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Cameron et al. BMC Neuroscience 2012, 13:90 http://www.biomedcentral.com/1471-2202/13/90

RESEARCH ARTICLE Open Access Hierarchical clustering of expression patterns in the Eomes + lineage of excitatory neurons during early neocortical development David A Cameron1, Frank A Middleton1, Anjen Chenn3 and Eric C Olson1,2,4*

Abstract Background: Cortical neurons display dynamic patterns of during the coincident processes of differentiation and migration through the developing cerebrum. To identify selectively expressed by the Eomes + (Tbr2) lineage of excitatory cortical neurons, GFP-expressing cells from Tg(Eomes::eGFP) Gsat embryos were isolated to > 99% purity and profiled. Results: We report the identification, validation and spatial grouping of genes selectively expressed within the Eomes + cortical excitatory neuron lineage during early cortical development. In these neurons 475 genes were expressed ≥ 3-fold, and 534 genes ≤ 3-fold, compared to the reference population of neuronal precursors. Of the up-regulated genes, 328 were represented at the Genepaint in situ hybridization database and 317 (97%) were validated as having spatial expression patterns consistent with the lineage of differentiating excitatory neurons. A novel approach for quantifying in situ hybridization patterns (QISP) across the cerebral wall was developed that allowed the hierarchical clustering of genes into putative co-regulated groups. Forty four candidate genes were identified that show spatial expression with Intermediate Precursor Cells, 49 candidate genes show spatial expression with Multipolar Neurons, while the remaining 224 genes achieved peak expression in the developing cortical plate. Conclusions: This analysis of differentiating excitatory neurons revealed the expression patterns of 37 transcription factors, many chemotropic signaling molecules (including the Semaphorin, Netrin and Slit signaling pathways), and unexpected evidence for non-canonical neurotransmitter signaling and changes in mechanisms of glucose metabolism. Over half of the 317 identified genes are associated with neuronal disease making these findings a valuable resource for studies of neurological development and disease. Keywords: Transcriptome, Migration, Cortex, Profiling, Excitatory neuron

Background neurons adopt multiple morphologies and display sev- Excitatory, or projection, neurons comprise ~85% of the eral distinct modes of migration [1]. neuronal population in the mature cerebral cortex. The period of radial migration and early differentiation These neurons share a stereotypical developmental tra- coincides with the sequential expression of a network of jectory, with most generated in the neocortical ventricu- transcription factors. Core elements of the network in- lar zone (VZ) and subsequently migrating outward, clude Pax6, Neurogenin, Neurod, Eomes (Tbr2), and through complex and changing micro-environments, to- Tbr1 [2-4]. Some of the network members are known to wards the cortical surface. During this radial migration be essential for proper migration [5,6], axogenesis [7,8], dendritogenesis [9], and adoption of neuronal subtype

* Correspondence: [email protected] [10-12]. The ability of neurons to simultaneously navi- 1Department of Neuroscience and Physiology, SUNY Upstate Medical gate through the developing cortex and acquire charac- University, Syracuse NY 13210, USA teristic phenotypes is thus likely to depend on dynamic 2Department of Electrical Engineering and Computer Science, Syracuse University, Syracuse NY 13210, USA patterns of gene expression during the early postmitotic Full list of author information is available at the end of the article period.

© 2012 Cameron et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and in any medium, provided the original work is properly cited. Cameron et al. BMC Neuroscience 2012, 13:90 Page 2 of 23 http://www.biomedcentral.com/1471-2202/13/90

Characterization of these cell-intrinsic, dynamic gene from Tg(Eomes::eGFP)Gsat embryos were rapidly dis- expression patterns remains incomplete. Transcriptional sected at E13.5 and E14.5. The meninges were removed profiling of microdissected samples derived from develop- and the cortical tissue was dissociated in 0.25% Trypsin- ing , although revealing regional and temporal differ- EDTA solution (Invitrogen) for subsequent fluorescence ences across the brain [13-15] is limited by the inclusion activated cell (FAC) sorting (Figure 1). Cortices from 2 of blood vessel endothelial cells, inhibitory neurons, and embryos were used per sorted sample and 3 samples glia within the dissected tissue: the derived transcriptional were generated per developmental time point. Disso- profile necessarily reflects contributions of multiple, and ciated cells were kept in ice-cold buffer (PBS + 2% BSA) often non-neuronal, cell types. These interpretive obsta- until FAC sorting, with the total elapsed time between cles can in principle be eliminated with a combined appli- dissection and sorting < 1 hr. Cell suspensions were cation of cell sorting from acutely dissociated brain [16] sorted into GFP + and GFP- populations on a Becton and subsequent transcriptional profiling [17,18]. Dickinson FACS Vantage Flow Cytometer Cell Sorter In this study fluorescence activated cell (FAC) sorting (SUNY Upstate Medical Flow Cytometry Unit). Sorting was used to purify neurons of the excitatory cortical continued until a minimum of 100,000 GFP + cells were lineage, from which RNA was isolated for transcriptional generated per sample. The total time required for sort- profiling. We focused on the neuronal lineage that ing the 3 samples was less than 30 min. Cells were expresses Eomes (Tbr2), a that is sorted directly into collection tubes containing RNALa- ™ expressed by intermediate precursor cells [19] and has ter (Qiagen Inc., Valencia, CA) to minimize post-sort been genetically linked to microcephaly in mouse [20] and RNA degradation. human [21]. The target neurons were acutely dissociated at E14.5 from a reporter transgenic mouse (Eomes::eGFP) RNA isolation and quality assessment ™ Gsat in which immature neurons of the excitatory lineage High quality total RNA was isolated using the RNAeasy express eGFP [22]. The Genepaint in situ database [23,24], (Qiagen) and flash frozen. From a total volume of which provides comprehensive coverage of gene expres- 35 μl, 1 μl of the RNAeasy fluid was run on an Agilent sion patterns in mouse embryos at E14.5 was then utilized 2100 Bioanalyzer using the RNA PicoChip to assess RNA to validate the transcriptional profiling, from which dy- structural quality and quantity. Peak areas for 28 S and namically expressed genes were identified. A simple but 18 S rRNA were determined. Degraded samples have robust technique was developed to quantify the optical reduced 28 S/18 S ratios and contain additional small density of the in situ patterns (QISPs) across the cerebral RNA fragments that appear as smears in the gel photo wall. These patterns were in turn hierarchically clustered and small humps in the electropherogram making them into putative functional groups. easily detectable. Samples were discarded if the total RNA profile displayed evidence of significant degradation, as Methods indicated by a ribosomal RNA peak ratio (28 S/18 S) Mice below 1.0 (Applied Biosystems/Ambion TechNote 11:2). All animal procedures were approved by the IACUC of No samples were lost due to RNA degradation pro- SUNY Upstate Medical University. Tg(Eomes::eGFP)Gsat vided the acutely dissociated cells were sorted into ™ mice (The Gene Expression Nervous System Atlas Project, collection tubes that contained RNALater .Thetyp- NINDS Contract # N01NS02331 to The Rockefeller Uni- ical yield of total RNA was ~700 ng per 100,000 cells. versity, New York, NY) were mated to C57Bl6 (Taconic Approximately 100 ng of total RNA was used for Labs, Germantown, NY) to generate embryos. The day of transcriptional profiling. plug discovery was designated embryonic day 0.5 (E0.5). (Eomes) – also known as Tbr2 – is a tran- Transcriptional profiling and quality assessment scription factor of the T-box family expressed by inter- The Affymetrix Gene 1.0 ST Array (Affymetrix, Santa mediate precursor cells in the excitatory neuron lineage of Clara, CA) provides whole-transcript coverage of 28,853 the cortex [3,25]. Eomes::eGFP mice contain a BAC trans- mouse genes. Each gene is represented on the array by gene with 185 kb of 5’ and 18 kb of 3’ sequence surround- 27 probes that interrogate the full length of the gene, ra- ing the Eomes . In these mice selective GFP ther than 3’-biased interrogations used in prior tech- expression is observed in developing excitatory neurons nologies (e.g., the Murine U74v2 set). Purification of indicating the fidelity of the large cis-regulatory elements RNA, probe generation, chip hybridization and array in the BAC transgene [26]. scanning was performed at the SUNY Microarray Core facility. Amplified and terminal-labeled cDNA were gen- Tissue dissociation and cell sorting erated by the WT Sense Target Labeling Protocol (Affy- To generate RNA from enhanced Green Fluorescent metrix). cDNA was hybridized to the array with the (eGFP) expressing neurons, dorsal neocortices Affymetrix Fluidics Station 450, and scanned with the Cameron et al. BMC Neuroscience 2012, 13:90 Page 3 of 23 http://www.biomedcentral.com/1471-2202/13/90

ab1) Dissect and Dissociate MZ CP

NCX IZ

LGE SVZ

VZ 2) FACS into GFP+ and GFP- populations

c

P4 P3 SSC-A 3) Profile w/ 1.0ST Arrays

GFP-A 4) Sort genes by RMA and Fold Up

5) Quantified In Situ Patterns (QISPs) 6) Hierarchical Clustering of QISPs

Figure 1 Isolation, profiling, and QISP analysis of GFP + (Eomes+) cortical neurons. a, Neocortex (NCX) containing GFP + neurons is dissected, and cells isolated with flourescence activated cell sorting (FACS). Following transcriptional profiling, genes are sorted by RMA and their level of fold up- or down-regulation compared to the FACS GFP- population. Verification of up-regulated genes is accomplished with quantified in situ hybridization (QISP), and the verified genes hierarchically clustered (see Methods). b, Fluorescence microscopic images of a section from an E14.5 Eomes::eGFP brain, displaying GFP + (green) and GFP- cells, that are blue due to Hoechst nuclear labeling. Subregions of Interest (sROIs) 1-20 are superimposed in white on the image. Scale bar, 25 μm. MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone; LGE, lateral ganglionic eminence. c, Representative outcome of FAC sort of GFP + and GFP- cells. A GFP + (P3) and GFP- (P4) were isolated based on fluorescence (GFP-A) and side scatter (SSC-A). The boxes denote the relevant sorted populations. All in situ hybridizations were obtained from Genepaint.

