Nucleus- and cell-specific gene expression in monkey thalamus

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.

Fig. 1.

RT-PCR measures of regional transcript expression levels confirm microarray results. Bar graph indicates relative expression between PT and VC for transcripts overrepresented in thalamus by ≥3-fold in microarray analysis. Dotted line indicates a ratio of 1, denoting no change in expression. Error bars indicate standard deviation of the mean. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and β-actin genes were included as internal controls. Asterisks indicate probe sets analyzed by in situ hybridization histochemistry.

Fig. 2.

Autoradiograms and immunocytochemical preporations from monkey thalamus and cortex. Images from film audioradiograms show abundance of transcripts of TCF7L2 (A, E, I, and M), PCP4 (B, F, and J), SPP1 (C, G, and K), and SPARC (D, H, and L) in thalamus (A–D and I–M) relative to cortex (E–H). Expression of TCF7L2 and SPP1 is restricted to thalamus, whereas PCP4 and SPARC are expressed in other subcortical regions as well. Higher-magnification images of the dorsal lateral geniculate nucleus (dLGN) illustrate the differential expression of TCF7L2, PCP4, and SPP1 in dLGN (I–L). Dashed line indicates separation between magnocellular (1 and 2) and parvocellular (–6) layers of the dLGN, as marked in adjacent Nissl section (L). In situ hybridization for TCF7L2 mRNA (M) and immunoreactivity for TCF7L2 (N and O) reveal expression in posterior division of the zona incerta (M), the deep lamina of the pregeniculate nucleus (N), and the field of Forel (O). (Scale bars: A–H, 3 mm; M, 1 mm; I–L, N, and O, 500 μm.) CN, caudate nucleus; FF, field of Forel; LP, lateral posterior nucleus; MB, midbrain; MD, mediodorsal nucleus; OT, optic tract; P, putamen; Prg, pregeniculate nucleus; R, reticular nucleus; SPf, sub-Pf; VL, ventral lateral nucleus; VMb, basal ventral medial nucleus; VP, ventral posterior nuclei; VPI, ventral posterior inferior nucleus; VPL, ventral posterior lateral nucleus; VPM, ventral posterior medial nucleus; ZI, zona incerta.

Fig. 3.

GeneSpring analysis of five thalamic nuclei. Nuclei of the dorsal thalamus display unique gene expression profiles. (A) Genes with significant expression (P < 0.05) in at least one of the five regions examined were hierarchically clustered by similarity in expression profile. The resulting heat map of the dendrogram tree reveals groups of genes with high (red) expression levels in one thalamic nucleus and moderate (black) or low (green) levels in the other four nuclei. (B) Genes with thalamic nucleus-specific expression profiles were identified by using the Drawn Gene function in the GeneSpring analysis program. For each region examined, sets of genes whose expression profiles significantly correlated (R² > 0.95) to a template pattern (red) were identified.

Fig. 4.

Microarray results confirmed by in situ hybridization histochemistry (A) and functional analysis of thalamus-enriched genes (B). In situ hybridization and functional annotation validate thalamic nucleus-specific gene expression. (A) Images from film autoradiograms show differential expression of CBLN1 (Upper) and PCP4 (Lower) in monkey dorsal thalamus, corroborating the microarray results (Right). (B) The Ingenuity Pathways Analysis program enabled biological/functional annotations of candidate genes identified by the Drawn Gene Function in GeneSpring analysis. The top five functions are compared across all regions. Abbreviations are as in Fig. 2.