Abstract

The subplate lays the foundation of the developing cerebral cortex, and abnormalities have been suggested to contribute to various brain developmental disorders. The causal relationship between cellular pathologies and cognitive disorders remains unclear, and therefore, a better understanding of the role of subplate cells in cortical development is essential. Only by determining the molecular taxonomy of this diverse class of neurons can we identify the subpopulations that may contribute differentially to cortical development. We identified novel markers for murine subplate cells by comparing gene expression of subplate and layer 6 of primary visual and somatosensory cortical areas of postnatal day (P)8 old mice using a microarray-based approach. We examined the utility of these markers in well-characterized mutants (reeler, scrambler, and p35-KO) where the subplate is displaced in relation to the cortical plate. In situ hybridization or immunohistochemistry confirmed subplate-selective expression of complexin 3, connective tissue growth factor, nuclear receptor–related 1/Nr4a2, and monooxygenase Dbh-like 1 while transmembrane protein 163 also had additional expression in layer 5, and DOPA decarboxylase was also present in the white matter. Localization of marker-positive cells in the reeler and p35-KO cortices suggests different subpopulations of subplate cells. These new markers open up possibilities for further identification of subplate subpopulations in research and in neuropathological diagnosis.

Introduction

Subplate neurons in the neocortex are an enigmatic group of early-generated, largely transient cells located directly above the white matter/intermediate zone and below layer 6. The subplate layer has been variously referred to as layer 6b, layer 7, interstitial, or even white matter cells, but the term subplate is also commonly used and will be used here. They were first described in primates (Kostović and Rakic 1980) and carnivores (Luskin and Shatz 1985a, 1985b). Functional equivalence of rodent subplate with that of primates or carnivores has not been established, and the subplate layer varies in size and proportions in different vertebrate species, with the subplate zone being considerably larger in primates than in rodents (Kostović and Rakic 1990). In rodents, subplate is a thin band of cells separating the white matter from layer 6 and derived from the primordial plexiform zone or preplate (Marin-Padilla 1971; De Carlos and O'Leary 1992). A large proportion of the early-generated subplate cells die by the end of the first postnatal week (Price et al. 1997), but a considerable population of cells remains visible in the location of the subplate in adulthood.

During development, subplate neurons are electrically active and capable of firing action potentials (Hanganu et al. 2001; Torres-Reveron and Friedlander 2007) while incorporating, at least transiently, into the cortical and subcortical circuitry (McConnell et al. 1989; Friauf and Shatz 1991; Allendoerfer and Shatz 1994; Higashi et al. 2002, 2005; Kanold et al. 2003). Murine subplate cells are born around embryonic day (E)11 (Price et al. 1997) and begin to extend axons toward the thalamus by E13 (De Carlos and O'Leary 1992; Molnár, Adams, and Blakemore 1998; Molnár, Adams, Goffinet, and Blakemore 1998). There is strong evidence that they play an important role in thalamocortical axon pathfinding. They are implicated both in the initial areal targeting (Ghosh et al. 1990; Molnár and Blakemore 1995; Catalano and Shatz 1998; Molnár 1998; Hevner et al. 2002) and the eventual thalamic innervation of cortical layer 4 (Ghosh and Shatz 1992). They are necessary for the establishment of ocular dominance and orientation columns (Kanold et al. 2003; Kanold and Shatz 2006) and for the maturation of inhibition in cortical layer 4 (Kanold and Shatz 2006). They also drive oscillations in the gap junction–coupled early cortical syncytium (Dupont et al. 2006). Little else is known about their role as part of cortical circuitry during development or in the mature cortex (Clancy et al. 2001; Torres-Reveron and Friedlander 2007).

