Slit Promotes Branching and Elongation of Neurites of Interneurons But Not Projection Neurons from the Developing Telencephalon

doi:10.1006/mcne.2002.1156

Molecular and Cellular Neuroscience

Volume 21, Issue 2, October 2002, Pages 250-265

https://doi.org/10.1006/mcne.2002.1156 Get rights and content

Abstract

Proper neuronal migration and establishment of circuitry are key processes for laying down the functional network of cortical neurons. A variety of environmental guidance cues, attractive or repulsive, have been shown to guide cell migration and axon arborization. One of these, Slit, appears to possess contrarian properties; it can either inhibit axon outgrowth or promote branching and elongation. The object of the present study was to assess the effect of Slit on MGE and neocortical neurons in culture and in the developing ventricle. When cocultured with a Slit source, E13.5 MGE explants displayed inhibited neurite outgrowth while GABA neuron dispersion away from Slit was increased. Similar inhibition of neurite outgrowth was seen in dissociated cells from E13.5 MGE, these cells were identified to be interneurons based upon their GABA staining. In contrast, E13.5 interneurons, after culture for another 5 days, were responsive to Slit by neurite branching and elongation. Projection neurons, identified by lack of GABA staining, did not respond to Slit, either by branching or elongation. Furthermore, GABA interneurons but not pyramidal neurons, appeared to avoid neocortical areas close to an implanted source of Slit in the ventricular wall. These results lead us to suggest that interneurons but not projection neurons are responsive to the chemorepellant effect of Slit. However, more mature interneurons appear to respond to Slit by neurite arborization. These results demonstrate a selective response to Slit by GABAergic neurons during neocortical development.

Key Words: slit; ganglion eminence; neocortex; axon branching; GABA; projection neuron.

