulation of neurite growth-promoting molecules on cortical plate cells controls the invasion of the cortical plate by thalamocortical and corticocortical axons.

Development of acetylcholinesterase-positive thalamic and basal forebrain afferents to embryonic rat neocortex.

By combining anterograde and retrograde axonal tracing with AChE histochemistry, we demonstrate the sources of AChE-positive afferents to embryonic neocortex, the pathways they use, their time of arrival into cortex, and their initial invasion of the cortical plate. Acetylcholinesterase (AChE) is expressed by two populations of cortical afferents: AChE is permanently present in basal forebrain fibers and has been reported to be transiently localized in axons of the principal sensory thalamic nuclei over the first few postnatal weeks beginning at the middle of the first week. We first detect AChE-positive afferents histochemically in neocortex on embryonic day seventeen (E17) and determine that they arise from the principal sensory thalamic nuclei. AChE histochemistry labels the entire length of developing thalamocortical axons, including their growth cones and branches. These AChE-positive afferents enter the neocortex by the internal capsule and take an intracortical pathway centered on the subplate layer. As soon as these axons are detected, some have already begun to extend AChE-positive collateral branches superficially toward the cortical plate. By E19, a few collaterals have entered the deep part of the cortical plate and by E21 have densely invaded all but its most superficial undifferentiated part. AChE-positive afferents from basal forebrain structures reach the neocortex by three routes: the external capsule, the internal capsule, and the cingulate bundle. Among basal forebrain components, only the substantia innominata and nucleus basalis of Meynert reach the cortex by the internal capsule. Afferents from these two sources reach neocortex on E18, but are a very minor component of the total population of AChE-positive afferents at this age. Afferents from other basal forebrain components do not reach neocortex until several days later. The spatial and temporal patterns of AChE expression in developing thalamocortical axons indicate that it is useful for delineating their innervation of the primary sensory areas of embryonic neocortex, and suggest that AChE may function in axon extension and cortical differentiation.

Development, critical period plasticity, and adult reorganizations of mammalian somatosensory systems.

This review covers recent progress in three major areas of investigation in somatosensory systems: development, developmental plasticity and functional reorganization. Important findings relate to the development of periphery-related patterning in thalamic afferents to somatosensory cortex, the controversial role of neural activity in the development and plasticity of periphery-related afferent patterning in the brainstem and cortex, experience-dependent reorganizations in adult somatosensory cortex, and the locus of these changes.

Early development of the somatotopic map and barrel patterning in rat somatosensory cortex.

Several lines of evidence implicate a crucial role for thalamic afferents from the ventroposterior nucleus (VP) in the development of barrels and their characteristic pattern in the primary somatosensory cortex (S1) of rodents. We sought to determine the stage in development when VP thalamocortical afferents are first distributed in a periphery-related pattern and the sequence of events that culminate in a mature pattern. Using acetylcholinesterase (AChE) histochemistry, an early marker for VP thalamocortical afferents, and the anterograde axon tracer DiI, we show that VP thalamocortical afferents become distributed into a periphery-related pattern earlier than was previously reported, including their parcellation into a barrel-related pattern that mirrors the distribution of sensory hairs on the face. The earliest periphery-related patterning observed is transiently present in the deep cortical layers prior to the emergence of layer 4, the layer in which barrels later develop. AChE histochemistry reveals a clear sequence of maturation of the barrel pattern in the distribution of VP afferents: An initially patternless distribution of AChE-reactive afferents is followed by their distribution in a nascent trigeminal representation, from which rows subsequently emerge; barrel-related clusters of afferents then emerge from the rows. This process begins before birth, and the transition from row-related to barrel-related distributions of VP afferents is evident during the first postnatal day (P0). This demonstration of a periphery-related pattern in developing rat S1 precedes by about 2 days that revealed by any other marker reported to delineate barrels. These findings confirm that VP thalamocortical afferents are the first barrel component to have a periphery-related pattern and support the hypothesis that thalamocortical afferents provide to immature S1 the patterning information that initiates the formation of barrels and their characteristic array. Furthermore because these findings show an earlier onset for barrel formation than was previously realized, they necessitate a reevaluation of conclusions drawn from experiments examining developmental plasticity in barrel patterning.

Thalamocortical axons extend along a chondroitin sulfate proteoglycan-enriched pathway coincident with the neocortical subplate and distinct from the efferent path.