Subplate cells are not a homogenous population; electrophysiological properties, connectivity, and cell morphology point to a high degree of diversity (Antonini and Shatz 1990; Kostović and Rakic 1990; Hanganu et al. 2001; Hevner and Zecevic 2006; Watakabe et al. 2007). The powerful cell separation and gene profiling methods have not yet been systematically employed for this particular group of cortical neurons (Nelson et al. 2006; Molyneaux et al. 2007). Although a number of subplate markers have been reported (Chun and Shatz 1989; Allendoerfer and Shatz 1994; McQuillen and Ferriero 2005; Bayatti et al. 2007; Kostović and Judas 2007; Watakabe et al. 2007), cellular diversity has not yet been correlated with distinct molecular markers expressed in subplate cells.

Subplate abnormalities have been implicated in the pathogenesis of various brain developmental disorders including autism, schizophrenia, and cerebral palsy (Volpe 2001; Eastwood and Harrison 2003; McQuillen and Ferriero 2005). The cells’ selective vulnerability is not fully understood, and the causal relationship between the cellular pathologies and cognitive disorders also remains unclear. Therefore, a better understanding of the role of subplate neurons in cortical development is of fundamental importance. Only by establishing the molecular taxonomy of these diverse neurons do we stand a chance of identifying the subpopulations that may have very different roles during development and in various pathologies.

In this study, we set out to identify novel markers for murine subplate cells by comparing gene expression in subplate and layer 6 of primary visual and somatosensory areas of the postnatal day (P)8 mouse cortex using a microarray-based approach. We examine the utility of these markers in well-characterized mutants (reeler, scrambler, and p35-KO) in which the subplate is displaced in relation to the cortical plate (Rakic and Caviness 1995; Rakić et al. 2006). We also study their colocalization with each other and with the neurotransmitter γ-aminobutyric acid (GABA).

Materials and Methods

Animals

All animal experiments were approved by a local ethical review committee and conducted in accordance with personal and project licenses under the UK Animals (Scientific Procedures) Act (1986). For the microarray analysis, a total of 16 brains of 4 separate postnatal litters (P8), that is, 4 brains from each litter, were immediately immersed in RNAlater (Ambion, Huntingdon, UK). Fixed, wild-type brains at E18, P2, P8, P21, and adult for immunohistochemistry were obtained by deeply anesthetizing with pentobarbitone (Euthatal 150 mg/kg intraperitoneally; Merial Animal Health Ltd, Harlow, UK) and perfusing through the heart with 4% paraformaldehyde (PFA) (TAAB, Reading, UK; n = 9 at P8, n = 2 for all other ages) or with 4% PFA (TAAB) with 0.25% glutaraldehyde (Agar Scientific, Stansted, UK; n = 3 at P8 only) in phosphate-buffered saline (PBS, 0.1 M; pH 7.4). Brains of P7 green fluorescent protein expression driven by glutamic acid decarboxylase 65 KDa prometer (Gad65-GFP) mice (n = 3) were immersion fixed in 4% PFA. Brains for in situ hybridization were dissected in ice-cold PBS and flash-frozen in TissueTek OCT compound (Sakura Finetek, Zoeterwoude, NL). For in situ hybridization, brains aged E18 (n = 2), P3 (n = 2), P8 (n = 5), P21 (n = 2), and adult (n = 2) were used. Four percent PFA fixed and fresh-frozen brains of P8 reeler (n = 2 each), scrambler mice (n = 2 each), and p35-KO (n = 2 PFA fixed, n = 3 fresh frozen) and 1 P9 p35-KO (4% PFA fixed) were used.

Tissue Preparation for Microarray

Hemispheres were embedded in 5% agarose (Bioline, London, UK) and sectioned on a vibrating microtome into 150 μm parasagittal sections in a 1:1 mixture of RNAlater (Ambion) and PBS. Thin strips of anterior subplate and adjacent layer 6 and posterior subplate and layer 6 were dissected out (see Supplementary Fig. 1B) under visual guidance (using transillumination on a dissecting microscope (MZFLIII, Leica Microsystems, Milton Keynes, UK) and stored separately in RNAlater at −20 °C. We aimed to include at least 8 fragments of each tissue type for each brain and pooled the fragments of 4 littermates per replicate. A total of 4 biological replicates were collected for each location.

