Nucleus- and cell-specific gene expression in monkey thalamus

Karl Murray

Prabhakara Choudary

Edward Jones

Center for Neuroscience and Department of Psychiatry and Behavioral Sciences, University of California, Davis, CA 95616

*To whom correspondence should be addressed. E-mail: ude.sivadcu@senoje

Contributed by Edward G. Jones, December 6, 2006

Author contributions: K.D.M., P.V.C., and E.G.J. designed research; K.D.M., P.V.C., and E.G.J. performed research; K.D.M., P.V.C., and E.G.J. analyzed data; and K.D.M., P.V.C., and E.G.J. wrote the paper.

Received 2006 Nov 16

Freely available online through the PNAS open access option.

Abstract

Nuclei of the mammalian thalamus are aggregations of neurons with unique architectures and input–output connections, yet the molecular determinants of their organizational specificity remain unknown. By comparing expression profiles of thalamus and cerebral cortex in adult rhesus monkeys, we identified transcripts that are unique to dorsal thalamus or to individual nuclei within it. Real-time quantitative PCR and in situ hybridization analyses confirmed the findings. Expression profiling of individual nuclei microdissected from the dorsal thalamus revealed additional subsets of nucleus-specific genes. Functional annotation using Gene Ontology (GO) vocabulary and Ingenuity Pathways Analysis revealed overrepresentation of GO categories related to development, morphogenesis, cell–cell interactions, and extracellular matrix within the thalamus- and nucleus-specific genes, many involved in the Wnt signaling pathway. Examples included the transcription factor TCF7L2, localized exclusively to excitatory neurons; a calmodulin-binding protein PCP4; the bone extracellular matrix molecules SPP1 and SPARC; and other genes involved in axon outgrowth and cell matrix interactions. Other nucleus-specific genes such as CBLN1 are involved in synaptogenesis. The genes identified likely underlie nuclear specification, cell phenotype, and connectivity during development and their maintenance in the adult thalamus.

Keywords: development, excitatory neurons, inhibitory neurons, thalamocortical, Wnt signaling pathway

Abstract

The mammalian thalamus is made up of groupings of neurons that reflect its evolutionary and developmental history, its function as a sensory relay, and its involvement in forebrain activities that underlie states of consciousness (1 –4). The three major subdivisions of thalamus (epithalamus, dorsal thalamus, and ventral thalamus) emerge during embryogenesis from the wall of the diencephalic alar plate (5 –7). Aggregation of fate-determined postmitotic neurons leads to the formation of multiple subnuclei within these divisions characterized by different chemo-, cyto-, and myeloarchitectures and differing patterns of connections.

The establishment of architecture and connections in all brain regions is modulated by molecular cues that govern cell aggregation, neurotransmitter phenotype, axon guidance, and synaptogenesis (8, 9). The unique architecture, connectivity, and transmitter/receptor characteristics of each thalamic nucleus are unlikely to be established or maintained in the absence of molecular genetic guidance. Clues to the nature of underlying mechanisms can be found by identifying genes that give a molecular identity to thalamic nuclei. Sets of regulatory genes distinguish the three major thalamic subdivisions in the developing and adult rodent and primate (10, 11), but expression occurs across multiple nuclei, in regional rather than nucleus-specific patterns. Some examples of nucleus-specific expression, however, do occur, e.g., Id-2 in the primate centre médian nucleus (CM; ref. 10). Expression of neurotransmitter- or receptor-related genes in the thalamus also tends to be regional rather than nucleus-specific, but there are exceptions to this rule as well (12, 13).

To determine the extent to which subnuclei of the major thalamic divisions are molecularly distinct, we used high-density oligonucleotide arrays to identify thalamus-specific genes in adult monkeys. By comparing expression profiles of thalamus and cerebral cortex, we identified genes not hitherto known to be expressed in thalamus. Further profiling of microdissected nuclei identified more comprehensive sets of genes with nucleus-specific expression. Confirmation of gene expression by RT-PCR, in situ hybridization histochemistry, and/or immunohistochemistry revealed remarkable cell- and nucleus-specific patterns of expression. Functionally, the genes are related to development and cell–cell interactions, implying their involvement in the establishment and maintenance of thalamic nuclear and cellular specificity.

Genes displaying region-specific expression were mapped to corresponding objects in the Ingenuity Pathways Knowledge Base. A score based on the number of mapped “focus” genes and network size is used for ranking purposes. The canonical Wnt/β-catenin signaling pathway was well represented among the top networks. An DT, anterior dorsal thalamus; CM, centre médian nucleus; Med DT, medial dorsal thalamus; Pulv, pulvinar; Ven DT, ventral dorsal thalamus.

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Acknowledgments

We thank Xiao-Hong Fan, Phong Nguyen, and Malalai Yusufzai for technical assistance. This work was supported by the National Institutes of Health (Grants NS21377 and NS39094) and the W. M. Keck Program in Neuroscience Imaging. K.D.M. is the recipient of a young investigator award from the National Alliance for Research in Schizophrenia and Depression and the Sunshine from Darkness Gala. The monoclonal antibody MPIIIB10₁ was developed by Michael Solursh and Ahnders Franzen under the auspices of the National Institute of Child Health and Human Development.

Acknowledgments

Abbreviations

ECM	extracellular matrix
CM	centre médian nucleus
FC	frontal cortex
GO	Gene Ontology
PT	posterior thalamus
Pf	parafascicular nucleus
VC	visual cortex
dLGN	dorsal lateral geniculate nucleus.

Abbreviations

Footnotes

The authors declare no conflict of interest.

Data deposition: The sequences reported in this paper have been deposited in the GEO database (accession no. {"type":"entrez-geo","attrs":{"text":"GSE6708","term_id":"6708","extlink":"1"}}GSE6708).

This article contains supporting information online at www.pnas.org/cgi/content/full/0610742104/DC1.

Footnotes

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