Affymetrix GeneChip® Scanner3000. Spike controls were Quantified in situ patterns (QISPs) and network analysis used to determine the quality of the hybridization. A QISPs were generated by measuring the optical density 3’/5’ probe signal ratio significantly greater or less than 1 of in situ images derived from Genepaint.org, a public was interpreted as indicating RNA degradation, in database of in situ hybridizations performed on E14.5 which case the experiment was repeated. mouse embryos [23]. The sampled regions of interest Cameron et al. BMC Neuroscience 2012, 13:90 Page 4 of 23 http://www.biomedcentral.com/1471-2202/13/90

(ROI) spanned the cerebral wall from the marginal zone cell populations, processed and profiled on the Affymetrix (MZ) to the VZ. A single ROI was first placed in rostral Gene 1.0 ST Array (Figure 1). To further improve the ana- cortex from an in situ section that included olfactory lytic stringency we restricted analysis to genes with an ex- bulb as an orienting landmark. This original ROI was pression level of RMA (robust multichip average) ≥ 7in then subdivided into 20 sub-regions (sROI1-20), each the GFP + population. This threshold was based on an in- sub-region corresponding to 5% of the cortical wall, and ternal standard; for 10 eye-specific genes ([29]; Additional the optical density of each sROI was measured. The file 1) we calculated from the GFP + population a RMA measured sROI optical density was corrected by sub- value of 5.5 ± 0.7 (mean ± SD). All analyzed genes in this tracting background optical density, and each sROI was report are thus expressed at a minimum of 2.8 fold over then averaged with adjacent sROIs to smooth the QISP this empirically-defined “not expressed” RMA level. profile (Figure 1). The total transcriptome of the E14.5 GFP + and E13.5 GFP + population was examined. We identified tran- Hierarchical clustering of QISPS using Pearson’s script that showed ≥ 3-fold difference (up or down) be- correlation tween E14.5 GFP + and E13.5 GFP + (Figure 2). The The 20 optical density measurements corresponding to gene expression was similar at the two time points: we each QISP (sROI1-20) were imported into Multiple Ex- found 9 up-regulated and 35 down-regulated transcripts periment Viewer [27,28] and normalized for grouping. (Figure 2a, 2a’ and Additional file 2 and Additional file Multiple Experiment Viewer can cluster data by Euclidean 3). We chose to focus our further analysis on the E14.5 distance or Pearson’s correlation. Pearson’scorrelation GFP + data, as this is the same developmental time point quantifies the covariance between samples producing used by the Genepaint mouse in situ database (www. values from -1 to 1. As such it is well suited to quan- genepaint.org). For the E14.5 excitatory neurons more tify the relationship between two QISPs. Importantly than 13,000 unique mRNAs, out of ~25,000 represented two QISP with identical profiles across sROIs1-20 but by the 1.0 ST Array, were above the defined threshold different magnitudes will have a correlation value of 1 (RMA = 7.0). Although numerous (i.e., 53% of all unique and be tightly clustered, thereby facilitating the analysis of mRNAs are expressed above threshold) it is similar to co-regulated genes independent of the absolute expression recent, independent estimates of cortical neuron tran- levels of each. scriptome size (66%, [30]; 60-70%, [31]). The difference between estimates may arise from distinct threshold IPA analysis of gene groups assignments as well as differences in sampling technique; The IPA network analysis tool (Ingenuity Systems, that is, purified FAC sorted embryonic neurons versus Redwood City, CA) was used to analyze clustered genes. dissections of postnatal cortical tissue. IPA is a literature-based analysis tool that uses relation- In an effort to focus analysis upon genes most robustly ships derived from reviewed journal articles as well as expressed by the E14.5 GFP + excitatory neuron popula- additional sources (e.g, KEGG, ) to gener- tion, we identified 475 transcripts up-regulated ≥ 3-fold ate networks of interactions within a data set. Clustered compared to the E13.5 GFP- reference sample genes were imported and network relationships were (Figure 2b and 2b’ and Additional file 4). An additional ascertained using the “Direct and Indirect interactions” 534 genes were identified as being down-regulated and included “Endogeneous Chemicals” (e.g., retinoic by ≥ 3-fold (Additional file 5). To validate the up- acid). regulated genes, Genepaint.org, a public database of in situ hybridizations performed on E14.5 mouse embryos, Results was queried [23]. Of the original 475 up-regulated tran- General analysis of the E14.5 excitatory neuron scripts, 352 were represented in the Genepaint in situ transcriptome database. Fort two of the up regulated transcripts not Previous work from our lab indicated that although represented by Genepaint in situs encoded small nu- Eomes (Tbr2) protein is transiently expressed in the sub- cleolar RNAs, 6 encoded RIKEN cDNA clones, 5 ventricular zone (SVZ), GFP from the transgene endures encoded predicted genes while the remainder of non- in post-mitotic neurons for several days after the downre- represented genes were not readily grouped and pre- gulation of endogenous Eomes [22]. Thus the GFP + sumably reflected Genepaint program priorities. neuron population in this system includes not only IPCs Twenty-four of the 352 (6.8%) in situ patterns either from the SVZ, but also migrating and differentiating exci- displayed no signal in the cerebral cortex or were tatory cortical neurons. FAC sorting purified this GFP + represented by a single in situ section with incom- population to near homogeneity; analytic re-sorting of plete sampling of the cortical wall. The remaining FAC sorted GFP + cells indicated a purity of > 99%. mRNA 328 in situ patterns were then processed for add- was successfully isolated from both the GFP + and GFP- itional validation and grouping. Cameron et al. BMC Neuroscience 2012, 13:90 Page 5 of 23 http://www.biomedcentral.com/1471-2202/13/90

Figure 2 Comparison of RMA values between samples. a, a’, Comparison of RMA values between E14.5 GFP + and E13.5 GFP + cells. b, b’) Comparison of RMA values between E14.5 GFP + and E13.5 GFP- cells.

Validation of the analytic grouping technique To objectively evaluate genes of interest we quantified A method to quantify the optical density of the in situ pat- the in situ pattern for Eomes itself, reasoning that up- terns (QISPs) across the cerebral wall was developed (see regulated genes would display a QISP equivalent to, or Methods). The method of QISP generation is potentially localized more superficially than, the QISP derived for subject to error due to variability of ROI positioning and Eomes (Tbr2). This assumption is based on the accepted histological heterogeneity between cortical areas and sec- observation that, at this developmental stage, the most tions. To assess the precision of the QISP method, mul- mature neurons are located more superficially in the tiple measurements along the rostral caudal axis were cortex than immature neurons [33]. Eomes displayed lit- performed using a set of E14.5 in situ hybridizations for tle expression in ventricular sROIs1-2 and higher expres- Dab1 (Additional file 6), which at E14.5 is strongly sion in the more superficial sROIs3-11. We therefore expressed in the VZ and cortical plate (CP) [32], providing compared the average in situ expression in sROIs1-2 ver- two distinct regions of peak signal. The Dab1 in situ pro- sus the average expression in sROIs3-20. Transcripts file was measured across the cerebral wall at 5 different where this ratio exceeded 1.0 (i.e., where expression was points within the rostral cortex on a single in situ image strongest in the deep VZ) were considered invalid. This (Additional file 6a). The Pearson’s correlation of these five procedure identified 11 invalid genes which were then distinct quantifications ranged from 0.94 to 0.99 with an removed from the initial, post-Genepaint list of 328 average value of 0.97 (n = 5). This indicates that values genes. Thus 97% (n = 317) of the ≥ 3-fold up-regulated, obtained with the ROI approach were consistent and RMA ≥ 7 transcripts displayed a Genepaint-derived in largely insensitive to the precise position of the ROI. Simi- situ hybridization pattern that was spatially consistent larly, we compared the Pearson’s correlation of Dab1 sig- with the Eomes + excitatory neuron lineage. nal from three different, sagittal in situ images derived from sections of rostral cortex along the lateral-to-medial Analytic grouping of the E14.5 excitatory neuron axis (Additional file 6b). These correlations ranged from transcriptome 0.87 to 0.97 with an average correlation of 0.93 (n =4). To explore in greater detail groups of spatially co- Importantly, for both the rostral-caudal and lateral-medial expressed genes, the 317 QISP-validated genes were planes the peak signals within the VZ and CP were always imported into the Multiple Experiment Viewer [27,28], restricted to within 10% of the cerebral wall (i.e., ± 2 the signal (expression) values normalized by gene, and sROI). These tests validated the QISP analysis by indicat- then hierarchically clustered. This procedure groups to- ing robust concordance of optical density measurements gether similar QISPs, and can reveal groups of genes between ROIs from different positions in the rostral cor- that are spatially co-expressed and thus potentially co- tex and along the lateral-to- medial axis. regulated [34]. Cameron et al. BMC Neuroscience 2012, 13:90 Page 6 of 23 http://www.biomedcentral.com/1471-2202/13/90

The clustering of similar QISPs revealed several dis- characterized by the expression of the transcriptional tinct spatial distributions of dynamically regulated genes regulators Neurod4, Bcl6, Tanc2 and Myt1. Earlier stud- in the radial dimension of the developing cerebral cortex ies focusing upon this deep area of cortex revealed (a) (Figure 3). Approximately 70% of up-regulated QISPs neurons with a long leading process that translocate out within the Eomes + lineage display peak expression in of the VZ [19,38,39] and (b) compact intermediate pre- the CP and upper intermediate zone (IZ). The majority cursor cells in the SVZ [40]. Thus, at this point in devel- of these QISPs displayed similar (i.e., tightly clustered) opment the GFP + cells in the VZ and SVZ are predicted expression patterns, characterized by gradually increas- to represent a mixture of intermediate precursors and ing expression in the IZ and peak expression in the CP. postmitotic neurons [41]. Consistent with this predic- The most heterogeneous expression patterns were tion, IPA analysis identified DNA Replication, Recom- observed in those QISPs with peak expression in the VZ bination and Repair as a functional category for these and IZ, with a number of QISPs displaying dual expres- group (p = 1.37E-04; Table 1, Additional file 7). Surpris- sion peaks (e.g., Gdap1, Bcl6) or expression restricted to ingly, Carbohydrate Metabolism was also identified (p = either the IZ or VZ (e.g., Neurod1 or Sstr2). 3.00E-05): the deep group includes genes involved in the Four groups of genes – that is, four groups of clus- synthesis (C1ql3 and Lpl) as well as level regulation tered QISPs – were examined using a network analysis (Chga, Gipr, Pcsk2 and Sstr2) of D-glucose. The top tool (IPA, Ingenuity Systems). The four groups were: (1) “Canonical Pathways” include Protein Kinase A Signa- all 317 genes; (2) 44 “deep group” genes that achieve ling and Cardiac β-Adrenergic Signaling. Additionally, a highest in situ expression in the VZ; (3) 49 “middle number of genes involved in intracellular calcium signa- group” genes that achieve highest expression in the IZ ling (Ryr3, Camk2b, Cacna1h) showed elevated expres- and lower CP; and (4) 224 “superficial group” genes with sion in this group. Network analysis of deep group genes greatest expression in the CP. This approach was thus suggests that the phenotypic transition from radial adopted because neurons in the deep, middle, and super- glial precursor to intermediate precursor/immature cor- ficial regions of the cortex have distinct morphologies tical neuron coincides with significant changes in and modes of migration [1] and thus can be expected to mechanisms of glucose metabolism and second messen- manifest dynamic regulation of genes related to morph- ger signaling. ology, navigation and migration. The “middle group” comprises 49 genes, or 16% of the ≥ 3 fold up data set. The middle group QISPs, com- Network analysis of all up-regulated genes prising clusters 9 and 10, are maximal in the IZ, where Network analysis of all 317 genes revealed 164 asso- developing excitatory neurons display a multipolar ciated with neurological disease and 85 associated with morphology. Multipolar neurons (MPNs) possess mul- psychological disorders; i.e., ≥ 52% of the genes have tiple fine processes that lack definitive orientation, and previously been implicated in human health (Table 1). migrate relatively slowly in the radial direction [1]. The One hundred thirteen of the genes were categorized as top “Molecular and Cellular Functions” in this group are Nervous System Development and Function, indicating Cellular Development and Cell Morphology (Table 1, a substantial number (36%) of genes in this group having Additional file 7). The middle group is dominated by the been previously identified and functionally characterized. basic helix loop helix (bHLH) transcription regulators The top “Molecular and Cellular Functions” (using the Neurod6 (71.5 fold), Neurod1 (30.9 fold), Nhlh1 (11.6 IPA terminology) include Cell to Cell Signaling and fold) and Neurod2 (7.3 fold), which represent some of Interactions, Cellular Assembly and Organization, and the most highly up-regulated genes in the entire data Cellular Movement, with the top “Canonical Pathways” set. These bHLH transcription factors have well- being Axonal Guidance Signaling and CDK5 signaling. described functions in excitatory cortical neuron differ- Both of these signaling pathways are known to be active entiation and development [6,42]. The top “Canonical during cortical neuron migration and differentiation Pathways” include Axonal Guidance Signaling and [35-37]. This network analysis showed that the Semaphorin Signaling in Neurons, as well as Inositol entire ≥ 3-fold up-regulated gene category contains a Phosphate Metabolism. Of particular interest within the large number of annotated genes associated with middle group genes is cluster 9, consisting of 26 genes neuronal development, migration, differentiation, and that includes seven with known chemotropic signaling neurological disease. functions in guidance. The expression of this set of The “deep group” of 44 up-regulated genes (14% of axonal guidance genes could promote the generation the total), which is spatially co-expressed with Eomes and extension of that are observed to the mi- (2.2 fold up), achieved highest expression in the VZ grating cortical neuron in the upper IZ and CP [39,43]. (sROIs 1-10). This group includes clusters 1, 5, 7, 8 and In addition to the highly up-regulated transcription fac- 11-15 (Figure 3). Along with Eomes, the deep group is tors, the top up-regulated genes include Vglut1/Slc17A6 Cameron et al. BMC Neuroscience 2012, 13:90 Page 7 of 23 http://www.biomedcentral.com/1471-2202/13/90