RNA Isolation and Microarray

For each step below, all tissue samples were processed at the same time to minimize variations in the experimental conditions. Total RNA was isolated using the RNeasy Micro kit (Qiagen, Crawley, UK) following the manufacturer's instructions. The quality and RNA integrity were assessed on a BioAnalyzer; all samples had a RNA Integrity Number ≥8 (Agilent Laboratories, Stockport, UK). Labeled cRNA for hybridization was generated with the Affymetrix “2 Cycle Target Labeling and Control” kit (Affymetrix, High Wycombe, UK) and MEGAscript T7 polymerase (Ambion) according to the manufacturer's instructions. Labeled anti-sense cRNA was fragmented and the distribution of fragment lengths was measured using a BioAnalyzer (Agilent).

Labeled and fragmented cRNA was hybridized to the Affymetrix 430 2.0 whole mouse genome microarray (Affymetrix). A total of 16 chips were used, all from the same batch. Chips were processed on an Affymetrix GeneChip Fluidics Station 450 and Scanner 3000.

Microarray Analysis

Arrays were normalized globally so that the average signal (target intensity) for each chip was 100. Genes with a signal difference between the perfect match (PM) and mismatch (MM) 25mer oligonucleotide probes are called present (P). The normalised arrays were filtered so that a probeset had to be called P in at least 1 out of 16 samples, to remove the background gene expression. The remaining genes were clustered using a condition tree with a Spearman Correlation in GeneSpring GX7 (Agilent). Arrays were Robust Multi-Array (RMA) normalized, and differentially expressed genes were identified using a paired t-test with a P value cut off of ≤0.05 and a fold change difference between any 2 comparisons of ≥1.5. Longer lists of ≥1.5-fold statistically differentially expressed genes were generated from Robust Multi-Array taking GC content into account (GCRMA) normalized data and sorted according to their gene ontology using GenMAPP's MAPPFinder (Salomonis et al. 2007). Only ontologies with ≥3 genes changing and a permuted P value ≤0.05 were selected. Gene interaction networks and canonical pathways were also analyzed using Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com).

Verification of Selected Microarray Results

The differential expression of selected genes with high fold changes in the RMA-normalized data set was confirmed by either in situ hybridization or immunohistochemistry.

In Situ Hybridization

We performed in situ hybridization to verify the expression of complexin 3 (Cplx3), connective tissue growth factor (CTGF), monooxygenase Dbh-like 1 (MoxD1), and transmembrane protein 163 (Tmem163) (Rik2610024A01) in the cortex of P8 mouse brains, and additional expression was studied for CTGF, MoxD1, and Tmem163 at E18, P3, P21, and adult. All in situ hybridization was performed on fresh-frozen brains, sectioned to 15 μm sagittally or coronally on a cryostat (Jung CM3000; Leica, Germany). Total RNA of P8 brain tissues was extracted, and first-strand cDNA was synthesized using Superscript III Reverse Transcriptase together with random hexamers (Invitrogen, Paisley, UK) following the manufacturer's instructions. DNA fragments corresponding to the regions of the mouse Cplx3, CTGF, MoxD1, and Tmem163 cDNAs were generated using the following sets of forward (F) and reverse (R) primers:

  • Cplx3: F = 5′-gaatctcatgtagctcaggc-3′, R = 5′-acatagtgttgctgcacatct-3′

  • CTGF: F = 5′-gactcagccagatccactcc-3′, R = 5′-gctgctttggaaggactcac-3′

  • MoxD1: F = 5′-tttccttcccaggctacc-3′, R = 5′-agtaagaaacacattcggct-3′

  • Tmem163: F = 5′-caacactaaaccggttcg-3′, R = 5′-ctgcccacaacgtgtact-3′.

