Notch Signaling Plays a Dual Role in Regulating the Neuron-to-Oligodendrocyte Switch in the Developing Dorsal Forebrain.

Neural progenitor cells in the developing dorsal forebrain generate excitatory neurons followed by oligodendrocytes (OLs) and astrocytes. However, the specific mechanisms that regulate the timing of this neuron-glia switch are not fully understood. In this study, we show that the proper balance of Notch signaling in dorsal forebrain progenitors is required to generate oligodendrocytes during late stages of embryonic development. Using ex vivo and in utero approaches in mouse embryos of both sexes, we found that Notch inhibition reduced the number of oligodendrocyte lineage cells in the dorsal pallium. However, Notch overactivation also prevented oligodendrogenesis and maintained a progenitor state. These results point toward a dual role for Notch signaling in both promoting and inhibiting oligodendrogenesis, which must be fine-tuned to generate oligodendrocyte lineage cells at the right time and in the right numbers. We further identified the canonical Notch downstream factors HES1 and HES5 as negative regulators in this process. CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated knockdown of Hes1 and Hes5 caused increased expression of the pro-oligodendrocyte factor ASCL1 and led to precocious oligodendrogenesis. Conversely, combining Notch overactivation with ASCL1 overexpression robustly promoted oligodendrogenesis, indicating a separate mechanism of Notch that operates synergistically with ASCL1 to specify an oligodendrocyte fate. We propose a model in which Notch signaling works together with ASCL1 to specify progenitors toward the oligodendrocyte lineage but also maintains a progenitor state through Hes-dependent repression of Ascl1 so that oligodendrocytes are not made too early, thus contributing to the precise timing of the neuron-glia switch.SIGNIFICANCE STATEMENT Neural progenitors make oligodendrocytes after neurogenesis starts to wind down, but the mechanisms that control the timing of this switch are poorly understood. In this study, we identify Notch signaling as a critical pathway that regulates the balance between progenitor maintenance and oligodendrogenesis. Notch signaling is required for the oligodendrocyte fate, but elevated Notch signaling prevents oligodendrogenesis and maintains a progenitor state. We provide evidence that these opposing functions are controlled by different mechanisms. Before the switch, Notch signaling through Hes factors represses oligodendrogenesis. Later, Notch signaling through an unknown mechanism promotes oligodendrogenesis synergistically with the transcription factor ASCL1. Our study underscores the complexity of Notch and reveals its importance in regulating the timing and numbers of oligodendrocyte production.

Dually innervated dendritic spines develop in the absence of excitatory activity and resist plasticity through tonic inhibitory crosstalk.

Dendritic spines can be directly connected to both inhibitory and excitatory presynaptic terminals, resulting in nanometer-scale proximity of opposing synaptic functions. While dually innervated spines (DiSs) are observed throughout the central nervous system, their developmental timeline and functional properties remain uncharacterized. Here we used a combination of serial section electron microscopy, live imaging, and local synapse activity manipulations to investigate DiS development and function in rodent hippocampus. Dual innervation occurred early in development, even on spines where the excitatory input was locally silenced. Synaptic NMDA receptor currents were selectively reduced at DiSs through tonic GABAB receptor signaling. Accordingly, spine enlargement normally associated with long-term potentiation on singly innervated spines (SiSs) was blocked at DiSs. Silencing somatostatin interneurons or pharmacologically blocking GABABRs restored NMDA receptor function and structural plasticity to levels comparable to neighboring SiSs. Thus, hippocampal DiSs are stable structures where function and plasticity are potently regulated by nanometer-scale GABAergic signaling.

TUBA1A tubulinopathy mutants disrupt neuron morphogenesis and override XMAP215/Stu2 regulation of microtubule dynamics.