Figure 3 (See legend on next page.) Cameron et al. BMC Neuroscience 2012, 13:90 Page 8 of 23 http://www.biomedcentral.com/1471-2202/13/90

(See figure on previous page.) Figure 3 Identification and hierarchical clustering of QISPs. a, Hierarchical clustering of 317 QISPs representing transcripts expressed ≥ 3 fold higher in Eomes + (GFP+) neurons, compared to GFP- cells. From left to right: gene clusters denoted numerically (1-15); expression levels in the MZ + CP, IZ, and VZ (red indicates high expression, green indicates low expression) and gene number (1-317). b, Gene lists of the clustered QISPs from panel a. QISPs were imported into Multiple Experiment Viewer (MeV) and clustered using Pearson’s correlation (see Methods). White and shaded areas demarcate the different gene clusters.

(19.4 fold) a glutamate transporter that identifies excita- “upper group” genes thus suggest maturing excitatory tory cortical neurons [44]. Network analysis of “middle neurons in the developing CP express a number of regu- group” genes thus indicated that developing excitatory latory and signaling factors, including the transcription neurons in the IZ are molecularly defined by a high ex- factors Tbr1, Bhlhb5, Hivep2/Schnurri2, Prdm8, Satb2, pression of canonical bHLH neuronal transcription fac- Sox5, and Bcl11b/Ctip2. tors and axonal guidance molecules, particularly those of the Semaphorin signaling pathway. Analysis of highly up- and down-regulated transcription The “upper group” of genes (n = 224) were observed in factors clusters 2, 3, 4 and 6. These genes, comprising 71% of To explore the spatial relationships of key transcription the up-regulated gene population, achieved highest ex- factors (TFs) in the cortical lineage, we identified all ≥ 3 pression in the developing cortical plate (CP), an area fold up- and down-regulated genes associated with the that contains migrating neurons with an elongated or bi- Gene Ontology descriptor “transcription, nuclear” and polar shape, and post-migratory differentiating neurons. clustered their respective QISPs (Figure 4). These tran- Within the upper group the most prominent transcrip- scription/nuclear QISPs span the cerebral wall at E14.5, tional regulators were Tbr1 (43.1 fold) [45], Bhlhb5 (19 with subsets of QISPs showing peak expression in the fold) [46], and Hivep2/Schnurri2 (18 fold). Additional VZ, IZ and CP (Figure 4a). We were initially surprised transcription factors in the upper group included Prdm8 that Eomes itself was upregulated only 2.2 fold, a value [47], Satb2 [48], Sox5 [11], and Bcl11b/Ctip2 [49]. All of below our threshold for analysis. There are several rea- these transcriptions factors, with the exception of sons for this low fold up value. The first is that GFP- Schnurri2, have demonstrated roles in corticogenesis. cells express Eomes mRNA (RMA = 10.7). The presence Schnurri2 is, however, a BMP signaling effector, with a of Eomes mRNA in the absence of GFP protein could neuronal function suggested by the observations that imply post-transcriptional regulation of the 5’ UTR of mice deficient in Schnurri2 are hyperactive and exhibit the Eomes message that is included in the Eomes: :eGFP hypersensitivity to stress [50]. transgene [26]. Similarly, the presence of Eomes mRNA Neurons within the forming CP migrate along radial in GFP- cells might reflect the rapid up regulation of glia fibers, from which they later detach and extend den- Eomes mRNA in a subpopulation of cells that has yet to drites. It is therefore not surprising that the top “Mo- express sufficient GFP protein for FAC detection and lecular and Cellular Functions” in the upper group are sorting into the GFP + group. Finally, Eomes mRNA is Cell-To-Cell Signaling and Interaction, Cellular Assem- sharply down regulated as neurons leave the SVZ [3], bly and Organization, and Cellular Movement (Table 1, while GFP protein expression persists and is expressed Additional file 7). The top “Canonical Pathways” include in all cortical zones superficial to the SVZ, at this devel- Corticotropin Releasing Hormone Signaling and CDK5 opmental time point. Thus the GFP + cell population is Signaling. A role for CDK5 signaling mechanisms is likely larger than the Eomes mRNA + cell population, ef- established for many neurodevelopmental functions, in- fectively reducing the average Eomes mRNA expression cluding the migration of cortical neurons along radial level in the GFP + population. Thus post-transcriptional glial fibers and subsequent cortical layer formation and regulation and/or the difference between sharp Eomes dendritogenesis [51,52]. In contrast the role(s) of mRNA regulation and the delayed and extended GFP Corticotropin Releasing Hormone Signaling in these protein expression may account for this relatively low neurons is less clear. The eight genes from the ≥ 3 fold fold up level for this critical TF. up-regulated category that populate the Corticotropin We first compared these genes to an earlier study of Releasing Hormone Signaling category include four with bHLH-family TFs denoted as the “Dorsal Telencephalon known functions in brain development, including the Network” [4]. Comparison to this independent data set of cannabinoid Cnr1 [53], the transcription factor 13 TFs revealed 7 (54%) to be members of our ≥ 3foldup- Mef2C [54], and nitric oxide synthase Nos1 [55]. The regulated category: Neurod1 (31 fold), Neurod2 (7.3 fold), remaining genes in this group include the protein Neurod6 (72 fold), Tbr1 (43 fold), Id2 (4.1 fold), Nhlh1 kinases Prkar1β, Prkcε and Prkcζ Network analysis of (12 fold) and Satb2 (12 fold) (Figure 4b). Other dorsal Cameron et al. BMC Neuroscience 2012, 13:90 Page 9 of 23 http://www.biomedcentral.com/1471-2202/13/90

Table 1 Summary of IPA Ingenuity analysis for the upper, middle, and deep cortical zones IPA Ingenuity Pathway Analysis317 GENES (ALL) Diseases and Disorders p-value #genes Neurological Disease 2.40E-21 - 1.99E-02 164 Psychological Disorder 3.15E-16 - 1.99E-02 85 Genetic Disorder 7.48E-16 - 1.99E-02 213 Skeletal and Muscular Disorders 3.73E-15 - 1.99E-02 123 Gastrointestinal Disorder 8.25E-15 - 1.99E-02 133 Molecular and Cellular Signaling p-value #genes Cell-To-Cell Signaling and Interaction 7.26E-20 - 1.99E-02 68 Cellular Assembly and Organization 5.56E-16 - 1.99E-02 82 Cellular Movement 1.28E-12 - 1.99E-02 44 Molecular Transport 2.71E-07 - 1.99E-02 67 Small Molecule 2.71E-07 - 1.99E-02 54 Canonical Pathways p-value Ratio Axonal Guidance Signaling 1.26E-06 23/433 CDK5 Signaling 3.87E-05 9/94 Cardiac b-adrenergic Signaling 4.55E-05 11/154 Corticotropin Releasing Hormone Signaling 2.09E-04 9/136 Signaling 6.05E-04 7/95 224 UPPER GENES Molecular and Cellular Signaling p-value #genes Cell-To-Cell Signaling and Interaction 4.31E-17 - 2.12E-02 51 Cellular Assembly and Organization 3.34E-11 - 2.12E-02 60 Cellular Movement 5.21E-10 - 1.42E-02 35 Cellular Function and Maintenance 4.02E-08 - 2.20E-02 49 Molecular Transport 4.02E-08 - 1.75E-02 49 Canonical Pathways p-value Ratio Corticotropin Releasing Hormone Signaling 1.16E-04 8/136 CDK5 Signaling 1.76E-04 7/94 Synaptic Long Term Depression 1.62E-03 7/147 Cardiac b-adrenergic Signaling 2.35E-03 7/154 Axonal Guidance Signaling 2.65E-03 13/433 49 MID GENES Molecular and Cellular Signaling p-value #genes Cellular Development 9.09E-07 - 4.47E-02 12 Cell Morphology 3.82E-06 - 3.88E-02 16 Cellular Assembly and Organization 1.16E-05 - 4.18E-02 16 Cellular Movement 4.59E-05 - 4.76E-02 9 Cell-To-Cell Signaling and Interaction 4.00E-04 - 4.76E-02 13 Canonical Pathways p-value Ratio Axonal Guidance Signaling 1.28E-07 10/433 Semaphorin Signaling in Neurons 1.54E-05 4/52 Inositol Phosphate Metabolism 7.95E-03 3/180 CXCR4 Signaling 9.66E-03 3/169 Regulation of Actin-based Motility by Rho 2.29E-02 2/91 Cameron et al. BMC Neuroscience 2012, 13:90 Page 10 of 23 http://www.biomedcentral.com/1471-2202/13/90