The resulting polymerase chain reaction products were ligated into the pST-Blue 1 plasmid (Novagen, Nottingham, UK). The antisense and sense (a negative control) cRNA probes were transcribed using either SP6 or T7 RNA polymerase with digoxygenin (DIG)-labeled RNA mixture, respectively (Roche, Penzberg, Germany). Frozen sections were postfixed with 4% PFA in PBS for 20 min, deproteinized with 0.1 N HCl for 5 min, acetylated with acetic anhydride (0.25% in 0.1 M triethanolamine hydrochloride) and prehybridized at room temperature (RT) for at least 1 h in a solution containing 50% formamide, 10 mM Tris (pH 7.6), 200 μg/mL Escherichia coli tRNA, 1× Denhardt's solution, 10% dextran sulfate, 600 mM NaCl, 0.25% sodium dodecyl sulfate, and 1 mM ethylenediaminetetraacetic acid. The sections were hybridized in the same buffer with the DIG-labeled probes overnight at 70 °C. After hybridization, sections were washed to a final stringency of 30 mM NaCl/3 mM sodium citrate at 70 °C and detected by anti-DIG–alkaline phosphatase antibody in conjunction with a mixture of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (Roche).

Immunohistochemistry

Immunohistochemistry was performed on sections from 4% PFA-fixed or glutaraldehyde-fixed (colocalization with GABA only) brains cut coronally or sagittally to 40 μm. Free-floating, permanent single, or fluorescent single or double immunohistochemistry was used on C57/BL6, reeler, scrambler, p35-KO, and Gad65-GFP brains for Cplx3, DOPA decarboxylase (DDC), GABA, nuclear receptor–related 1 (Nurr1), NeuN, and tyrosine hydroxylase (TH as a dopaminergic neuronal marker). FoxP2 and Er81 were used to identify cortical layers in the p35-KO and scrambler cortex (Yoneshima et al., 2006). For fluorescent immunohistochemistry, sections were blocked for 2 h at RT with 2% (pH 7.4) donkey or goat serum (Sigma, Gillingham, UK) in Tris-buffered saline with 0.1% Triton-X100 (BDH, Poole, UK). Sections were incubated with primary antibody in blocking solution (see Table 1 for concentration, manufacturer, and combinations used) for 48 h at 4 °C. Secondary antibody (see Table 2 for list of species - reactivity and manufacturer) in blocking solution was applied for 2 h at RT before the sections were counterstained with bisbenzimide (2.5 μg/100 mL, Hoechst33258; Sigma) or 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) to reveal the cytoarchitecture. Where relevant, streptavidin-cy3 or streptavidin-cy2 (Jackson ImmunoResearch, Newmarket, UK) was applied in blocking solution for 2 h. For permanent immunohistochemistry, sections were additionally quenched in 1.5% hydrogen peroxide prior to the blocking step. Biotinylated secondary antibodies were used and reacted with avidin-biotinylated enzyme complex (ABC) using the Vectastain Elite kit (Vector, Peterborough, UK) and diaminobenzidene (DAB) or DAB with nickel solution (Vector) according to the manufacturer's instructions.

Table 1

This table gives the primary antibodies used including manufacturer and product code (where applicable), concentration for fluorescence (F) or permanent immunohistochemistry (P) and the secondary antibodies with which it was combined

Primary antibody Manufacturer Product # Concentration Double labeling Secondary antibody 
Cplx3 Reim et al. (2005) 1:2000 (F, P) Nurr1 D α-rb Al555 
DDC Abcam ab3905 1:500 (F) NeuN and Nurr1 D α-rb Al555 
1:2000 (P) G α-rb biot 
Er81 Gift from Thomas Jessell 1:32000 (P)  G α-rb biot 
FoxP2 Abcam (Cambridge, UK) Ab16046 1:4000 (P)  G α-rb biot 
GABA Abcam ab5097 1:1500 (F) Cplx3, DDC, and Nurr1 G α-gp cy3 
D α-gp cy3 
NeuN Chemicon (Millipore, Waterford, UK) MAB377 1:1000 (F) DDC G α-m cy3 
Nurr1 R&D Systems AF2156 1:100 (F, P) Cplx3 and DDC D α-g Al488 
D α-g biot 
TH Abcam (Abingdon, UK) ab113 1:200 (F) DDC D α-s biot 
Primary antibody Manufacturer Product # Concentration Double labeling Secondary antibody 
Cplx3 Reim et al. (2005) 1:2000 (F, P) Nurr1 D α-rb Al555 
DDC Abcam ab3905 1:500 (F) NeuN and Nurr1 D α-rb Al555 
1:2000 (P) G α-rb biot 
Er81 Gift from Thomas Jessell 1:32000 (P)  G α-rb biot 
FoxP2 Abcam (Cambridge, UK) Ab16046 1:4000 (P)  G α-rb biot 
GABA Abcam ab5097 1:1500 (F) Cplx3, DDC, and Nurr1 G α-gp cy3 
D α-gp cy3 
NeuN Chemicon (Millipore, Waterford, UK) MAB377 1:1000 (F) DDC G α-m cy3 
Nurr1 R&D Systems AF2156 1:100 (F, P) Cplx3 and DDC D α-g Al488 
D α-g biot 
TH Abcam (Abingdon, UK) ab113 1:200 (F) DDC D α-s biot 