Heterozygous, missense mutations in α- or ß-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutation's impact at the molecular and cellular levels scale with the severity of brain malformation. Here, we focus on two mutations at the valine 409 residue of TUBA1A, V409I, and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and a decrease in the number of neurite retraction events in primary rat neuronal cultures. These neuronal phenotypes are accompanied by increased microtubule acetylation and polymerization rates. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends in budding yeast and ablate tubulin binding to TOG (tumor overexpressed gene) domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations disrupt microtubule regulation typically conferred by XMAP215 proteins during neuronal morphogenesis and migration, and this impact on tubulin activity at the molecular level scales with the impact at the cellular and tissue levels.

Proteins are molecules made up of long chains of building blocks called amino acids. When a mutation changes one of these amino acids, it can lead to the protein malfunctioning, which can have many effects at the cell and tissue level. Given that human proteins are made up of 20 different amino acids, each building block in a protein could mutate to any of the other 19 amino acids, and each mutations could have different effects. Tubulins are proteins that form microtubules, thin tubes that help give cells their shape and allow them to migrate. These proteins are added or removed to microtubules depending on the cell's needs, meaning that microtubules can grow or shrink depending on the situation. Mutations in the tubulin proteins have been linked to malformations of varying severities involving the formation of ridges and folds on the surface of the brain, including lissencephaly, pachygyria or polymicrogyria. Hoff et al. wanted to establish links between tubulin mutations and the effects observed at both cell and tissue level in the brain. They focused on two mutations in the tubulin protein TUBA1A that affect the amino acid in position 409 in the protein, which is normally a valine. One of the mutations turns this valine into an amino acid called isoleucine. This mutation is associated with pachygyria, which leads to the brain developing few ridges that are broad and flat. The second mutation turns the valine into an alanine, and is linked to lissencephaly, a more severe condition in which the brain develops no ridges, appearing smooth. Hoff et al. found that both mutations interfere with the development of the brain by stopping neurons from migrating properly, which prevents them from forming the folds in the brain correctly. At the cellular level, the mutations lead to tubulins becoming harder to remove from microtubules, making microtubules more stable than usual. This results in longer microtubules that are harder for the cell to shorten or destroy as needed. Additionally, Hoff et al. showed that the mutant versions of TUBA1A have weaker interactions with a protein called XMAP215, which controls the addition of tubulin to microtubules. This causes the microtubules to grow uncontrollably. Hoff et al. also established that the magnitude of the effects of each mutation on microtubule growth scale with the severity of the disorder they cause. Specifically, cells in which TUBA1A is not mutated have microtubules that grow at a normal rate, and lead to typical brain development. Meanwhile, cells carrying the mutation that turns a valine into an alanine, which is linked to the more severe condition lissencephaly, have microtubules that grow very fast. Finally, cells in which the valine is mutated to an isoleucine ­ the mutation associated with the less severe malformation pachygyria ­ have microtubules that grow at an intermediate rate. These findings provide a link between mutations in tubulin proteins and larger effects on cell movement that lead to brain malformations. Additionally, they also link the severity of the malformation to the severity of the microtubule defect caused by each mutation. Further work could examine whether microtubule stabilization is also seen in other similar diseases, which, in the long term, could reveal ways to detect and treat these illnesses.

Csmd2 Is a Synaptic Transmembrane Protein that Interacts with PSD-95 and Is Required for Neuronal Maturation.

Mutations and copy number variants of the CUB and Sushi multiple domains 2 (CSMD2) gene are associated with neuropsychiatric disease. CSMD2 encodes a single-pass transmembrane protein with a large extracellular domain comprising repeats of CUB and Sushi domains. High expression of CSMD2 in the developing and mature brain suggests possible roles in neuron development or function, but the cellular functions of CSMD2 are not known. In this study, we show that mouse Csmd2 is expressed in excitatory and inhibitory neurons in the forebrain. Csmd2 protein exhibits a somatodendritic localization in the neocortex and hippocampus, with smaller puncta localizing to the neuropil. Using immunohistochemical and biochemical methods, we demonstrate that Csmd2 localizes to dendritic spines and is enriched in the postsynaptic density (PSD). Accordingly, we show that the cytoplasmic tail domain of Csmd2 interacts with synaptic scaffolding proteins of the membrane-associated guanylate kinase (MAGUK) family. The association between Csmd2 and MAGUK member PSD-95 is dependent on a PDZ-binding domain on the Csmd2 tail, which is also required for synaptic targeting of Csmd2. Finally, we show that knock-down of Csmd2 expression in hippocampal neuron cultures results in reduced complexity of dendritic arbors and deficits in dendritic spine density. Knock-down of Csmd2 in immature developing neurons results in reduced filopodia density, whereas Csmd2 knock-down in mature neurons causes significant reductions in dendritic spine density and dendrite complexity. Together, these results point toward a function for Csmd2 in development and maintenance of dendrites and synapses, which may account for its association with certain psychiatric disorders.

