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.     

Type III neuregulin 1 regulates pathfinding of sensory axons in the developing spinal cord and periphery

Sensory axons must develop appropriate connections with both central and peripheral targets. Whereas the peripheral cues have provided a classic model for neuron survival and guidance, less is known about the central cues or the coordination of central and peripheral connectivity. Here we find that type III Nrg1, in addition to its known effect on neuron survival, regulates axon pathfinding. In type III Nrg1(-/-) mice, death of TrkA(+) nociceptive/thermoreceptive neurons was increased, and could be rescued by Bax elimination. In the Bax and type III Nrg1 double mutants, axon pathfinding abnormalities were seen for TrkA(+) neurons both in cutaneous peripheral targets and in spinal cord central targets. Axon guidance phenotypes in the spinal cord included penetration of axons into ventral regions from which they would normally be repelled by Sema3A. Accordingly, sensory neurons from type III Nrg1(-/-) mice were unresponsive to the repellent effects of Sema3A in vitro, which might account, at least in part, for the central projection phenotype, and demonstrates an effect of type III Nrg1 on guidance cue responsiveness in neurons. Moreover, stimulation of type III Nrg1 back-signaling in cultured sensory neurons was found to regulate axonal levels of the Sema3A receptor neuropilin 1. These results reveal a molecular mechanism whereby type III Nrg1 signaling can regulate the responsiveness of neurons to a guidance cue, and show that type III Nrg1 is required for normal sensory neuron survival and axon pathfinding in both central and peripheral targets.     

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.     

Temporal regulation of neuropilin-1 expression and sensitivity to semaphorin 3A in NGF- and NT3-responsive chick sensory neurons

The extracellular molecule semaphorin 3A (Sema3A) is proposed to be a negative guidance cue that participates in patterning DRG sensory axons in the developing chick spinal cord. During development Sema3A is first expressed throughout the spinal cord gray matter, but Sema3A expression later disappears from the dorsal horn, where small-caliber cutaneous afferents terminate. Sema3A expression remains in the ventral horn, where large-muscle proprioceptive afferents terminate. It has been proposed that temporal changes in the sensitivity of different classes of sensory afferents to Sema3A contribute to the different pathfinding of these sensory afferents. This study compared the expression of the semaphorin 3A receptor subunit, neuropilin-1, and the collapse response of growth cones to semaphorin 3A for NGF (cutaneous)- and NT3 (proprioceptive)-dependent sensory axons extended from E6-E10 chick embryos. Growth cones extended from E6 DRGs in NT3-containing medium expressed neuropilin-1 and collapsed in response to Sema3A. From E7 until E10 NT3-responsive growth cones expressed progressively lower levels of neuropilin-1, and were less sensitive to Sema3A. On the other hand, growth cones extended from DRGs in NGF-containing medium expressed progressively higher levels of neuropilin-1 and higher levels of collapse response to Sema3A over the period from E6-E10. Thus, developmental patterning of sensory terminals in the chick spinal cord may arise from changes in both Sema3A expression in the developing spinal cord and accompanying changes in neuronal expression of the Sema3A receptor subunit, neuropilin-1.     

Temporal regulation of neuropilin‐1 expression and sensitivity to semaphorin 3A in NGF‐ and NT3‐responsive chick sensory neurons

The extracellular molecule semaphorin 3A (Sema3A) is proposed to be a negative guidance cue that participates in patterning DRG sensory axons in the developing chick spinal cord. During development Sema3A is first expressed throughout the spinal cord gray matter, but Sema3A expression later disappears from the dorsal horn, where small‐caliber cutaneous afferents terminate. Sema3A expression remains in the ventral horn, where large‐muscle proprioceptive afferents terminate. It has been proposed that temporal changes in the sensitivity of different classes of sensory afferents to Sema3A contribute to the different pathfinding of these sensory afferents. This study compared the expression of the semaphorin 3A receptor subunit, neuropilin‐1, and the collapse response of growth cones to semaphorin 3A for NGF (cutaneous)‐ and NT3 (proprioceptive)‐dependent sensory axons extended from E6‐E10 chick embryos. Growth cones extended from E6 DRGs in NT3‐containing medium expressed neuropilin‐1 and collapsed in response to Sema3A. From E7 until E10 NT3‐responsive growth cones expressed progressively lower levels of neuropilin‐1, and were less sensitive to Sema3A. On the other hand, growth cones extended from DRGs in NGF‐containing medium expressed progressively higher levels of neuropilin‐1 and higher levels of collapse response to Sema3A over the period from E6–E10. Thus, developmental patterning of sensory terminals in the chick spinal cord may arise from changes in both Sema3A expression in the developing spinal cord and accompanying changes in neuronal expression of the Sema3A receptor subunit, neuropilin‐1. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 43–53, 2002     

