ORIGINAL RESEARCH ARTICLE published: 26 June 2012 NEURAL CIRCUITS doi: 10.3389/fncir.2012.00037 Developmental coordination of gene expression between synaptic partners during GABAergic circuit assembly in cerebellar cortex Anirban Paul 1,Ying Cai 2,3, Gurinder S. Atwal 2 and Z. Josh Huang 1* 1 Cold Spring Harbor Laboratory, Neuroscience, Cold Spring Harbor, New York, NY, USA 2 Cold Spring Harbor Laboratory, Quantitative Biology, Cold Spring Harbor, NY, USA 3 Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY, USA Edited by: The assembly of neural circuits involves multiple sequential steps such as the specifica- Edward M. Callaway, California tion of cell-types, their migration to proper brain locations, morphological and physiological Institute of Technology, USA differentiation, and the formation and maturation of synaptic connections. This intricate Reviewed by: David J. Margolis, University of and often prolonged process is guided by elaborate genetic mechanisms that regulate Zurich, Switzerland each step. Evidence from numerous systems suggests that each cell-type, once speci- Yuanquan Song, University of fied, is endowed with a genetic program that unfolds in response to, and is regulated by, California San Francisco, USA extrinsic signals, including cell–cell and synaptic interactions. To a large extent, the exe- *Correspondence: cution of this intrinsic program is achieved by the expression of specific sets of genes Z. Josh Huang, Cold Spring Harbor Laboratory, One Bungtown Road, that support distinct developmental processes. Therefore, a comprehensive analysis of Cold Spring Harbor, New York, NY the developmental progression of gene expression in synaptic partners of neurons may 11724, USA. provide a basis for exploring the genetic mechanisms regulating circuit assembly. Here e-mail: [email protected] we examined the developmental gene expression profiles of well-defined cell-types in a stereotyped microcircuit of the cerebellar cortex. We found that the transcriptomes of Purkinje cell and stellate/basket cells are highly dynamic throughout postnatal develop- ment. We revealed “phasic expression” of transcription factors, ion channels, receptors, cell adhesion molecules, gap junction proteins, and identified distinct molecular pathways that might contribute to sequential steps of cerebellar inhibitory circuit formation. We fur- ther revealed a correlation between genomic clustering and developmental co-expression of hundreds of transcripts, suggesting the involvement of chromatin level gene regulation during circuit formation. Keywords: cerebellum, circuit assembly, development, GABAergic, gene expression, microcircuit, Purkinje cell, stellate basket cell INTRODUCTION All cerebellar neurons derive from progenitors that prolifer- The cerebellum, a primary center for motor coordination, is an ate in two germinal neuroepithelia: the ventricular zone (VZ) excellent system to study neural circuit assembly in the CNS due generates GABAergic neurons, whereas the rhombic lip is the to its highly stereotyped cytoarchitecture. For example, the cere- origin of glutamatergic neurons. Among VZ-derivatives, GABAer- bellar cortex is organized as a near lattice-like circuit architecture gic projection neurons and interneurons are generated according with a protracted period of circuit formation. At the focal position to different strategies. PC are produced at the onset of cerebel- in cerebellar cortex and as its sole output are the Purkinje neurons, lar neurogenesis by discrete progenitor pools located in distinct which receive multiple sets of excitatory and inhibitory inputs. The VZ microdomains; they are specified within the VZ and acquire glutamatergic parallel fibers synapse onto the slender spines of the mature phenotypes largely according to cell-autonomous pro- distal dendrite, while the climbing fibers prefer the stubby spines grams. On the other hand, the different classes of inhibitory of the more proximal dendrite. In addition, the GABAergic bas- interneurons, including basket (BkC) and stellate (StC) cells, ket interneurons target Purkinje cell (PC) soma and axon initial derive from a single population of precursors that delaminate segments (AIS), whereas the stellate interneurons innervate the into the prospective white matter (PWM), where they continue dendritic shafts. The integration of these excitatory and inhibitory to divide until the end of the first postnatal week (Altman and inputs shapes PC outputs, which are transmitted to deep cerebel- Bayer, 1997; Leto et al., 2012). lar nuclei and regulate motor coordination (D’Angelo et al., 2011). Following their generation in the PWM, basket, and stellate While significant progress have been made in understanding the cells migrate toward cerebellar