Loss of Shh signaling in the neocortex reveals heterogeneous cell recovery responses from distinct oligodendrocyte populations.

The majority of oligodendrocytes in the neocortex originate from neural progenitors that reside in the dorsal forebrain. We recently showed that Sonic Hedgehog (Shh) signaling in these dorsal progenitors is required to produce normal numbers of neocortical oligodendrocytes during embryonic development. Conditional deletion of the Shh signaling effector, Smo, in dorsal progenitors caused a dramatic reduction in oligodendrocyte numbers in the embryonic neocortex. In the current study, we show that the depleted oligodendrocyte lineage in Smo conditional mutants is able to recover to control numbers over time. This eventual recovery is achieved in part by expansion of the ventrally-derived wild-type lineage that normally makes up a minority of the total oligodendrocyte population. However, we find that the remaining dorsally-derived mutant cells also increase in numbers over time to contribute equally to the recovery of the total population. Additionally, we found that the ways in which the dorsal and ventral sources cooperate to achieve recovery is different for distinct populations of oligodendrocyte-lineage cells. Oligodendrocyte precursor cells (OPCs) in the neocortical white matter recover completely by expansion of the remaining dorsally-derived Smo mutant cells. On the other hand, mature oligodendrocytes in the white and gray matter recover through an equal contribution from dorsal mutant and ventral wild-type lineages. Interestingly, the only population that did not make a full recovery was OPCs in the gray matter. We find that gray matter OPCs are less proliferative in Smo cKO mutants compared to controls, which may explain their inability to fully recover. Our data indicate that certain populations of the dorsal oligodendrocyte lineage are more affected by loss of Shh signaling than others. Furthermore, these studies shed new light on the complex relationship between dorsal and ventral sources of oligodendrocytes in the developing neocortex.

Lmx1a drives Cux2 expression in the cortical hem through activation of a conserved intronic enhancer.

During neocortical development, neurons are produced by a diverse pool of neural progenitors. A subset of progenitors express the Cux2 gene and are fate restricted to produce certain neuronal subtypes; however, the upstream pathways that specify these progenitor fates remain unknown. To uncover the transcriptional networks that regulate Cux2 expression in the forebrain, we characterized a conserved Cux2 enhancer that recapitulates Cux2 expression specifically in the cortical hem. Using a bioinformatic approach, we identified putative transcription factor (TF)-binding sites for cortical hem-patterning TFs. We found that the homeobox TF Lmx1a can activate the Cux2 enhancer in vitro Furthermore, we showed that Lmx1a-binding sites were required for enhancer activity in the cortical hem in vivo Mis-expression of Lmx1a in hippocampal progenitors caused an increase in Cux2 enhancer activity outside the cortical hem. Finally, we compared several human enhancers with cortical hem-restricted activity and found that recurrent Lmx1a-binding sites are a top shared feature. Uncovering the network of TFs involved in regulating Cux2 expression will increase our understanding of the mechanisms pivotal in establishing Cux2 lineage fates in the developing forebrain.

The Dorsal Wave of Neocortical Oligodendrogenesis Begins Embryonically and Requires Multiple Sources of Sonic Hedgehog.