Table 1 Summary of IPA Ingenuity analysis for the upper, middle, and deep cortical zones (Continued) 44 DEEP GENES Molecular and Cellular Signaling p-value #genes Carbohydrate Metabolism 3.00E-05 - 3.88E-02 7 Molecular Transport 3.00E-05 - 3.88E-02 11 Small Molecule Biochemistry 3.00E-05 - 4.34E-02 12 DNA Replication, Recombination and Repair 1.37E-04 - 1.81E-02 2 Nucleic Acid Metabolism 1.37E-04 - 2.32E-02 7 Canonical Pathways p-value Ratio Protein Kinase A Signaling 8.73E-05 6/328 Cardiac b-adrenergic Signaling 3.18E-04 4/154 G-Protein Coupled Receptor Signaling 1.43E-03 6/530 cAMP-mediated signaling 1.94E-03 4/219 Synaptic Long Term Potentiation 2.08E-03 3/114 In each panel the most salient “Molecular and Cellular Signaling” and “Canonical Pathways” categories are indicated, along with the associated p values. a, “all”. b, “upper group.” c, “middle group.” d, “deep group.”. telencephalon TFs outside the ≥ 3-fold up-regulated cat- We hypothesized that the highly expressed “middle” and egory include the early genes Pax6 (7.6 fold down-regu- “deep” group TFs are critical molecular regulators, per- lated), Neurog 1 (1.3 fold down-regulated), Neurog 2 (1.1 haps functioning to simultaneously repress precursor- fold down-regulated) and Eomes itself (2.2 fold up-regu- specific TFs and promote the expression of TFs lated). Although Pax6 expression in the GFP + population required for proper differentiation of cortical excitatory was below threshold (RMA = 6.2) Neurog1, Neurog2, and neurons. To test this idea, a network analysis tool (IPA; Eomes were not (RMA values of 7.6, 10.1, and 11.9, re- see Methods) was used to explore within our dataset pos- spectively). This suggests that the GFP + neurons in our sible regulatory interactions among the dynamically study substantially down-regulate Pax6 relative to neuronal expressed TFs (Figure 5). All TFs from the ≥ 3foldup- precursors, but do express the neuronal specifying TFs and down-regulated populations were imported into the Neurog1, Neurog2 and Eomes. Additionally, two genes pathway analysis and the network of direct and indirect specific to cortical layer V-VI, Etv1 and Otx1 [56], were interactions was identified. All interacting genes that were down-regulated in our data (5 fold and 2 fold, respec- identified, but absent from the populations of ≥ 3foldup- tively), an observation supported by strong Genepaint in and down-regulated TFs, were assigned to a group situ hybridization signals at E14.5intheventricularzone denoted “IPA Interacting Genes.” for both genes. Because postnatal expression of Etv1 and Approximately 430 connections were identified for TFs Otx1 is specific to cortical layer V-VI, this result suggests in the up- and down-regulated groups. temporally differential (i.e., developmentally early and late) Only 4 direct interactions were reported between the requirements for these TFs during cortical development. In “deep group” or “middle group” genes and either the “upper contrast to the “Dorsal Telencephalon Network” we identi- group” genes or the down-regulated genes (Figure 5). Nu- fied the layer II-IV marker Satb2 within our ≥ 3foldup- merous direct interactions, however (50 of 54), were regulated data set (12 fold), an observation confirmed by detected that connected “middle” and “deep” genes to the Genepaint-derived in situ hybridization. Consistent with “IPA Interacting Genes” group. Included in this group are the dorsal telencephalic identity of the Eomes + population, signaling molecules that function non-cell autonomously in none of the nine genes associated with the “Ventral Telen- neuronal development: Notch signaling [58], Wnt-βcatenin cephalon Network” [4] were observed in the ≥ 3foldup- signaling [59], VEGF [60], TGF β [37] and retinoic acid sig- regulated population. By comparison to published studies naling [61]. Each of these signaling pathways has been of excitatory neuron differentiation [2-4,57], this TF expres- shown to regulate either the production and/or differenti- sion analysis developmentally positions the “maturation ation of excitatory cortical neurons. Surprisingly, only 14, or spectrum” of the isolated E14.5 GFP + neurons to a period ~3%, of all detected interactions were direct between any after the expression of early dorsal specification genes (e.g., group of dynamically regulated TF genes (i.e., between the Pax6), coincident with neural specification genes (e.g., Neu- 4lowerboxesinFigure5).Theresultsofthisnetwork rog1, Neurog2), and prior to the expression of late layer- analysis support models of neurogenesis that predict com- specific markers (e.g., Etv1 and Otx1). plex interactions between specific cell autonomous (e.g. Cameron et al. BMC Neuroscience 2012, 13:90 Page 11 of 23 http://www.biomedcentral.com/1471-2202/13/90

a CP+MZ IZ VZ Fold Zone Gene Name Affy ID 10373680 D 4.1 Neurod4 neurogenic differentiation 4 (Neurod4), mRNA. 10479698 M 3.1 Myt1 myelin transcription factor 1 (Myt1), mRNA. 10395074 M 6.9 Myt1l myelin transcription factor 1-like (Myt1l), transcript variant 1, mRNA. 10360202 M 11.6 Nhlh1 nescient helix loop helix 1 (Nhlh1), mRNA. 10358272 M 3.4 Lhx9 LIM protein 9 (Lhx9), transcript variant 3, mRNA. 10390618 M 7.3 Neurod2 neurogenic differentiation 2 (Neurod2), mRNA. 10484276 M 30.9 Neurod1 neurogenic differentiation 1 (Neurod1), mRNA. 10503259 M 3.8 Trp53inp1 transformation related protein 53 inducible nuclear protein 1 (Trp53inp1) 10544936 M 71.5 Neurod6 neurogenic differentiation 6 (Neurod6), mRNA. 10354111 M 5.6 Aff3 AF4/FMR2 family, member 3, mRNA 10352092 U 3.8 Zfp238 protein 238 (Zfp238), transcript variant 1, mRNA. 10545045 D U 3.5 D430015B01 RIKEN cDNA D430015B01 gene (D430015B01Rik), mRNA. 10454235 U 3.3 Asxl3 gene:ENSMUSG00000045215 10402554 U 4.5 Bcl11b B-cell leukemia/lymphoma 11B (Bcl11b), transcript variant 1, mRNA. 10361807 U 17.8 Hivep2 human immunodeficiency type I enhancer binding protein 2 (Hivep2) 10504838 U 12.0 Nr4a3 subfamily 4, group A, member 3 (Nr4a3), mRNA 10344679 U 3.9 St18 suppression of tumorigenicity 18 (St18), mRNA. 10606174 U 3.1 Nap1l2 nucleosome assembly protein 1-like 2 (Nap1l2), mRNA. 10423902 U 3.1 Zfpm2 zinc finger protein, multitype 2 (Zfpm2), mRNA. 10406434 U 3.1 Mef2c myocyte enhancer factor 2C (Mef2c), mRNA. 10529875 U 4.8 Ldb2 LIM domain binding 2 (Ldb2), transcript variant 2, mRNA. 10429944 U 3.8 Scrt1 scratch homolog 1, zinc finger protein (Drosophila) (Scrt1), mRNA. 10399691 U 4.1 Id2 inhibitor of DNA binding 2 (Id2), mRNA 10490950 U 19.3 Bhlhb5 basic helix-loop-helix domain containing, class B5 (Bhlhb5), mRNA 10472313 U 43.1 Tbr1 T-box brain gene 1 (Tbr1), mRNA. 10354777 U 11.6 Satb2 special AT-rich sequence binding protein 2 (Satb2), mRNA. 10549200 U 5.2 Sox5 SRY-box containing gene 5 (Sox5), mRNA. 10381006 U 3.4 Thra thyroid alpha (Thra), mRNA. 10469906 U 3.1 Nelf nasal embryonic LHRH factor (Nelf), transcript variant 1, mRNA. 10478145 U 4.0 Ppp1r16b protein phosphatase 1, regulatory (inhibitor) subunit 16B (Ppp1r16b) 10365428 U 3.3 Btbd11 BTB (POZ) domain containing 11 (Btbd11), transcript variant 1, mRNA. 10523483 U 12.8 Prdm8 PR domain containing 8 (Prdm8) Transcription factor, mRNA. 10538275 U 5.7 Nfe2l3 nuclear factor, erythroid derived 2, like 3 (Nfe2l3), mRNA. 10358978 U 3.6 Ier5 immediate early response 5 (Ier5), mRNA. 10365344 U 4.1 Tcp11l2 t-complex 11 (mouse) like 2 (Tcp11l2), mRNA. 10438738 D U 3.9 Bcl6 B-cell leukemia/lymphoma 6 (Bcl6), mRNA. 10381934 D U 3.4 Tanc2 tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 (Tanc2) b 10522712 D 0.3 Rest RE1-silencing transcription factor (Rest), mRNA 10450519 D 0.3 Tcf19 transcription factor 19 (Tcf19), mRNA. 10478355 D 0.3 Mybl2 myeloblastosis oncogene-like 2 (Mybl2), mRNA. 10437687 D 0.2 Litaf LPS-induced TN factor (Litaf), mRNA 10359624 D 0.2 Prrx1 paired related homeobox 1 (Prrx1), GRADIENT transcript variant 2, mRNA. 10498284 D 0.2 Wwtr1 WW domain containing transcription regulator 1 (Wwtr1), mRNA. 10510172 D 0.3 Hmgb2 high mobility group box 2 (Hmgb2), mRNA. 10415396 D 0.3 Nfatc4 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4 (Nfatc4) 10594404 D 0.2 Smad3 MAD homolog 3 (Drosophila) (Smad3), mRNA 10421950 D 0.3 Dach1 dachshund 1 (Drosophila) (Dach1), transcript variant 1, mRNA. 10514466 D 0.2 Jun Jun oncogene (Jun), mRNA. 10382328 D 0.1 Sox9 SRY-box containing gene 9 (Sox9), mRNA 10474312 D 0.1 Pax6 paired box gene 6 (Pax6), mRNA. PAX6 10424213 D 0.3 Zhx2 zinc fingers and protein 2 (Zhx2), mRNA. 10515090 D 0.2 Cdkn2c cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) (Cdkn2c), mRNA. 10464084 D 0.3 Tcf7l2 transcription factor 7-like 2, T-cell specific, HMG-box (Tcf7l2), mRNA. 10391461 D 0.2 Brca1 breast 1 (Brca1), mRNA. 10403727 D 0.1 Gli3 GLI-Kruppel family member GLI3 (Gli3), mRNA. 10385325 D 0.2 Pttg1 pituitary tumor-transforming 1 (Pttg1), mRNA 10500630 D 0.3 Ttf2 transcription termination factor, RNA polymerase II (Ttf2), mRNA 10469936 D 0.2 Nrarp Notch-regulated ankyrin repeat protein (Nrarp), mRNA. 10395457 D 0.2 Etv1 ets variant gene 1 (Etv1), mRNA. 10361771 D 0.3 Plagl1 pleiomorphic adenoma gene-like 1, mRNA (cDNA clone IMAGE:6856210). 10491477 D 0.1 SRY-box containing gene 2 (Sox2), mRNA. 10366266 D 0.2 Pawr PRKC, apoptosis, WT1, regulator (Pawr), mRNA. 10545458 D 0.1 Tcf3 transcription factor 3 (Tcf3), transcript variant 1, mRNA. 10404975 D 0.1 Id4 inhibitor of DNA binding 4 (Id4), mRNA. 10428534 D 0.2 Trps1 4 days neonate male adipose cDNA,clone:B430110C06 product:hypothetical protein 10580510 D 0.1 Sall1 sal-like 1 (Drosophila) (Sall1), mRNA. 10452633 D 0.3 Tgif1 TG interacting factor 1 (Tgif1), mRNA. 10363901 D 0.1 Etv5 cdna:Genscan :NCBIM37:10:71167116:71168033:1 10527801 D 0.3 Brca2 2 (Brca2), transcript variant 1, mRNA. 10499366 D 0.3 Pmf1 polyamine-modulated factor 1 (Pmf1), mRNA. 10422272 D 0.2 Sox21 SRY-box containing gene 21 (Sox21), mRNA; SRY-box containing gene 21 (Sox21) 10600755 D 0.2 Arx aristaless related homeobox gene GRADIENT (Drosophila) (Arx), mRNA. 10550593 D 0.3 Six5 sine oculis-related homeobox 5 homolog (Drosophila) (Six5), mRNA. 10543058 D 0.1 Dlx5 distal-less homeobox 5 (Dlx5), transcript variant 1, mRNA.

