Thalamocortical axons control the cytoarchitecture of neocortical layers by area-specific supply of VGF.

Neuronal abundance and thickness of each cortical layer are specific to each area, but how this fundamental feature arises during development remains poorly understood. While some of area-specific features are controlled by intrinsic cues such as morphogens and transcription factors, the exact influence and mechanisms of action by cues extrinsic to the cortex, in particular the thalamic axons, have not been fully established. Here, we identify a thalamus-derived factor, VGF, which is indispensable for thalamocortical axons to maintain the proper amount of layer 4 neurons in the mouse sensory cortices. This process is prerequisite for further maturation of the primary somatosensory area, such as barrel field formation instructed by a neuronal activity-dependent mechanism. Our results provide an actual case in which highly site-specific axon projection confers further regional complexity upon the target field through locally secreting signaling molecules from axon terminals.

Nucleocytoplasmic Shuttling of Histone Deacetylase 9 Controls Activity-Dependent Thalamocortical Axon Branching.

During development, thalamocortical (TC) axons form branches in an activity-dependent fashion. Here we investigated how neuronal activity is converted to molecular signals, focusing on an epigenetic mechanism involving histone deacetylases (HDACs). Immunohistochemistry demonstrated that HDAC9 was translocated from the nucleus to the cytoplasm of thalamic cells during the first postnatal week in rats. In organotypic co-cultures of the thalamus and cortex, fluorescent protein-tagged HDAC9 also exhibited nuclueocytoplasmic translocation in thalamic cells during culturing, which was reversed by tetrodotoxin treatment. Transfection with a mutant HDAC9 that interferes with the translocation markedly decreased TC axon branching in the culture. Similarly, TC axon branching was significantly decreased by the mutant HDAC9 gene transfer in vivo. However, axonal branching was restored by disrupting the interaction between HDAC9 and myocyte-specific enhancer factor 2 (MEF2). Taken together, the present results demonstrate that the nucleocytoplasmic translocation of HDAC9 plays a critical role in activity-dependent TC axon branching by affecting transcriptional regulation and downstream signaling pathways.

Thalamus-derived molecules promote survival and dendritic growth of developing cortical neurons.

The mammalian neocortex is composed of various types of neurons that reflect its laminar and area structures. It has been suggested that not only intrinsic but also afferent-derived extrinsic factors are involved in neuronal differentiation during development. However, the role and molecular mechanism of such extrinsic factors are almost unknown. Here, we attempted to identify molecules that are expressed in the thalamus and affect cortical cell development. First, thalamus-specific molecules were sought by comparing gene expression profiles of the developing rat thalamus and cortex using microarrays, and by constructing a thalamus-enriched subtraction cDNA library. A systematic screening by in situ hybridization showed that several genes encoding extracellular molecules were strongly expressed in sensory thalamic nuclei. Exogenous and endogenous protein localization further demonstrated that two extracellular molecules, Neuritin-1 (NRN1) and VGF, were transported to thalamic axon terminals. Application of NRN1 and VGF to dissociated cell culture promoted the dendritic growth. An organotypic slice culture experiment further showed that the number of primary dendrites in multipolar stellate neurons increased in response to NRN1 and VGF, whereas dendritic growth of pyramidal neurons was not promoted. These molecules also increased neuronal survival of multipolar neurons. Taken together, these results suggest that the thalamus-specific molecules NRN1 and VGF play an important role in the dendritic growth and survival of cortical neurons in a cell type-specific manner.

Laminar and areal expression of unc5d and its role in cortical cell survival.

The UNC-5 family of netrin receptors is known to regulate axon guidance, cell migration, and cell survival. We have previously demonstrated that unc5d, one of the UNC-5 family member genes, is specifically expressed in layer 4 of the developing rat neocortex (Zhong Y, Takemoto M, Fukuda T, Hattori Y, Murakami F, Nakajima D, Nakayama M, Yamamoto N. 2004. Identification of the genes that are expressed in the upper layers of the neocortex. Cereb Cortex. 14:1144-1152). However, the role of UNC5D in cortical development is still unknown. In this study, we revealed that unc5d was highly expressed in the primary sensory areas of the mouse neocortex at around postnatal day 7. Netrin-4 was also found to be predominantly expressed in layer 4 of the sensory cortex and sensory thalamic nuclei. Cell surface binding assay showed that netrin-4 protein bound to UNC5D-expressing cells. An in vitro study further demonstrated that cell death of unc5d-expressing layer 4 cells was reduced by exogenous application of netrin-4 protein, whereas UNC5D is not sufficient to mediate the effect of netrin-4 in deep layer cells. Taken together, these results suggest that UNC5D is primarily expressed by layer 4 cells in the primary sensory areas of the developing neocortex and may mediate the effect of netrin-4 on cortical cell survival in a lamina-specific manner.