Neural progenitor cells in the developing dorsal forebrain give rise to excitatory neurons, astrocytes, and oligodendrocytes for the neocortex. While we are starting to gain a better understanding about the mechanisms that direct the formation of neocortical neurons and astrocytes, far less is known about the molecular mechanisms that instruct dorsal forebrain progenitors to make oligodendrocytes. In this study, we show that Sonic hedgehog (Shh) signaling is required in dorsal progenitors for their late embryonic transition to oligodendrogenesis. Using genetic lineage-tracing in mice of both sexes, we demonstrate that most oligodendrocytes in the embryonic neocortex derive from Emx1+ dorsal forebrain progenitors. Deletion of the Shh signaling effector Smo specifically in Emx1+ progenitors led to significantly decreased oligodendrocyte numbers in the embryonic neocortex. Conversely, knock-out of the Shh antagonist Sufu was sufficient to increase neocortical oligodendrogenesis. Using conditional knock-out strategies, we found that Shh ligand is supplied to dorsal progenitors through multiple sources. Loss of Shh from Dlx5/6+ interneurons caused a significant reduction in oligodendrocytes in the embryonic neocortex. This phenotype was identical to that observed upon Shh deletion from the entire CNS using Nestin-Cre, indicating that interneurons migrating into the neocortex from the subpallium are the primary neural source of Shh for dorsal oligodendrogenesis. Additionally, deletion of Shh from migrating interneurons together with the choroid plexus epithelium led to a more severe loss of oligodendrocytes, suggesting that the choroid plexus is an important non-neural source of Shh ligand. Together, our studies demonstrate that the dorsal wave of neocortical oligodendrogenesis occurs earlier than previously appreciated and requires highly regulated Shh signaling from multiple embryonic sources.SIGNIFICANCE STATEMENT Most neocortical oligodendrocytes are made by neural progenitors in the dorsal forebrain, but the mechanisms that specify this fate are poorly understood. This study identifies Sonic hedgehog (Shh) signaling as a critical pathway in the transition from neurogenesis to oligodendrogenesis in dorsal forebrain progenitors during late embryonic development. The timing of this neuron-to-glia "switch" coincides with the arrival of migrating interneurons into the dorsal germinal zone, which we identify as a critical source of Shh ligand, which drives oligodendrogenesis. Our data provide evidence for a new model in which Shh signaling increases in the dorsal forebrain late in embryonic development to provide a temporally regulated mechanism that initiates the third wave of neocortical oligodendrogenesis.

Pejvakin, a Candidate Stereociliary Rootlet Protein, Regulates Hair Cell Function in a Cell-Autonomous Manner.

Mutations in the Pejvakin (PJVK) gene are thought to cause auditory neuropathy and hearing loss of cochlear origin by affecting noise-induced peroxisome proliferation in auditory hair cells and neurons. Here we demonstrate that loss of pejvakin in hair cells, but not in neurons, causes profound hearing loss and outer hair cell degeneration in mice. Pejvakin binds to and colocalizes with the rootlet component TRIOBP at the base of stereocilia in injectoporated hair cells, a pattern that is disrupted by deafness-associated PJVK mutations. Hair cells of pejvakin-deficient mice develop normal rootlets, but hair bundle morphology and mechanotransduction are affected before the onset of hearing. Some mechanotransducing shorter row stereocilia are missing, whereas the remaining ones exhibit overextended tips and a greater variability in height and width. Unlike previous studies of Pjvk alleles with neuronal dysfunction, our findings reveal a cell-autonomous role of pejvakin in maintaining stereocilia architecture that is critical for hair cell function.SIGNIFICANCE STATEMENT Two missense mutations in the Pejvakin (PJVK or DFNB59) gene were first identified in patients with audiological hallmarks of auditory neuropathy spectrum disorder, whereas all other PJVK alleles cause hearing loss of cochlear origin. These findings suggest that complex pathogenetic mechanisms underlie human deafness DFNB59. In contrast to recent studies, we demonstrate that pejvakin in auditory neurons is not essential for normal hearing in mice. Moreover, pejvakin localizes to stereociliary rootlets in hair cells and is required for stereocilia maintenance and mechanosensory function of the hair bundle. Delineating the site of the lesion and the mechanisms underlying DFNB59 will allow clinicians to predict the efficacy of different therapeutic approaches, such as determining compatibility for cochlear implants.