Figure 4 Transcripts encoding transcription factors. Functional groupings of QISPs based upon Gene Ontology (GO) molecular function and biological process annotations. a, Clustered QISPs of transcripts expressed ≥ 3 fold higher in Eomes + neurons compared to GFP- cells. The left- most dendrogram reveals the hierarchical arrangement of QISPs. b, Clustered QISPs of transcripts expressed ≥ 3 fold lower in Eomes + neurons compared to GFP- cells. a’ and b’. Cameron et al. BMC Neuroscience 2012, 13:90 Page 12 of 23 http://www.biomedcentral.com/1471-2202/13/90

a IPA Interacting Genes

14 13 12 11 10 9 8 7 6 5

4 Upper Genes CP+MZ 3 2 1 Middle Genes IZ 3 Fold Down Genes Deep Genes VZ VZ

Interact. # Genes Relationship Ref. b 1 BCL6 / SMAD3 Bcl6 binds Smad3 and decreases Smad3 and p300 binding 1 2 BCL6 / JUN Bcl6 binds Jun 1 3 NEUROD1 / PAX6 Neurod1 increase Pax6 mRNA expression in duct cells 1 4 LHX9 / LDB2 Lhx9 binds Ldb2 1 5 SOX2 / ID2 Sox2 protein decrease Id2 mRNA expression 1 6 SMAD3 / ID2 Id2 binds Smad3 1 7 ID2 / WWTR1 Suppression of WWTR1 by siRNA decreases Tgfb1-dependent Id2 expression 1 8 ID2 / JUN Jun protein is i nvolved in the expression of Id2 mRNA 1 9 SOX2 / MEF2C Sox2, and Oct4 protein increase Mef2c mRNA expression 1 10 MEF2C / DACH1 Dach1 decreases Tgfb-dependent Mef2c mRNA expression 1 11 MEF2C / JUN Acti vation of Mef2c protein increases t ransacti vation of Jun gene promoter 1 12 SOX9 / S OX5 Sox9 protein increases S ox5 mRNA expression in mouse limb bud 1 13 ZFPM2 / S OX 9 ZFPM2 deficiency decreases S ox9 mRNA in mouse gonad 1 14 THRA / JUN Thra protein increases Jun protein expression in dermal fibroblasts 1 Figure 5 Network analyses of dynamically regulated transcription factors (TFs) in differentiating cortical excitatory neurons, derived from the IPA-Ingenuity Network Tool (see Methods). a, ≥ 3 fold up- and ≥ 3 fold down-regulated TFs were imported along with endogenous chemicals (e.g., retinoic acid) to populate the network. Grey boxes collect the up-regulated genes expressed in the “upper,”“mid” and “deep” cortical regions (CP + MZ, IZ, VZ alone) as well as the down-regulated genes (VZ) and the group of IPA Interacting Genes. Gray lines that are numbered indicate interactions between cortical zones (upper, middle, and deep); blue lines indicate direct interactions of upper, middle, and deep up-regulated genes, and down-regulated genes, with IPA interacting genes. Dark blue lines indicate interactions between IPA interacting genes (see Results). b, Tabular summary of interactions between cortical zones. 1, Interaction identified by IPA Ingenuity analysis. Cameron et al. BMC Neuroscience 2012, 13:90 Page 13 of 23 http://www.biomedcentral.com/1471-2202/13/90

transcription regulatory networks) and non-cell autono- Chrm3. Cnr1 has multiple devel- mous signaling mechanisms (e.g., Notch, TGFβ signaling) opmental roles including axonal guidance [78]. Specific during the maturation of cortical excitatory neurons. developmental functions of Mcr4, Gipr, and Chrm3 are unknown, although Mcr4 interacts with Cnr1 to regulate Analysis of highly up- and down-regulated receptor genes food intake in adults [79], Gipr regulates insulin secre- To more fully explore the role(s) of dynamically regu- tion after feeding [80], and Chrm3 is required for pan- lated receptors in the differentiation and migration of creatic insulin and glucagon release [81] and neuronal cortical neurons, we identified within the ≥ 3 fold up- endocannabinoid release [82]. The up-regulated Gprs and down-regulated gene populations those with the also include the Gpr21, as well as a Gene Ontology descriptor “receptor activity,” and clus- highly conserved receptor, Gpr85 (also known as Sreb2), tered their respective QISPs (Figure 6). The down- known to control brain size [83]. The homolog regulated receptors were enriched for signaling systems Celsr3, which is required for neuronal projections to the with established functions in neuronal precursors anterior commissure as well as subcerebral targets, is (Figure 6b, 6b’), including the Wnt-βcatenin signaling also up-regulated [84]. family members Sfrp2, Fzd8, Lrp4, and Wnt7a. Wnt- βcatenin signaling controls the production of neurons by Analysis of highly up- and down-regulated neurotransmitter neural precursors [62] and regulates intermediate pre- receptors and synaptic genes cursor proliferation [59,63]. The FGF receptors Fgfr2 Neurotransmitters and their receptors have important and Fgfr3, and Notch family members Notchr1, Notchr2 functions in the developing nervous system [85]. Al- and Notchr3 were also down-regulated. Notch receptors though our dataset contained no neurotransmitter have multiple roles in neurogenesis and differentiation, receptors down-regulated ≥ 3 fold, the up-regulated cat- including the regulation of neuronal precursor differenti- egory contained a variety of neurotransmitter receptor ation [58,64], the promotion of glial fate [65], and den- subunits. Cholinergic receptor subunit β2 (Chrnb2) and dritic growth [66,67]. Fgfr2 is required for excitatory Chrna7 are up-regulated in the IZ and CP by 4.5 fold neuron development [68], with Fgfr3 required for attain- and 2.8 fold, respectively. These receptor subunits form ment of normal cortical and hippocampal volume and heteromeric receptor complexes in the developing caudal cortex development [69]. These results suggest that, brain, suggesting an early role for cholinergic signaling along with a dynamic enhancement of pro-differentiation as excitatory neurons migrate out of the VZ. Although genes, genes responsible for the maintenance of precursor/ deletion of Chrna7 in mice does not cause overt struc- progenitor/glial phenotypes are dynamically suppressed at tural abnormalities [86], recent studies indicate that E14.5 in the lineage of excitatory cortical neurons upon haploinsufficiency in CHRNA7 may cause a range of neu- exiting the VZ. rodevelopmental phenotypes associated with the 15q13.3 Thirty four receptors were identified in the ≥ 3foldup syndrome, including developmental delay, mental retard- category (Figure 6a, 6a’). The top category of up-regulated ation and seizure [87]. Although no glutamate receptors receptors includes those with established roles in axono- were upregulated ≥ 3 fold, N-methyl-D-aspartate (NMDA) genesis (8.14E-11 p-value): members of the Semaphorin receptor subunits Grina and Grin1 were mildly up- signaling family (Nrp1, Sema6d, Plxna2, Sema7a, Plxna4, regulated (2.6 fold and 2.0 fold, respectively; data not Sema4g, Plxnd1); Netrin receptors (Unc5d, Dcc); the Slit shown). In addition to mediating adult forms of synaptic signaling receptor Robo1. Because they are observed in plasticity, NMDA receptors may mediate forms of spon- clusters 9 and 10 (Figure 3), many of these canonical che- taneous activity in the developing cortex [88] as well as motropic signaling receptors are co-expressed in the IZ. structural plasticity in the late embryonic and early post- Specifically, Semaphorin signaling molecules are expressed natal period [89,90]. mRNA encoding GABA (gamma- by excitatory neurons in the IZ soon after exit from the amino-butyric-acid) receptor subunits were also up-regu- VZ, with Netrin receptors and Robo1 expressed in more lated, including Gabarg2 (4.6 fold) and Gabbrb2 (3.9 fold). superficial domains. These chemotropic signaling systems Gabarb3 was upregulated 2.8 fold. Deficiency in Gabarg2 have multiple roles and may be enabled for axonal growth may be causal in Dravet syndrome, a form of childhood [70-72], neuronal migration [73], dendritic growth [74-76] [91]. GABA functions as an excitatory neuro- and cell survival [77]. transmitter in early development, xand deficiency in Also included among the most highly up-regulated GABAA receptor subunits alter Inhibitory Post Synaptic receptors were eight GTP-binding protein-coupled Potentials (IPSPs) but not synapse number [92]. During receptors (Gprs) (2.87 E-5 p-value), including Mcr4 early periods GABA can function as a depolarizing ( 4), Cnr1 (cannabinoid recep- neurotransmitter and may relieve Mg2+ blockade of tor1), Sstr2 ( 2), Gipr (gastric in- NMDA receptors [93,94]. One glycine receptor subunit, hibitory polypeptide receptor), and the muscarinic Glra2 (10.7 fold up-regulated) was identified in this Cameron et al. BMC Neuroscience 2012, 13:90 Page 14 of 23 http://www.biomedcentral.com/1471-2202/13/90