GAP-43 is critical for normal targeting of thalamocortical and corticothalamic, but not trigeminothalamic axons in the whisker barrel system

Mice lacking the growth-associated protein GAP-43 (KO) show disrupted cortical topography and no barrels. Whisker-related patterns of cells are normal in the KO brainstem trigeminal complex (BSTC), while the pattern in KO ventrobasal thalamus (VB) is somewhat compromised. To better understand the basis for VB and cortical abnormalities, we used small placements of DiI to trace axonal projections between BSTC, VB, and barrel cortex in wildtype (WT) and GAP-43 KO mice. The trigeminothalamic (TT) pathway consists of axons from cells in the Nucleus Prinicipalis that project to the contralateral VB thalamus. DiI-labeled KO TT axons crossed the midline from BSTC and projected to contralateral VB normally, consistent with normal BSTC cytoarchitecture. By contrast, the KO thalamocortical axons (TCA) projection was highly abnormal. KO TCAs showed delays of 1-2 days in initial ingrowth to cortex. Postnatally, KO TCAs showed multiple pathfinding errors near intermediate targets, and were abnormally fasciculated within the internal capsule (IC). Interestingly, most individually labeled KO TCAs terminated in deep layers instead of in layer IV as in WT. This misprojection is consistent with birthdating analysis in KO mice, which revealed that neurons normally destined for layer IV remain in deep cortical layers. Early outgrowth of KO corticofugal (CF) axons was similar for both genotypes. However, at P7 KO CF fibers remained bundled as they entered the IC, and exhibited few terminal branches in VB. Thus, the establishment of axonal projections between thalamus and cortex are disrupted in GAP-43 KO mice.     

Immunohistochemical localization of Pax6 in the developing tooth germ of mice

Recent studies have reported that supernumerary teeth were observed in the maxillary incisor area in several Pax6 homozygous mutant mouse and rat strains. To date, it remains unknown whether Pax6 is expressed during tooth development in any species. The study aimed to analyze the expression of Pax6 during mouse incisor and molar development. C57BL/6J mouse embryos on days E12.5, E13.5, E14.5, E16.5 and E18.5 were produced. Heads from these embryos, as well as from P1.5 mice, were processed for paraffin wax embedding (N ≥ 3 for each stage) and prepared for immunohistochemistry. Pax6 immunostaining was found in all tooth germs examined. At the E12.5 dental placode, E13.5 bud stage, E14.5 cap stage and E16.5 early bell stage, Pax6 was expressed in ectodermally derived tissues of tooth germs and oral epithelia adjacent to the tooth germs. Cells in the underlying dental ectomesenchyme that showed Pax9 expression were Pax6 negative. At E18.5 and P1.5, Pax6 was expressed in more differentiated ameloblasts and cells of the stratum intermedium and stellate reticulum that were derived from the oral epithelium, as well as in mesenchyme-derived differentiated odontoblasts. Pax6 expression was also observed in the submandibular gland, tongue filiform papilla and hair follicle at E16.5 and P1.5. The present study demonstrated that Pax6 was expressed in incisor and molar germs during mouse tooth development. The results provide a basis for exploring the function of Pax6 during tooth development.     

Tlx and Pax6 co-operate genetically to establish the pallio-subpallial boundary in the embryonic mouse telencephalon

We have examined the role of Tlx, an orphan nuclear receptor, in dorsal-ventral patterning of the mouse telencephalon. Tlx is expressed broadly in the ventricular zone, with the exception of the dorsomedial and ventromedial regions. The expression spans the pallio-subpallial boundary, which separates the dorsal (i.e. pallium) and ventral (i.e. subpallium) telencephalon. Despite being expressed on both sides of the pallio-subpallial boundary, Tlx homozygous mutants display alterations in the development of this boundary. These alterations include a dorsal shift in the expression limits of certain genes that abut at the pallio-subpallial boundary as well as the abnormal formation of the radial glial palisade that normally marks this boundary. The Tlx mutant phenotype is similar to, but less severe than, that seen in Small eye (i.e. Pax6) mutants. Interestingly, removal of one allele of Pax6 on the homozygous Tlx mutant background significantly worsens the phenotype. Thus Tlx and Pax6 cooperate genetically to regulate the establishment of the pallio-subpallial boundary. The patterning defects in the Tlx mutant telencephalon result in a loss of region-specific gene expression in the ventral-most pallial region. This correlates well with the malformation of the lateral and basolateral amygdala in Tlx mutants, both of which have been suggested to derive from ventral portions of the pallium.     