Cadherin 2/4 signaling via PTP1B and catenins is crucial for nucleokinesis during radial neuronal migration in the neocortex.

Cadherins are crucial for the radial migration of excitatory projection neurons into the developing neocortical wall. However, the specific cadherins and the signaling pathways that regulate radial migration are not well understood. Here, we show that cadherin 2 (CDH2) and CDH4 cooperate to regulate radial migration in mouse brain via the protein tyrosine phosphatase 1B (PTP1B) and α- and ß-catenins. Surprisingly, perturbation of cadherin-mediated signaling does not affect the formation and extension of leading processes of migrating neocortical neurons. Instead, movement of the cell body and nucleus (nucleokinesis) is disrupted. This defect is partially rescued by overexpression of LIS1, a microtubule-associated protein that has previously been shown to regulate nucleokinesis. Taken together, our findings indicate that cadherin-mediated signaling to the cytoskeleton is crucial for nucleokinesis of neocortical projection neurons during their radial migration.

Lineage Tracing Using Cux2-Cre and Cux2-CreERT2 Mice.

Using genetic fate-mapping with Cux2-Cre and Cux2-CreERT2 mice we demonstrated that the neocortical ventricular zone (VZ) contains radial glial cells (RGCs) with restricted fate potentials (Franco et al., 2012). Using the same mouse lines, Guo et al. (2013) concluded that the neocortical VZ does not contain lineage-restricted RGCs. We now show that the recombination pattern in Cux2-Cre/CreERT2 mice depends on genetic background and breeding strategies. We provide evidence that Guo et al. likely reached different conclusions because they worked with transgenic sublines with drifted transgene expression patterns. In Cux2-Cre and Cux2-CreERT2 mice that recapitulate the endogenous Cux2 expression pattern, the vast majority of fate-mapped neurons express Satb2 but not Ctip2, confirming that a restricted subset of all neocortical projection neurons belongs to the Cux2 lineage. This Matters Arising paper is in response to Guo et al. (2013), published in Neuron. See also the Matters Arising Response paper by Eckler et al. (2015), published concurrently with this Matters Arising in Neuron.

Dab1 is required for synaptic plasticity and associative learning.

Disabled-1 (Dab1) is an adaptor protein that is an obligate effector of the Reelin signaling pathway, and is critical for neuronal migration and dendrite outgrowth during development. Components of the Reelin pathway are highly expressed during development, but also continue to be expressed in the adult brain. Here we investigated in detail the expression pattern of Dab1 in the postnatal and adult forebrain, and determined that it is expressed in excitatory as well as inhibitory neurons. Dab1 was found to be localized in different cellular compartments, including the soma, dendrites, presynaptic and postsynaptic structures. Mice that are deficient in Dab1, Reelin, or the Reelin receptors ApoER2 and VLDLR exhibit severely perturbed brain cytoarchitecture, limiting the utility of these mice for investigating the role of this signaling pathway in the adult brain. In this study, we developed an adult forebrain-specific and excitatory neuron-specific conditional knock-out mouse line, and demonstrated that Dab1 is a critical regulator of synaptic function and hippocampal-dependent associative and spatial learning. These dramatic abnormalities were accompanied by a reduction in dendritic spine size, and defects in basal and plasticity-induced Akt and ERK1/2 signaling. Deletion of Dab1 led to no obvious changes in neuronal positioning, dendrite morphology, spine density, or synaptic composition. Collectively, these data conclusively demonstrate an important role for Reelin-Dab1 signaling in the adult forebrain, and underscore the importance of this pathway in learning and memory.