a CP+MZ IZ VZ Fold Affy ID Zone Name Gene 10382341 D U 9.7 Sstr2 (Sstr2), transcript variant 1, mRNA. 10560443 D U 4.1 Gipr gastric inhibitory polypeptide receptor (Gipr), mRNA. 10576639 M 9.7 Nrp1 1 (Nrp1) semaphorin receptor, mRNA 10475544 M 4.0 Sema6d sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D 10585778 M 4.9 Sema7a sema domain, (Ig), and GPI membrane anchor, (semaphorin) 7A 10543802 M 13.5 Plxna4 plexin A4 (Plxna4), mRNA. 10577999 M 5.0 Unc5d unc-5 homolog D (C. elegans) (Unc5d), mRNA. 10440344 M 3.1 Robo2 roundabout homolog 2 (Drosophila) (Robo2), mRNA. 10352867 M 4.0 Plxna2 plexin A2 (Plxna2), mRNA. 10499643 M 4.5 Chrnb2 cholinergic receptor, nicotinic, beta polypeptide 2 (neuronal) (Chrnb2) 10470529 M 3.4 Olfm1 olfactomedin 1 (Olfm1), transcript variant 1, mRNA. 10589130 M 4.5 Celsr3 EGF LAG seven-pass G-type receptor 3 (Celsr3), mRNA. 10485840 D M U 4.1 Ryr3 3 (Ryr3), mRNA. 10459512 U 14.4 Mc4r (Mc4r), mRNA. 10547100 U 3.8 Plxnd1 plexin D1 (Plxnd1), mRNA. 10607792 U 10.7 Glra2 glycine receptor, alpha 2 subunit (Glra2), mRNA. 10436519 U 5.8 Robo1 roundabout homolog 1 (Drosophila) (Robo1), mRNA. 10463430 U 3.9 Sema4g sema, immunoglobulin, transmembrane and short cytoplasmic domain (semaphorin) 4G 10532542 U 5.7 Sez6l seizure related 6 homolog like (Sez6l), mRNA. 10436865 U 6.1 Ifngr2 interferon gamma receptor 2 (Ifngr2), mRNA. 10399892 U 3.1 Gpr22 G protein-coupled receptor 22 (Gpr22), mRNA 10596095 U 3.0 Ephb1 Eph receptor B1 (Ephb1), mRNA. 10542414 U 4.1 Ptpro protein tyrosine phosphatase, receptor type, O (Ptpro), mRNA. 10375261 U 3.9 Gabrb2 gamma-aminobutyric acid (GABA-A) receptor, subunit beta 2 (Gabrb2), mR 10385283 U 4.6 Gabrg2 gamma-aminobutyric acid (GABA-A) receptor, subunit gamma 2 (Gabrg2), t 10407591 U 3.0 Chrm3 cholinergic receptor, muscarinic 3, cardiac (Chrm3), mRNA. 10543219 U 7.8 Gpr85 G protein-coupled receptor 85 (Gpr85) 10522530 U 11.2 Kit kit oncogene (Kit), mRNA. 10503902 U 10.6 Cnr1 1 (brain) (Cnr1), mRNA. 10557535 U 3.7 Sez6l2 seizure related 6 homolog like 2 (Sez6l2), mRNA. 10504375 U 4.2 Npr2 natriuretic peptide receptor 2 (Npr2), mRNA. 10459671 U 5.1 Dcc deleted in colorectal carcinoma (Dcc), mRNA. 10563487 U 7.1 Abcc8 ATP-binding cassette, sub-family C (CFTR/MRP), member 8 (Abcc8), mRNA. 10471814 U 3.5 Gpr21 G protein-coupled receptor 21 (Gpr21), mRNA. b 10407173 D 0.33 Il6st interleukin 6 signal transducer (Il6st), mRNA. 10492798 D 0.14 Sfrp2 secreted -related protein 2 (Sfrp2), mRNA 10526566 D 0.25 Ephb4 Eph receptor B4 (Ephb4), mRNA 10521111 D 0.16 Fgfr3 fibroblast growth factor receptor 3 (Fgfr3), mRNA. 10453738 D 0.11 Fzd8 frizzled homolog 8 (Drosophila) (Fzd8), mRNA 10500666 D 0.11 Ptgfrn prostaglandin F2 receptor negative regulator (Ptgfrn), mRNA. 10494595 D 0.09 Notch2 Notch gene homolog 2 (Drosophila) (Notch2), mRNA. 10473880 D 0.15 Lrp4 low density lipoprotein receptor-related protein 4 (Lrp4), mRNA 10512279 D 0.30 Cntfr ciliary neurotrophic factor receptor (Cntfr), mRNA 10513256 D 0.12 Edg2 (Lpar1) endothelial differentiation, G-protein-coupled receptor, 2 (Edg2) 10449775 D 0.21 Notch3 Notch gene homolog 3 (Drosophila) (Notch3), mRNA. 10423520 D 0.25 Sema5a sema, transmembrane and short cytoplasmic domain, (semaphorin) 5A 10380896 D 0.32 Erbb2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, mRNA. 10561104 D 0.20 Axl AXL receptor tyrosine kinase (Axl), mRNA. 10381898 D 0.27 Mrc2 , C type 2 (Mrc2), mRNA. 10473281 D 0.23 Itgav alpha V (Itgav), mRNA. 10516789 D 0.30 Ptpru protein tyrosine phosphatase, receptor type, U (Ptpru), transcript variant 2, mRNA. 10546339 D 0.18 Wnt7a wingless-related MMTV integration site 7A (Wnt7a), mRNA. 10481056 D 0.13 Notch1 Notch gene homolog 1 (Drosophila) (Notch1), mRNA 10422164 D 0.05 Ednrb type B (Ednrb), mRNA. 10439766 D 0.19 Pvrl3 poliovirus receptor-related 3 (Pvrl3), transcript variant beta, mRNA 10468452 D 0.24 Sorcs1 VPS10 domain receptor protein SORCS 1 (Sorcs1), mRNA. 10568436 D 0.19 Fgfr2 fibroblast growth factor receptor 2 (Fgfr2), transcript variant 1

Figure 6 Transcripts encoding receptors. Functional groupings of QISPs based on Gene Ontology molecular function and biological process annotations. Graphical representation as in Figure 4. Clustered QISPs of transcripts expressed (a) ≥ 3 fold higher and (b) ≥ 3 fold lower in Eomes:: GFP + cells compared to eGFP- cells. group, although mice lacking Glra2 display no overt ab- IV (SytIV), a calcium sensing protein normalities of cortical structure [95]. involved in neurotransmitter release [98], and synaptic Synaptogenesis begins quite early in cortical develop- vesicle Sv2a and Sv2b that may also be ment, at ~ E15 in subplate neurons in the rodent tem- involved in regulated secretion [99]. Additionally detected poral cortex [96], and continues well into the postnatal were three members of the Leucine Rich Repeat Trans- period [97]. At the developmental point of our analysis, membrane family (Lrrtm1-3), which function as synap- E14.5, few if any synapses are present in the developing tic organizers [100]. These results admit the possibilities CP. Despite this ultrastructural feature, we detected that neurotransmitter receptors and synaptic mRNAs not only multiple mRNAs encoding neurotransmitter expressed by these immature excitatory neurons either receptors, but also message for 22 other synaptic genes (a) do not encode functional , or (b)encode identified by the Gene Ontology descriptor “synapse” proteins involved in non-canonical functions. In either or “synaptic” (Figure 7). Included in this group are case, the results are consistent with important roles for Cameron et al. BMC Neuroscience 2012, 13:90 Page 15 of 23 http://www.biomedcentral.com/1471-2202/13/90

classical neurotransmitter signaling molecules in shap- toward shaping the development of cortical neurons (and ing the developmental trajectory of differentiating exci- presumably their synaptic/functional networks), but also tatory cortical neurons. implicates specific channel types and sub-units in the mani- festation of these early activity-dependent processes by cor- Analysis of highly up- and down-regulated voltage- tical excitatory neurons. dependent channels Early patterns of electrical activity and calcium transients Analysis of highly up- and down-regulated adhesion areknowntoshapethedevelopingnervoussystem molecules [101-104]. For this reason we queried the grouping of Spatial distributions of adhesion molecules, and changes QISPs associated with known voltage-dependent channels in such distributions, can in principal convey substantial (Figure 8). One down-regulated, and ten up-regulated, tran- morphologic and migratory changes upon developing scripts were identified and grouped. Of the up-regulated neurons. To group expression patterns of known adhe- transcripts, four encode subunits of voltage-dependent cal- sion molecules within the Eomes + neuronal lineage, we cium channels (Cacna1e, Cacna1h, Cacnb3, Cacna2d1), sorted within the ≥ 3 fold up-regulated category for the four encode voltage-dependent potassium channels (Kcna1, Gene Ontology descriptor “adhesion” (Figure 9). Twenty Kcnj3, Kcnq3, Kcna6), and two encode voltage-dependent one genes were identified that were verified by screening sodium channels (Scn3b, Scn2a1). Hierarchical clustering the Genepaint database. These genes include several Im- of these transcripts’ in situ hybridization profiles revealed munoglobulin Super Family Members (IGSFMs), such as peak expression within the upper-most ten sROIs, corre- L1cam, Dscam, Nfasc, Cadm3, Cntn1 and Cntn2. All of sponding to the IZ, CP and MZ. This expression pattern these IGSFM genes have confirmed neurodevelopmental is spatially consistent with the acquisition of voltage- functions: L1cam is involved in neuronal migration and dependent ionic currents in multipolar and migrating neu- neurite outgrowth [107]; Dscam is a co-receptor for rons [103]. Only one gene (Kcnj10), encoding the weakly Netrin signaling and is required for commissural axon inwardly rectifying subunit Kir4.1, was pathfinding [108]; Cadm3, also known as -like 1 down-regulated (3.3 fold), and this observation was consist- or Syncam3, is a molecule involved in ent with the corresponding in situ hybridization data. Al- axonal myelination [109]; Nfasc and Cntn1 interact to though robustly down-regulated at this developmental time promote the formation of paranodal axo-glial junctions point by differentiating excitatory cortical neurons, we note during axonal myelination [110]; Cntn2, also known as that deficiency in KCNJ10 produces seizures, sensorineural Tag-1, is highly expressed on axons and can serve as a deafness, ataxia, mental retardation, and electrolyte imbal- substrate for interneuron migration [111] (although Tag- ance (SeSAME syndrome) [105,106]. This analysis is con- 1 knockout mice display no overt cortical phenotype sistent with an important contribution of electrical activity [112], there is a disruption of paranodal myelin junctions

a CP+MZ IZ VZ Zone Gene Name Affy ID Fold 10457106 D 5.6 Cbln2 cerebellin 2 precursor protein, mRNA complete cds. 10554900 M 3.2 Dlg2 discs, large homolog 2 (Drosophila) (Dlg2), mRNA. 10540880 M 3.9 Syn2 synapsin II (Syn2), mRNA. 10457942 U 6.7 Syt4 synaptotagmin IV (Syt4), mRNA. 10476512 U 5.0 Snap25 synaptosomal-associated protein 25 (Snap25), mRNA. 10603843 U 3.1 Syn1 synapsin I (Syn1), mRNA. 10539169 U 3.5 Lrrtm1 leucine rich repeat transmembrane neuronal 1 (Lrrtm1) 10532180 U 3.9 Cplx1 complexin 1 (Cplx1), mRNA. 10532784 U 3.3 Svop SV2 related protein (Svop), mRNA. 10438358 U 3.9 Gp1bb / Sept5 septin 5 (Sept5), mRNA. 10413461 U 3.2 Erc2 ELKS/RAB6-interacting/CAST family member 2 (Erc2). 10593671 U 3.9 Dmxl2 premature mRNA for mKIAA0856 protein. 10539211 U 4.1 Lrrtm4 leucine rich repeat transmembrane neuronal 4, mRNA. 10545086 U 4.3 Snca synuclein, alpha (Snca), transcript variant 1, mRNA. 10369752 U 2.8 Lrrtm3 leucine rich repeat transmembrane neuronal 3 (Lrrtm3) 10503054 U 4.7 Lrrc7 leucine rich repeat containing 7 (Lrrc7), mRNA. 10363786 U 6.1 Ank3 ankyrin 3, epithelial (Ank3), transcript variant 2, mRNA. 10579554 U 3.7 Unc13a unc-13 homolog A (C. elegans) (Unc13a), mRNA. 10400866 U 3.8 Trim9 tripartite motif protein 9 (Trim9), transcript variant 1, mRNA. 10481711 U 5.0 Stxbp1 syntaxin binding protein 1 (Stxbp1), mRNA 10564646 U 6.5 Sv2b synaptic vesicle 2 b (Sv2b), transcript variant 2. 10494372 U 3.4 Sv2a synaptic vesicle glycoprotein 2 a (Sv2a), mRNA.