Effects of canonical Wnt signaling on dorso-ventral specification of the mouse telencephalon

Wnt signaling is involved in numerous processes during vertebrate CNS development. In this study, we used conditional Cre/loxP system in mouse to ablate or activate beta-catenin in the telencephalon in two time windows: before and after the onset of neurogenesis. We show that beta-catenin mediated Wnt signals are required to maintain the molecular identity of the pallium. Inactivation of beta-catenin in the telencephalon before neurogenesis results in downregulated expression of dorsal markers Emx1, Emx2 and Ngn2, and in ectopic up-regulation of ventral markers Gsh2, Mash1 and Dlx2 in the pallium. In contrast, ablation of ss-catenin after the onset of cortical neurogenesis (E11.5) does not result in a dorso-ventral fate shift. In addition, activation of canonical Wnt signaling in the subpallium leads to a repression of ventral telencephalic cell identities as shown by the down-regulation of subpallial markers Dlx2, Nkx2.1, Gsh2, Olig2 and Mash1. This was accompanied with an expansion of dorsal identities ventrally as shown by the expanded expression domains of pallial markers Pax6 and Ngn2. Thus, our data suggest that canonical Wnt signals are involved in maintaining the identity of the pallium by controlling expression of dorsal markers and by suppressing ventral programs from being activated in pallial progenitor cells.     

Dual functional activity of semaphorin 3B is required for positioning the anterior commissure

Chemorepulsion by semaphorins plays a critical role during the development of neuronal projections. Although semaphorin-induced chemoattraction has been reported in vitro, the contribution of this activity to axon pathfinding is still unclear. Using genetic and culture models, we provide evidence that both attraction and repulsion by Sema3B, a secreted semaphorin, are critical for the positioning of a major brain commissural projection, the anterior commissure (AC). NrCAM, an immunoglobulin superfamily adhesion molecule of the L1 subfamily, associates with neuropilin-2 and is a component of a receptor complex for Sema3B and Sema3F. Finally, we show that activation of the FAK/Src signaling cascade distinguishes Sema3B-mediated attractive from repulsive axonal responses of neurons forming the AC, revealing a mechanism underlying the dual activity of this guidance cue.     

A novel approach to selectively target neuronal subpopulations reveals genetic pathways that regulate tangential migration in the vertebrate hindbrain

Vertebrate genes often play functionally distinct roles in different subsets of cells; however, tools to study the cell-specific function of gene products are poorly developed. Therefore, we have established a novel mouse model that enables the visualization and manipulation of defined subpopulations of neurons. To demonstrate the power of our system, we dissected genetic cascades in which Pax6 is central to control tangentially migrating neurons of the mouse brainstem. Several Pax6 downstream genes were identified and their function was analyzed by over-expression and knock-down experiments. One of these, Pou4f2, induces a prolonged midline arrest of growth cones to influence the proportion of ipsilaterally versus contralaterally settling neurons. These results demonstrate that our approach serves as a versatile tool to study the function of genes involved in cell migration, axonal pathfinding, and patterning processes. Our model will also serve as a general tool to specifically over-express any gene in a defined subpopulation of neurons and should easily be adapted to a wide range of applications.     

The prenatal development of alkaline phosphatase activity in the hypothalamus of the rat

The localization of alkaline phosphatase (E.C. 3.1.3.1.) positivity during prenatal development of the hypothalamus of the rat is described. At E12 all layers of the prosencephalon display alkaline phosphatase (AP) positivity. The AP positivity increases from dorsal to ventral. Within the hypothalamic area a second, rostro-ventral gradient exists from E14 onwards. At E18 both gradients have decreased. At E20 almost all AP positivity has disappeared from the hypothalamus, with the exception of some reaction product in the dorsal ventricular matrix of the hypothalamus. The significance of this pattern in relation to the differentiation of the hypothalamus and to the formation of hypothalamic connections is discussed. It is suggested that AP activity is related to the formation of connections.