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A subset of chicken statoacoustic ganglion neurites are repelled by Slit1 and Slit2
2014, Hearing Research
Citation Excerpt :
Not only was the average neurite length similar between control and Slit-treated groups, but the outgrowth density was also comparable. Slits are also known to have effects on branching (Sang et al., 2002). If there were differences in neurite complexity or other features, some of those may have been revealed quantitatively by the pixel measurements.
Mechanosensory hair cells in the chicken inner ear are innervated by bipolar afferent neurons of the statoacoustic ganglion (SAG). During development, individual SAG neurons project their peripheral process to only one of eight distinct sensory organs. These neuronal subtypes may respond differently to guidance cues as they explore the periphery in search of their target. Previous gene expression data suggested that Slit repellants might channel SAG neurites into the sensory primordia, based on the presence of robo transcripts in the neurons and the confinement of slit transcripts to the flanks of the prosensory domains. This led to the prediction that excess Slit proteins would impede the outgrowth of SAG neurites. As predicted, axonal projections to the primordium of the anterior crista were reduced 2–3 days after electroporation of either slit1 or slit2 expression plasmids into the anterior pole of the otocyst on embryonic day 3 (E3). The posterior crista afferents, which normally grow through and adjacent to slit expression domains as they are navigating towards the posterior pole of the otocyst, did not show Slit responsiveness when similarly challenged by ectopic delivery of slit to their targets. The sensitivity to ectopic Slits shown by the anterior crista afferents was more the exception than the rule: responsiveness to Slits was not observed when the entire E4 SAG was challenged with Slits for 40 h in vitro. The corona of neurites emanating from SAG explants was unaffected by the presence of purified human Slit1 and Slit2 in the culture medium. Reduced axon outgrowth from E8 olfactory bulbs cultured under similar conditions for 24 h confirmed bioactivity of purified human Slits on chicken neurons. In summary, differential sensitivity to Slit repellents may influence the directional outgrowth of otic axons toward either the anterior or posterior otocyst.
Midbrain dopaminergic axons are guided longitudinally through the diencephalon by Slit/Robo signals
2011, Molecular and Cellular Neuroscience
Citation Excerpt :
Growth medium (50% F12/40% optimem/10% fetal calf serum/1× pen-strep/1× glutamax) was added to the well and explants were allowed to grow for 48 h. After fixation, explants and cell aggregates were immunolabeled using standard technique (Lin et al., 2005; Sang et al., 2002). Aggregates transfected with Slit2 were labeled to verify Slit2 expression with mouse anti-c-myc (Santa Cruz Biotechnology, 1:200 dilution) and Cy2-conjugated secondary antibody (1:200).
Dopaminergic neurons from the ventral mesencephalon/diencephalon (mesodiencephalon) form vital pathways constituting the majority of the brain's dopamine systems. Mesodiencephalic dopaminergic (mdDA) neurons extend longitudinal projections anteriorly through the diencephalon, ascending toward forebrain targets. The mechanisms by which mdDA axons initially navigate through the diencephalon are poorly understood. Recently the Slit family of secreted axon guidance proteins, and their Robo receptors, have been identified as important guides for descending longitudinal axons. To test the potential roles of Slit/Robo guidance in ascending trajectories, we examined tyrosine hydroxylase-positive (TH+) projections from mdDA neurons in mutant mouse embryos. We found that mdDA axons grow out of and parallel to Slit-positive ventral regions within the diencephalon, and that subsets of the mdDA axons likely express Robo1 and possibly also Robo2. Slit2 was able to directly inhibit TH axon outgrowth in explant co-culture assays. The mdDA axons made significant pathfinding errors in Slit1/2 and Robo1/2 knockout mice, including spreading out in the diencephalon to form a wider tract. The wider tract resulted from a combination of invasion of the ventral midline, consistent with Slit repulsion, but also axons wandering dorsally, away from the ventral midline. Aberrant dorsal trajectories were prominent in Robo1 and Robo1/2 knockout mice, suggesting that an aspect of Robo receptor function is Slit-independent. These results indicate that Slit/Robo signaling is critical during the initial establishment of dopaminergic pathways, with roles in the dorsoventral positioning and precise pathfinding of these ascending longitudinal axons.
The role of Slit-Robo signaling in the generation, migration and morphological differentiation of cortical interneurons
2008, Developmental Biology
Cortical interneurons in rodents are generated in the ventral telencephalon and migrate tangentially into the cortex. This process requires the coordinated action of many intrinsic and extrinsic factors. Here we show that Robo1 and Robo2 receptor proteins are dynamically expressed throughout the period of corticogenesis and colocalize with interneuronal markers, suggesting that they play a role in the migration of these cells. Analysis of Robo mutants showed a marked increase in the number of interneurons in the cortices of Robo1^−/−, but not Robo2^−/−, animals throughout the period of corticogenesis and in adulthood; this excess number of interneurons was observed in all layers of the developing cortex. Using BrdU incorporation in dissociated cell cultures and phosphohistone-3 labeling in vivo, we demonstrated that the increased number of interneurons in Robo1^−/− mice is, at least in part, due to increased proliferation. Interestingly, a similar increase in proliferation was observed in Slit1^−/−/Slit2^−/− mutant mice, suggesting that cell division is influenced by Slit-Robo signaling mechanisms. Morphometric analysis of migrating interneurons in Robo1^−/−, Robo2^−/− and Slit1^−/−/Slit2^−/−, but not in Slit1^−/− mice, showed a differential increase in neuronal process length and branching suggesting that Slit-Robo signaling also plays an important role in the morphological differentiation of these neurons.