Figure 7 Transcripts encoding synaptic proteins. Functional groupings of QISPs based on Gene Ontology molecular function and biological process annotations. Graphical representation as in Figure 4. Clustered QISPs of transcripts expressed (a) ≥ 3 fold higher in Eomes-GFP + cells compared to GFP- cells. Note: there were no downregulated synaptic transcripts. Cameron et al. BMC Neuroscience 2012, 13:90 Page 16 of 23 http://www.biomedcentral.com/1471-2202/13/90

a CP+MZ IZ VZ Zone Gene Name Affy ID Fold

10462313 U 6.4 Slc1a1 solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, Xag), member 1 (Slc1a1) 10584549 U 3.9 Scn3b , voltage-gated, type III, beta (Scn3b), transcript variant 2, mRNA. 10358928 U 7.9 Cacna1e , voltage-dependent, R type, alpha 1E subunit (Cacna1e), mRNA. 10472400 U 5.3 Scn2a1 sodium channel, voltage-gated, type II, alpha 1 (Scn2a1), mRNA. 10552857 U 12.0 Slc17a7 solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 7 (Slc17a7),Vglut1 10429083 U 7.3 Kcnq3 potassium voltage-gated channel, subfamily Q, member 3 (Kcnq3), mRNA. 10548051 U 4.0 Kcna6 potassium voltage-gated channel, -related, subfamily, member 6 (Kcna6), mRNA 10472155 U 4.8 Kcnj3 potassium inwardly-rectifying channel, subfamily J, member 3 (Kcnj3), mRNA 10453233 U 5.0 Slc8a1 solute carrier family 8 (sodium/calcium exchanger), member 1 (Slc8a1), mRNA. 10548047 U 4.1 Kcna1 potassium voltage-gated channel, shaker-related subfamily, member 1 (Kcna1), mRNA. 10448925 D U 3.0 Cacna1h calcium channel, voltage-dependent, T type, alpha 1H subunit (Cacna1h), mRNA. 10553501 M 19.4 Slc17a6 solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 6 (Slc17a6), Vglut2 10519815 U 6.5 Cacna2d1 calcium channel, voltage-dependent, alpha2/delta subunit 1 (Cacna2d1), mRNA. 10426611 U 3.1 Cacnb3 calcium channel, voltage-dependent, beta 3 subunit (Cacnb3), transcript variant 1, mRNA. 10511789 D U 3.3 Nkain3 Na+/K+ transporting ATPase interacting 3 (Nkain3), mRNA.

b 10455873 D 0.33 Slc12a2 solute carrier family 12, member 2 (Slc12a2), mRNA. 10543031 D 0.20 Slc25a13 solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 13 (Slc25a13) 10427590 D 0.13 Slc1a3 solute carrier family 1 (glial high affinity glutamate transporter), member 3 (Slc1a3) 10351781 D 0.30 Kcnj10 potassium inwardly-rectifying channel, subfamily J, member 10 (Kcnj10), mRNA. 10550332 D 0.30 Slc1a5 solute carrier family 1 (neutral transporter), member 5 (Slc1a5), mRNA. 10439321 D 0.29 Slc15a2 solute carrier family 15 (H+/peptide transporter), member 2 (Slc15a2), mRNA Figure 8 Transcripts encoding ion channels and solute carriers. Functional groupings of QISPs based on Gene Ontology molecular function and biological process annotations. Graphical representation as in Figure 4. Clustered QISPs of transcripts expressed (a) ≥ 3 fold higher and (b) ≥ 3 fold lower in Eomes-GFP + cells compared to GFP- cells.

[113]). Several (Cadh8, Cadh11 and Cadh13) dynamically regulated at the transcriptional level in the as well as 20 were observed to be up- Eomes + lineage at E14.5. regulated, although to date none of these Cadherin genes In contrast other adhesion molecules, such as Fibronectin have identified roles in cortical development. Surpris- (Fn1), were down-regulated in the developing Eomes + ingly, no members of the integrin family were observed lineage. Two that via heterodimerization form in the ≥ 3 fold, or the ≥ 2 fold, up-regulated groups, sug- a fibronectin receptor (and an activating integrin for gesting this important family of adhesion proteins is not TGFβ signaling), Itgv and Itgβ8, were also down-

CP+MZ IZ VZ a Affy ID Zone Fold Gene Name 10580990 M 2.9 Cdh8 cadherin 8 (Cdh8), transcript variant 1, mRNA. 10584024 D U 3.1 Opcml opioid binding protein/-like (Opcml), mRNA. 10421911 M 3.1 Pcdh20 protocadherin 20 (Pcdh20), mRNA. 10605113 M 7.9 L1cam cell adhesion molecule (L1cam), mRNA. 10545538 U 3.3 Ctnna2 catenin (cadherin associated protein), alpha 2 (Ctnna2), transcript variant 2 10379482 U 3.3 Cdk5r1 cyclin-dependent kinase 5, regulatory subunit (p35) 1 (Cdk5r1), mRNA. 10584561 U 8.1 9030425E11Rik RIKEN cDNA 9030425E11 gene (9030425E11Rik), mRNA. 10397633 U 4.0 Flrt2 fibronectin leucine rich transmembrane protein 2 (Flrt2), mRNA 10357705 U 32.9 Cntn2 contactin 2 (Cntn2) 10441195 U 7.8 Dscam Down syndrome cell adhesion molecule (Dscam), mRNA. 10536483 U 5.2 Tes testis derived transcript (Tes), transcript variant 1, mRNA. 10360349 U 3.0 Cadm3 cell adhesion molecule 3 (Cadm3), mRNA. 10395961 U 9.1 Lrfn5 leucine rich repeat and fibronectin type III domain containing 5 (Lrfn5) 10561927 U 3.8 Aplp1 amyloid beta (A4) precursor-like protein 1 (Aplp1), mRNA. 10453518 U 7.0 Nrxn1 neurexin I (Nrxn1), mRNA. 10581013 U 3.8 Cdh11 cadherin 11 (Cdh11), mRNA. 10575844 U 10.8 Cdh13 cadherin 13 (Cdh13), mRNA. 10357736 U 5.7 Nfasc neurofascin (Nfasc), mRNA. 10595871 U 5.1 Clstn2 calsyntenin 2 (Clstn2), mRNA. 10423258 U 4.7 Cdh12 cadherin 12 (Cdh12), mRNA. 10465619 U 3.6 Flrt1 fibronectin leucine rich transmembrane protein 3 (Flrt1), mRNA b 10439766 D 0.19 Pvrl3 poliovirus receptor-related 3 (Pvrl3), transcript variant beta, mRNA 10407955 D 0.30 Epdr1 ependymin related protein 1 () (Epdr1), mRNA 10454786 D 0.30 Ctnna1 catenin (cadherin associated protein), alpha 1 (Ctnna1), mRNA. 10516789 D 0.30 Ptpru protein tyrosine phosphatase, receptor type, U (Ptpru), transcript variant 2, mRNA. 10355403 D 0.25 Fn1 fibronectin 1 (Fn1), mRNA. 10564713 D 0.13 Mfge8 milk fat globule-EGF factor 8 protein (Mfge8), transcript variant 1, mRNA. 10556426 D 0.20 Parva parvin, alpha (Parva), mRNA 10473281 D 0.23 Itgav (Itgav), mRNA. 10549594 D 0.15 Ttyh1 tweety homolog 1 (Drosophila) (Ttyh1), transcript variant 2, mRNA. 10403229 D 0.10 Itgb8 integrin beta 8 (Itgb8), mRNA. 10584208 D 0.23 Cdon cell adhesion molecule-related/down-regulated by oncogenes (Cdon), mRNA 10439612 D 0.34 Boc biregional CAM-related/down-regulated by oncogenes (Cdon) binding protein (Boc) 10455826 D 0.11 Megf10 multiple EGF-like-domains 10 (Megf10), mRNA. 10571530 D 0.14 Fat1 FAT tumor suppressor homolog 1 Gradient (Fat1), mRNA.

Figure 9 Transcripts encoding adhesion molecules. Functional groupings of QISPs based on Gene Ontology molecular function and biological process annotations. Graphical representation as in Figure 4. Clustered QISPs of transcripts expressed (a) ≥ 3 fold higher and (b) ≥ 3 fold lower in Eomes-GFP + cells compared to GFP- cells. Cameron et al. BMC Neuroscience 2012, 13:90 Page 17 of 23 http://www.biomedcentral.com/1471-2202/13/90

regulated. Conditional deletion of Itgαvinradialgliaand and few neurons have “settled” or been displaced to dee- neurons causes cerebral hemorrhage [114] while reduced per positions by subsequent, later-born neurons. expression of αvβ8 integrin is associated with arterior- One of the initially surprising features of the data set venous malformations [115]. Other down-regulated genes is the large number of genes that exceeded our empirical encoding adhesion molecules were: Jam3, a tight-junction expression threshold (RMA 7.0). We found that 53% of protein essential for maintaining the integrity of the cere- all unique mRNAs are expressed above this threshold. brovascular endothelium [116]; Cdh6, which is expressed Although initially surprising, this result is consistent in the VZ where it may serve to assign the cortico-striatal with prior studies of more mature cortical tissues [30,31] boundary [117]; two additional IGSFM genes, Boc and that contain a mixture of fully differentiated neurons Cdon, that are evident in the VZ, where they function with differing molecular properties. Although mRNA ex- in Sonic Hedgehog signaling and are implicated in pression is not equivalent to protein expression, the fact forms of holoprosencephaly [118,119]. Taken together that newly-generated excitatory cortical neurons display these groupings suggest that expression (and suppressed a complex transcriptome points to the potential plasti- expression) of numerous adhesion molecules, particularly city of these cells from their earliest developmental ori- specific members of the cadherin and IGSF families, are gins. The broad spectrum of expressed receptors required elements for migration, neurite formation, and indicates chemosensitivity to major chemotropic signal- subsequent myelination of developing excitatory cortical ing mechanisms (Semaphorin, Ephrin, Netrin, and Slit) neurons. and classical neurotransmitters including GABA, Glu- tamate, Acetylcholine and Glycine. Immature excitatory Discussion neurons are therefore potentially broadly sensitive to en- At E14.5 the cerebral neocortex is composed primarily vironmental cues, and could harness these cues to guide, of neuronal precursors and immature neurons, with a and ultimately define, their early differentiation and mi- small number of differentiated preplate neurons. To bet- gration across the cerebral wall. While the large number ter identify specific patterns of gene expression within of signaling pathways underscores the potential plasticity the excitatory neuron lineage during early cortical devel- and adaptability of immature neurons, this observation opment, we combined FAC sorting and transcriptional also highlights their potential susceptibility to toxic in- profiling with a new, comprehensive in situ hybridization sult and drugs of abuse. validation procedure, termed QISP. This approach The Eomes::eGFP transgene causes GFP expression for revealed 317 validated genes in the ≥ 3 fold up category a defined interval (approximately 3 days) in the early of transcripts. We then clustered the QISPs into groups postmitotic life of excitatory neurons. Our analyses of spatially co-expressed genes that can contribute to the showed that the gene expressed by neurons during this combined processes of migration and differentiation. ~ 3 day period of postmitotic life, is similar regardless of This approach allowed us to identify 44 genes expressed whether the GFP + cells were isolated from E13.5 or in the deep VZ that are likely to be IPC specific. These E14.5 embryos (Figure 2a and 2a’). This presumably deep genes would be difficult to isolate by a microdissec- reflects the shared initial developmental trajectory of tion based profiling and in situ approach [13-15] because these early born neurons, regardless of birthdate. This of the mixture of cell types and maturity. Similarly, we finding supports the observation of a core excitatory identified a group of 49 genes that are expressed in the neuron developmental program centered on the tran- IZ and may contribute to the recently identified multi- scription factors Pax6, Eomes and Tbr1 [57]. Thus, while polar neuron stage cortical development [1], as well our analyses focused on the comparison of E14.5 GFP + identifying 224 genes expressed in the developing cor- cells to GFP- precursors, we expect that many (but not tical plate that in part contribute to the early morpho- all) of the genes we identified as upregulated in the tran- logic differentiation of cortical excitatory neurons sition from precursor to immature deep layer neuron (Figure 10). This approach allows inferences about the will also be upregulated during the differentiation of temporal sequence of neuronal development: While the later born, upper layer cortical neurons. in situ analysis was performed at one time point (E14.5) The complex interplay between the developing neuron and is formally interpreted as a spatial pattern of gene and its environment was highlighted in our analysis of the expression at that time point, an approximate develop- network of transcription factors (TFs) that drive excitatory mental timeline of gene expression within the lineage neurondifferentiation.Althoughwewereabletoidentifya can be inferred, based on the well-established observa- total of 74 dynamically regulated TFs in the excitatory tion that more superficial neurons in the developing lineage, only 3% of the IPA literature-based interactions mammalian cortex are more mature [33,120]. This infer- connected these TFs between different spatial zones (i.e., ence is particularly valid at early developmental time “deep,” middle,” and “upper” cortical plate). The remaining points, such as E14.5, when the CP is starting to form ~97% of interactions connected dynamically regulated TFs Cameron et al. BMC Neuroscience 2012, 13:90 Page 18 of 23 http://www.biomedcentral.com/1471-2202/13/90