cortex and reach Purkinje cell layer development of excitatory connectivity (i.e., parallel fibers and (PCL) by approximately P7 and P10, respectively. During the climbing fibers), the development of inhibitory circuitry remains subsequent weeks, PC, BkC, and StC undergo profound morpho- poorly understood. logical and physiological differentiation while establishing specific Frontiers in Neural Circuits www.frontiersin.org June 2012 | Volume 6 | Article 37 | 1 Paul et al. Cell-type-specific transcriptome during circuit assembly synaptic connectivity (Cameron et al., 2009). For example, the amplified by two rounds of linear isothermal amplification steps PCs elaborate their dendrites with characteristic branching pat- using the MessageAmpII aRNA amplification kit (Ambion, USA terns while the BskCs extend their axon branches onto PCs. Each Cat#AM1751) followed by biotin labeling. BskC innervates 7–10 PCs at their soma and AIS (Figure 1A). The maturation of BskC axon arbor and pinceau synapses con- GENE EXPRESSION tinue into the fourth postnatal week (Ango et al., 2004, 2008). Labeled aRNA were hybridized to Affymetrix mouse 30 expres- The development of StCs lags behind that of BskCs by a few days. sion array MOE430.2 according to manufacturers fluid han- StC axons extend along the Bergmann glia fibers with characteris- dling, hybridization, and scanning protocols. CEL files from each tic trajectories to innervate PC dendrites. Using a candidate gene hybridization was converted to normalized expression values using approach, we have previously demonstrated that members of the Bioconductor package1 in R2 using GCRMA method. Further L1 family immunoglobulin cell adhesion molecules (IgCAMs), differential analysis, Principal Component Analysis (PCA), cross- neurofascin, and CHL1, contribute to the subcellular organization correlation analysis, and downstream visualization was done in of BskC and StC innervation,respectively (Ango et al.,2004,2008). dCHIP software3. Microarray data submitted to GEO; accession However, the genetic mechanisms that control the concerted dif- GSE37055. ferentiation and synaptic connectivity during inhibitory circuit formation remain poorly understood. IN SITU HYBRIDIZATION To explore the intrinsic genetic program that direct the dif- To generate in situ probes Trizol extracted total mouse brain RNA ferentiation and connectivity between PC and BskC/StC(S/BC) was used to perform RT-PCR using gene specific primers (Super- cells, here we examined the developmental dynamics of their spe- script III, Invitrogen, USA). RT product was subjected to nested cific gene expression profiles. We found (i) phasic developmental PCR with T3 tagged forward and T7 tagged reverse primers (see expression of transcription factors (TFs), ion channels, recep- primer list in Table A2 in Appendix). In vitro transcription using tors, cell adhesion molecules (CAMs), gap junction proteins, and with T7 and T3 driven RNA polymerase and DIG-labeled rNTPs novel cell-type specific transcripts,(ii) distinct molecular pathways generate the probes that was run on Bioanalyzer to ensure sin- that may contribute to different developmental stages for these gle RNA product of expected size. T7 produced antisense probes cell-types; (iii) correlation between genomic clustering and devel- and T3 generated the control sense probes. In situ hybridization opmental co-expression of hundreds of transcripts, suggesting the was performed at 61˚C on 15 mm thick sagittal cryo-sectioned involvement of chromatin level gene regulation during circuit for- brains from C57B6 male animals. Detection was done using anti- mation. Our dataset also provides a valuable resource for further DIG antibody and VectaRed detection reagent (Vector Labs, USA studying the molecular and developmental genetic mechanisms Cat#SK-5100). underlying cerebellar circuit formation. PERMUTATION TEST MATERIALS AND METHODS Permutation test was performed on normalized expression values MANUAL SORTING of PC and S/BC cells to find genes that are differentially expressed Amplified and labeled mRNA were prepared from 50 to 75 PC or across different time points. For each probe, a t-statistic T obs was between 125 and 150 S/BC cells that were manually sorted from computed as each developmental stage. While PCs are 40 mm in diameter and lie along the PCL the StC and the BskC are both 8–10 mm in diam- .Xi/ − Xj Tobs D r 2 eter and are distributed in the molecular layer. Although the BskC S2 S i C j cell bodies lie relatively closer to the PC it is not always apparent ni nj since StC and BskC are not uniformly distinct in layer separation. Also there is no molecular marker that distinguishes Bsk from Where <xi> is the mean of expression values of replicates at the StC. This prevented us from