Cytoplasmic distribution of T-box transcription factor Tbr-1 in adult rodent brain
2007, Journal of Chemical Neuroanatomy
T-brain-1 (Tbr-1), a brain-specific T-box transcription factor, plays a critical role in cerebral cortex and olfactory bulb development. The expression levels of Tbr-1 are highest at the embryonic stage and are gradually reduced during the developmental process. In adult brain, Tbr-1 is expressed at a lower, but still significant level. Tbr-1 transcriptional activity is enhanced via interaction with CASK, a membrane-associated guanylate kinase, but it is not clear whether any other mechanism regulates Tbr-1 activity. We examined the subcellular distribution of Tbr-1 in adult and postnatal rat brains using DAB stain and confocal imaging analysis. In contrast to the embryonic stage, Tbr-1 was distributed in P3 and adult rat brain in the nucleus as well as the cytoplasm of neurons in the cerebral cortex and hippocampus. Confocal analysis clearly showed dendritic distribution of Tbr-1 in pyramidal neurons. In the cerebellum of P15, P22, and adult rats, Tbr-1 was specifically expressed in Purkinje cells, where Tbr-1 was localised in the cytoplasm, including the dendritic tree. In addition, biochemical fractionation of adult cerebral cortex and hippocampus showed that cytoplasmic Tbr-1 is highly enriched in the lysed synaptosomal fraction, further indicating a synaptic distribution of cytoplasmic Tbr-1 in adult brain. Our study suggests that translocation from synapse to the nucleus is involved in regulation of Tbr-1 function in postnatal and adult brains.
Robo4 signaling in endothelial cells implies attraction guidance mechanisms
2006, Journal of Biological Chemistry
Citation Excerpt :
These results support the notion that Robo1 and Robo4 might be co-expressed in varying amounts on endothelial cells (10), and regulation of signals from each could determine the ultimate path of the endothelial growing tip. In fact, precedence for different combinations of robo receptors mediating attractive versus repulsive signals does exist in Drosophila tracheal branching network (35) and is suggestive in studies where slit promotes branching and elongation of neurites of selective neuron while inhibiting others (36). Our study extends this observation to perhaps vascular guidance as well and whether these signals arise from common or separate ligand is unclear.
Roundabouts (robo) are cell-surface receptors that mediate repulsive signaling mechanisms at the central nervous system midline. However, robos may also mediate attraction mechanisms in the context of vascular development. Here, we have performed structure-function analysis of roundabout4 (Robo4), the predominant robo expressed in embryonic zebrafish vasculature and found by gain of function approaches in vitro that Robo4 activates Cdc42 and Rac1 Rho GTPases in endothelial cells. Indeed, complementary robo4 gene knockdown approaches in zebrafish embryos show lower amounts of active Cdc42 and Rac1 and angioblasts isolated from these knockdown embryos search actively for directionality and guidance cues. Furthermore, Robo4-expressing endothelial cells show morphology and phenotype, characteristic of Rho GTPase activation. Taken together, this study suggests that Robo4 mediates attraction-signaling mechanisms through Rho GTPases in vertebrate vascular guidance.
Netrin-1 and slit-2 regulate and direct neurite growth of ventral midbrain dopaminergic neurons
2005, Molecular and Cellular Neuroscience
We investigated the roles of netrin-1 and slit-2 in regulation and navigation of dopamine (DA) axon growth using an explant culture preparation of embryonic ventral midbrain (embryonic day 14) and a co-culture system. We found that netrin-1 protein significantly enhanced DA axonal outgrowth and promoted DA axonal outgrowth in a co-culture system of netrin-1 expressing cells. Such effects were mediated by the receptor DCC as demonstrated by antibody perturbation of the DCC receptor. In contrast, slit-2 inhibited DA neuron extensions and repelled DA neurite growth. These slit-2 activities required robo receptors since the reduced neurite extension was abolished by addition of excess robo receptors. In this system, netrin-1 stimulated and slit-2 opposed DA neurite growth. Such regulation may be important for DA axonal maintenance, regeneration, and phenotypic target recognition.

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Regular ArticleSlit Promotes Branching and Elongation of Neurites of Interneurons But Not Projection Neurons from the Developing Telencephalon

Abstract

Neuron

Cell

Neuroscience

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Neuron

Cell

Cell

Cell

Trends Neurosci.

Trends Neurosci.

Neuron

Neuroscience

Cell

Neuron

Cell

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Dev. Biol.

Neuron

Interneuron migration from basal forebrain to neocortex: Dependence on Dlx genes

Science

Differential origins of neocortical projection and local circuit neurons: Role of Dlx genes in neocortical interneuronogenesis

Cereb. Cortex

Distinct cortical migrations from the medial and lateral ganglionic eminences

Development

Semaphorins act as attractive and repulsive guidance signals during the development of cortical projections

Development

The N-terminal leucine-rich region in Slit are sufficient to repel olfactory bulb axons and subventricular zone neurons

J. Neurosci.

Glutamate-positive neurons in the somatic sensory cortex of rats and monkeys

J. Neurosci.

Regular Article
Slit Promotes Branching and Elongation of Neurites of Interneurons But Not Projection Neurons from the Developing Telencephalon