14 1 Molecules Fold abc13 2 TBR1 43.1 CNTN2 32.9 12 3 SLA 21.9 Upper TIAM2 21 11 4 BHLHE22 19.3 CP HIVEP2 17.8 10 5 ELMOD1 17.1 PMN FAM49A 16 6 9 Top Upper Genes 7 DYNC1I1 15 8 MC4R 14.4 BPN 14 Molecules Fold 1 13 NEUROD6 71.5 NEUROD1 30.9 2 SLC17A6 19.4 12 PLXNA4 13.5 Mid 3 MPN 10 NHLH1 11.6 9 NUAK1 10.2 IZ 8 NRP1 9.7 L1CAM 7.9 4 Top Mid Genes 7 KIF21B 7.6 5 NEUROD2 7.3 IPC 6 Molecules Fold up 14 1 NRN1 16.8 2 CLVS1 11 13 3 SSTR2 9.7 4 TTC39B 8.8 Deep C8orf46 7.2 5 RASGEF1B 6.5 12 6 CHGA 6.4 ADAMTSL3 6.2 Top Deep Genes IPC SEC24D 5.9 7 CBLN2 5.6 11 9 8 VZ Classification Upper Mid Deep n=224 n=49 n=44 1 Transcription 10.7 16.3 11.4 2 Adhesion 7.6 6.1 2.3 3 Phosphatases 3.1 2.0 2.3 4 Receptors 9.4 20.4 6.8 5 Ion channels / Solute carriers 5.4 2.0 4.5 6 Kinases and Adaptors 5.8 8.2 6.8 7 Extracellular 7.1 12.2 13.6 RGC 8 Small G-protein signaling 6.7 2.0 2.3 9 Synaptic 8.5 4.1 2.3 10 Cytoskeletal 7.6 2.0 0.0 11 2.2 0.0 6.8 12 Other enzymes 8.0 8.2 18.2 13 Other 11.6 10.2 13.6 14 Unknown 6.3 6.1 9.1 Figure 10 Schematic of excitatory neuron migration, differentiation, and patterns of gene expression. a, Illustration of radial migration, and general morphological characteristics, of excitatory neurons. RGC, radial glial cell; IPC, intermediate precursor cell; MPN, multipolar neuron; BPN, bipolar neuron; PMN, postmigratory neuron; VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone. b, Pie charts representing gene clusters 1-14 (see Figure 3) as a function of their relative contribution to the population of ≥ 3 fold up-regulated genes as a function of cortical location (upper, middle, deep). The accompanying table (bottom) indicates the Gene Ontology (GO) classification for each of the gene clusters, and the percentage of genes within each GO classification in each zone (D, M, U). c, The individual, greatest up-regulated genes in the Eomes lineage for each cortical zone. to a group of “IPA Interacting Genes,” a collection that interactions. So instead of a network composed exclusively included a number of molecules involved in non-cell au- of sequentially active (or activated) TFs, our data is consist- tonomous signaling, such as Tgfβ,Vegf,andWnt-βcatenin ent with an emerging model of early cortical neuron differ- signaling systems. While this analysis only reports known entiation that predicts interaction between cell autonomous interactions, and is therefore likely to under represent the and non-cell autonomous mechanisms [102,121-124]. This true number of functional TF-TF interactions, it is presum- model helps account for one of the paradoxical features of ably also likely to under represent indirect molecular neuronal development: the stochastic cell type composition Cameron et al. BMC Neuroscience 2012, 13:90 Page 19 of 23 http://www.biomedcentral.com/1471-2202/13/90

of clonally related neurons observed in, for example, the Conclusion neural retina [125-127] and cortex [128,129]. In contrast to In summary, this study provides a rigorously validated the invariant lineages that would be expected from a strictly list of transcripts expressed by immature excitatory neu- deterministic developmental program, the variable mixture rons that have been grouped by in situ expression pat- of neuronal subtypes within each clone may indicate a tern. We identified the transcriptional complexity of “noisy” deterministic program and/or a developmental pro- these cells, and highlighted the potential functionality of gram that relies on non-cell autonomous cues (e.g., growth these neurons with regards to the expression of specific factors or neurotransmitters) that may change in functional chemotropic receptors, neurotransmitter receptors, ion abundance and/or localization during development. channels, adhesion molecules, and synaptic proteins. Another important result emerging from recent work The established association of many of the validated is that specific members of TF families control restricted genes with human neurological disorders revealed new sets of phenotypic attributes. For example, Neurog2 molecular bases for understanding how specific genes, enacts a program of motility in migrating neurons in the and functional networks of such genes, control neuronal IZ through activation of the Rho family GTPase Rnd2 development and contribute to pathology. Future chal- [130]. Later in development a second bHLH TF, Ascl1, lenges include relating identified TFs and TF networks regulates the transition to locomotory migration on ra- to specific developmental events, and identifying the ex- dial glial fibers via Rnd3 activation [131]. Additionally, ogenous cues that coordinate these events. the subcortical projection patterns of cortical neurons depends on the expression of Sox5 [11] and may involve Additional files other TFs as well [7,132,133]. This stepwise control of neuronal phenotype may enable the appropriate matur- Additional file 1: Comparison of QISPs from repeated measurement ation of neurons, independent of the size of the develop- of Dab1 in situ expression in rostral neocortex along (a, b) the ing cerebral cortex or the time required for neurons to rostral-caudal axis and (c, d) the lateral-medial axis. Panels b and d plot strength of the in situ hybridization label (“intensity”) as a function of migrate across the cerebral wall. During early cortico- cortical position. All in situ hybridizations are derived from Genepaint. genesis, mouse layer 6 neurons can transition from cell Additional file 2: Table S1. RMA values for eye-specific genes that cycle exit to a postmigratory differentiating neuron in were used to establish not-expressed threshold for this study. ~1.5 days [22,134], whereas layer 2/3 cortical neurons Additional file 3: Table S2. Complete list of all ≥ 3 fold up-regulated require 4-5 days for this transition [135]. Nevertheless, a genes in E14.5 GFP + Eomes lineage compared to E13.5 GFP + Eomes lineage. similar, if not identical, TF program is thought to under- Additional file 4: Table S3. Complete list of all ≥ 3 fold down-regulated lie the maturation of both classes of neurons [3,57]. genes in E14.5 GFP + Eomes lineage compared to E13.5 GFP + Eomes Developing neurons’ interactions with external cues that lineage. promote (or suppress) the expression of stage-specific Additional file 5: Table S4. Complete list of all ≥ 3 fold up-regulated TFs would thus permit the coordinated transitions of genes in the E14.5 GFP + Eomes lineage compared to E13.5 GFP- precursors. Blue cells indicate genes that were unavailable or could not neuronal phenotype (e.g., multipolar neuron to bipolar be analyzed by Genepaint in situ. migrating neuron) in a manner independent of a cell- Additional file 6: Table S5. Complete list of all ≥ 3 fold down-regulated intrinsic “timer” of differentiation. genes in the E14.5 GFP + Eomes lineage compared to GFP- precursors. In this regard the cluster of genes expressed in the IZ Additional file 7: Functional groupings of QISPs based on Gene may be important for coordinating the multipolar Ontology (GO) molecular function and biological process annotations. Genes are grouped according to zone of expression: deep neuron stage, an early stage of migration, and later axon (D, dark gray), middle (M, light gray), upper (U, white). Listed genes are initiation. In clusters 9 and 10 we observed a significant ranked by fold up-regulation. enrichment of the Gene Ontology categories Cellular Movement Signaling (p = 4.59E-05) as well as Axonal Abbreviations Guidance Signaling Molecules (p = 1.28E-07) and bHLH BAC: Bacterial Artificial Chromosome; CP: Cortical Plate; eGFP: enhanced TFs including Neurod6 (72 fold) and Neurod1 (31 fold). Green Fluorescent Protein; Eomes: Eomesodermin; Gsat: Gensat; IACUC: Institutional Animal Care and Use Committee; IPC: Intermediate Neurod6 in particular may have a role in regulating the Precursor Cell; IZ: Intermediate Zone; MPN: Multipolar Neuron; MZ: Marginal mitochondrial mass increase and cytoskeletal rearrange- Zone; QISP: Quantified In Situ Pattern; ROI: Region of Interest; sROI: sub – ments prior to axonal growth [136], while other bHLHs, Region of Interest; sVZ: sub-Ventricular Zone; Tg: Transgene; VZ: Ventricular Zone. including Neurod1, promote the expression of genes critical for cortical migration, including the microtubule Competing interests associated protein Dcx, the Cdk5 regulatory subunit The authors declare that they have no competing interests. p35, and the small GTPase RhoA [137]. The multipolar neuron phenotype could thus be explained, in part, by Authors’ contributions EO and FM designed the study. EO performed the study and co-authored cellular specification provided by Neurod1 and Neurod6, the manuscript with DC and AC. All authors read and approved the final two of the most highly upregulated TFs in our data set. manuscript. Cameron et al. BMC Neuroscience 2012, 13:90 Page 20 of 23 http://www.biomedcentral.com/1471-2202/13/90

Acknowledgements National Academy of Sciences of the United States of America 2011, The authors thank the following: Dr. Nick Gonchoroff, Director of Flow 108(36):14950–14955. Cytometery at SUNY Upstate for FAC sorting, Karen Gentile, Technical and 15. Kang HJ, Kawasawa YI, Cheng F, Zhu Y, Xu X, Li M, Sousa AM, Pletikos M, Adminstrative Director of the SUNY Microarray Core Facility for Affymetrix Meyer KA, Sedmak G, et al: Spatio-temporal transcriptome of the human profiling and Dr. Robert Quinn and the staff in the Department of Laboratory brain. Nature 2011, 478(7370):483–489. Animal Resources for animal care. 16. Hempel CM, Sugino K, Nelson SB: A manual method for the purification of fluorescently labeled neurons from the mammalian brain. Nature protocols 2007, 2(11):2924–2929. Funding 17. 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