Cajal-Retzius cells instruct neuronal migration by coincidence signaling between secreted and contact-dependent guidance cues.

Cajal-Retzius (CR) cells are a transient cell population of the CNS that is critical for brain development. In the neocortex, CR cells secrete reelin to instruct the radial migration of projection neurons. It has remained unexplored, however, whether CR cells provide additional molecular cues important for brain development. Here, we show that CR cells express the immunoglobulin-like adhesion molecule nectin1, whereas neocortical projection neurons express its preferred binding partner, nectin3. We demonstrate that nectin1- and nectin3-mediated interactions between CR cells and migrating neurons are critical for radial migration. Furthermore, reelin signaling to Rap1 promotes neuronal Cdh2 function via nectin3 and afadin, thus directing the broadly expressed homophilic cell adhesion molecule Cdh2 toward mediating heterotypic cell-cell interactions between neurons and CR cells. Our findings identify nectins and afadin as components of the reelin signaling pathway and demonstrate that coincidence signaling between CR cell-derived secreted and short-range guidance cues direct neuronal migration.

Shaping our minds: stem and progenitor cell diversity in the mammalian neocortex.

The neural circuits of the mammalian neocortex are crucial for perception, complex thought, cognition, and consciousness. This circuitry is assembled from many different neuronal subtypes with divergent properties and functions. Here, we review recent studies that have begun to clarify the mechanisms of cell-type specification in the neocortex, focusing on the lineage relationships between neocortical progenitors and subclasses of excitatory projection neurons. These studies reveal an unanticipated diversity in the progenitor pool that requires a revised view of prevailing models of cell-type specification in the neocortex. We propose a "sequential progenitor-diversification model" that integrates current knowledge to explain how projection neuron diversity is achieved by mechanisms acting on proliferating progenitors and their postmitotic offspring. We discuss the implications of this model for our understanding of brain evolution and pathological states of the neocortex.

Fate-restricted neural progenitors in the mammalian cerebral cortex.

During development of the mammalian cerebral cortex, radial glial cells (RGCs) generate layer-specific subtypes of excitatory neurons in a defined temporal sequence, in which lower-layer neurons are formed before upper-layer neurons. It has been proposed that neuronal subtype fate is determined by birthdate through progressive restriction of the neurogenic potential of a common RGC progenitor. Here, we demonstrate that the murine cerebral cortex contains RGC sublineages with distinct fate potentials. Using in vivo genetic fate mapping and in vitro clonal analysis, we identified an RGC lineage that is intrinsically specified to generate only upper-layer neurons, independently of niche and birthdate. Because upper cortical layers were expanded during primate evolution, amplification of this RGC pool may have facilitated human brain evolution.

Extracellular matrix functions during neuronal migration and lamination in the mammalian central nervous system.

Extracellular matrix (ECM) glycoproteins are expressed in the central nervous system (CNS) in complex and developmentally regulated patterns. The ECM provides a number of critical functions in the CNS, contributing both to the overall structural organization of the CNS and to control of individual cells. At the cellular level, the ECM affects its functions by a wide range of mechanisms, including providing structural support to cells, regulating the activity of second messenger systems, and controlling the distribution and local concentration of growth and differentiation factors. Perhaps the most well known role of the ECM is as a substrate on which motile cells can migrate. Genetic, cell biological, and biochemical studies provide strong evidence that ECM glycoproteins such as laminins, tenascins, and proteoglycans control neuronal migration and positioning in several regions of the developing and adult brain. Recent findings have also shed important new insights into the cellular and molecular mechanisms by which reelin regulates migration. Here we will summarize these findings, emphasizing the emerging concept that ECM glycoproteins promote different modes of neuronal migration such as radial, tangential, and chain migration. We also discuss several studies demonstrating that mutations in ECM glycoproteins can alter neuronal positioning by cell nonautonomous mechanisms that secondarily affect migrating neurons.

Reelin regulates cadherin function via Dab1/Rap1 to control neuronal migration and lamination in the neocortex.