The distinct axonal tracts of the mature nervous system are defined during development by sets of substrate-bound and diffusible molecular signals that promote or restrict axonal elongation. In the adult cerebral cortex, efferent and afferent axons are segregated within the white matter. To define the relationship of growing efferent and afferent axons in the developing murine cortex to chondroitin sulfate proteoglycans (CSPGs) in the pericellular and extracellular matrix, we used the fluorescent tracer Dil to determine axonal trajectories and immunolabeling to disclose the distribution of CSPGs. Axons of neurons in the preplate are the first to leave the cortex; they arise in the CSPG-rich preplate and extend obliquely across it to enter the CSPG-poor intermediate zone. Slightly later, axons of cortical plate neurons extend directly across the CSPG-rich subplate, and then turn abruptly to run in the upper intermediate zone. In contrast, once afferent axons from the thalamus reach the developing cortical wall, their intracortical trajectory is centered on the CSPG-rich subplate, above the path taken by efferent axons. Our findings demonstrate a molecular difference between the adjacent but distinct efferent and afferent pathways in developing neocortex. Early efferents cross the subplate and follow a pathway that contains very little CSPG, while afferents preferentially travel more superficially within the CSPG-rich subplate. Thus, CSPGs and associated extracellular matrix (ECM) components in the preplate/subplate do not form a barrier to axonal initiation or outgrowth in the neocortex as they may in other locations. Instead, their distribution suggests a role in defining discrete axonal pathways during early cortical development.

Acetylcholinesterase as an early marker of the differentiation of dorsal thalamus in embryonic rats.

The enzyme acetylcholinesterase (AChE) is transiently expressed in rats by neurons of the principal sensory thalamic nuclei, although these neurons do not use acetylcholine as a neurotransmitter. Reports that AChE expression begins at late embryonic stages led to the proposal that AChE may function in the establishment of connections, but not in earlier events. However, we find AChE reactivity in rat dorsal thalamus 5 days earlier than previously described. Cells that form the ventrobasal complex (VB), the dorsal lateral geniculate nucleus (dLG) and the medial geniculate nucleus, express AChE as they migrate and aggregate into definitive nuclei. AChE-positive cells are occasionally observed in the dorsal thalamic neuroepithelium, but are more common in others regions of the diencephalic neuroepithelium. AChE reactivity delineates VB and dLG earlier than Nissl-stained cytoarchitecture. These findings indicate that AChE is an early marker of neuronal differentiation. Certain properties of AChE, together with its early detection, are consistent with a proposed role in the migration of principal sensory neurons and their organization into discrete nuclei.

Growth and targeting of subplate axons and establishment of major cortical pathways.

In the developing mammalian neocortex, the first postmitotic neurons form the "preplate" superficial to the neuroepithelium. The preplate is later split into a marginal zone (layer 1) and subplate by cortical plate neurons that form layers 2-6. Cortical efferent axons from layers 5 and 6 and cortical afferent axons from thalamus pass between cortex and subcortical structures through the internal capsule. Here, we identify in rats the axonal populations that establish the internal capsule, and characterize the potential role of subplate axons in the development of cortical efferent and afferent projections. The early growth of cortical efferent and afferent axons was studied using 1-1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) as an anterograde and retrograde tracer in aldehyde-fixed brains of embryonic rats. Cortical axons first enter the nascent internal capsule on embryonic day (E) 14 and originate from lateral and anterior cortex; axons from posterior cortex extend rostrally but do not yet exit cortex. The labeled axons, tipped by growth cones with complex morphologies, take a pathway deep to the preplate. Preplate neurons extend these early cortical efferents, based on the developmental stage of the cortex, and on their location and morphology. Most of these cells later occupy the subplate. Cortical plate neurons extend axons into the internal capsule by E16. En route to the internal capsule, cortical plate axons take the same path as the earlier-growing preplate axons, through the intermediate zone deep to subplate. Subplate axons reach thalamus by E16; the first cortical plate axons enter thalamus about a day later. Thalamic axons enter cortex by E16, prior to other cortical afferents. On E15, both preplate and thalamic axons reach the midpoint of the internal capsule. To determine the subcortical distribution of subplate axons, we used Dil as a retrograde tracer in aldehyde-fixed brains and fast blue and rhodamine-B-isothiocyanate as in vivo retrograde markers in neonatal rats. Tracers were injected into the superior colliculus, the principal midbrain target of layer 5 neurons, at times before, during, and after the arrival of cortical axons, or into the subcortical pathway of primary layer 5 axons at two points, the cerebral peduncle caudal to the internal capsule, and the pyramidal decussation at the junction of the hindbrain and spinal cord, at times shortly after the passing of cortical axons. In every case, the labeled neurons are confined to layer 5; subplate neurons are not labeled.(ABSTRACT TRUNCATED AT 400 WORDS)