Neuronal migration is critical for establishing neocortical cell layers and migration defects can cause neurological and psychiatric diseases. Recent studies show that radially migrating neocortical neurons use glia-dependent and glia-independent modes of migration, but the signaling pathways that control different migration modes and the transitions between them are poorly defined. Here, we show that Dab1, an essential component of the reelin pathway, is required in radially migrating neurons for glia-independent somal translocation, but not for glia-guided locomotion. During migration, Dab1 acts in translocating neurons to stabilize their leading processes in a Rap1-dependent manner. Rap1, in turn, controls cadherin function to regulate somal translocation. Furthermore, cell-autonomous neuronal deficits in somal translocation are sufficient to cause severe neocortical lamination defects. Thus, we define the cellular mechanism of reelin function during radial migration, elucidate the molecular pathway downstream of Dab1 during somal translocation, and establish the importance of glia-independent motility in neocortical development.

Extracellular matrix: functions in the nervous system.

An astonishing number of extracellular matrix glycoproteins are expressed in dynamic patterns in the developing and adult nervous system. Neural stem cells, neurons, and glia express receptors that mediate interactions with specific extracellular matrix molecules. Functional studies in vitro and genetic studies in mice have provided evidence that the extracellular matrix affects virtually all aspects of nervous system development and function. Here we will summarize recent findings that have shed light on the specific functions of defined extracellular matrix molecules on such diverse processes as neural stem cell differentiation, neuronal migration, the formation of axonal tracts, and the maturation and function of synapses in the peripheral and central nervous system.

Talin1 regulates TCR-mediated LFA-1 function.

The leukocyte integrin LFA-1 plays a critical role in T cell trafficking and T cell adhesion to APCs. It is known that integrin-mediated adhesion is regulated by changes in integrin ligand-binding affinity and valency through inside-out signaling. However, the molecular mechanisms involved in TCR-mediated LFA-1 regulation are not well understood. In this study, we show that the cytoskeletal protein talin1 is required for TCR-mediated activation of LFA-1 through regulation of LFA-1 affinity and clustering. Depletion of talin1 from human T cells by small interfering RNAs impairs TCR-induced adhesion to ICAM-1 and T cell-APC conjugation. TCR-induced LFA-1 polarization, but not actin polarization, is defective in talin1-deficient T cells. Although LFA-1 affinity is also reduced in talin1-deficient T cells, rescue of LFA-1 affinity alone is not sufficient to restore LFA-1 adhesive function. Together, our findings indicate that TCR-induced up-regulation of LFA-1-dependent adhesiveness and resulting T cell-APC conjugation require talin1.

The conserved C-terminal I/LWEQ module targets Talin1 to focal adhesions.

The cytoskeletal protein Talin1 is a critical link between integrins and the actin cytoskeleton, where it is required for the structural and signaling functions of integrin-containing adhesion complexes. However, the elements in Talin1 that are responsible for localizing it to adhesion complexes are not known. In this report we have used a series of constructs based on the modular structure of Talin1 to determine the structural elements that specify the subcellular localization of Talin1. We show that the conserved actin-binding I/LWEQ module at the C-terminus of Talin1 is necessary and sufficient for targeting to focal adhesion complexes. We also used truncation and site-directed mutagenesis to demonstrate that this novel targeting function correlates with, but is separable from, the actin-binding properties of the Talin1 I/LWEQ module. In addition, we have shown that focal adhesion targeting, unlike actin binding, is not conserved among I/LWEQ module proteins. Finally, we have demonstrated that the subcellular localization of the Talin1 I/LWEQ module is regulated by an intrasteric interaction with an upstream alpha-helix, suggesting that both the actin binding and adhesion-targeting elements are masked in full-length Talin1. Our results define a novel role for the I/LWEQ module as the primary adhesion-complex targeting determinant of Talin1 and suggest that pathways that can relieve inhibition of I/LWEQ module function will be important for regulating the structural and signaling properties of adhesion complexes.