Note: D, donkey; G, goat; Al, Alexa; rb, rabbit; m, mouse; g, goat; gp, guinea pig; s, sheep, biot. biotinylated.

Table 2

This table gives the secondary antibodies used including the manufacturer and product code and the concentration at which the secondary antibody was used

 Manufacturer Product # Concentration 
Donkey α-goat Alexa488 Molecular Probes (Invitrogen; Paisley, UK) A11055 1:500 
Donkey α-goat biotinylated Abcam Ab6884 1:100 
Goat α-guinea pig cy3 Jackson Laboratories 106-165-003 1:500 
Goat α-mouse cy3 Jackson Laboratories 115-165-003 1:500 
Goat α-rabbit biotinylated Abcam Ab6720 1:100 
Goat α-rabbit biotinylated Vector BA-1000 1:200 
Donkey α-rabbit Alexa488 Molecular Probes A21206 1:500 
Donkey α-rabbit Alexa555 Molecular Probes A31572 1:500 
Donkey α-sheep biotinylated Abcam Ab6895 1:100 
 Manufacturer Product # Concentration 
Donkey α-goat Alexa488 Molecular Probes (Invitrogen; Paisley, UK) A11055 1:500 
Donkey α-goat biotinylated Abcam Ab6884 1:100 
Goat α-guinea pig cy3 Jackson Laboratories 106-165-003 1:500 
Goat α-mouse cy3 Jackson Laboratories 115-165-003 1:500 
Goat α-rabbit biotinylated Abcam Ab6720 1:100 
Goat α-rabbit biotinylated Vector BA-1000 1:200 
Donkey α-rabbit Alexa488 Molecular Probes A21206 1:500 
Donkey α-rabbit Alexa555 Molecular Probes A31572 1:500 
Donkey α-sheep biotinylated Abcam Ab6895 1:100 

Note: Biotinylated secondary antibodies were combined with steptavidin-cy3 or –Alexa488 when used for double immunofluorescence.

Imaging and Analysis

Fluorescently labeled sections were imaged and analyzed for cell distribution and double labeling using a fluorescent microscope (DMR; Leica Microsystems). Quantification for double labeling was performed using a 40× objective, and selected examples were confirmed on confocal data sets. No bleedthrough of fluorochromes was noticed. Images for publication were obtained using single laser line scanning confocal microscope (Zeiss, Welwyn Garden City, UK). Permanently labeled sections were imaged on a DMR transmission light microscope (Leica). Images for publication were contrast adjusted and compiled using Adobe Photoshop CS3.

Results

We conducted a microarray screen for subplate markers in order to investigate the diversity of cell types contributing to the mouse subplate at P8. Comparing gene expression in the subplate with the adjacent layer 6 in the somatosensory and visual cortices, we identified 601 probe sets (corresponding to 383 genes and hypothetical genes) that were expressed at a higher (≥1.5-fold) level in the subplate compared with layer 6 in both comparisons. The gene expression patterns of subplate and layer 6 samples from somatosensory and visual cortices were analyzed for condition tree clustering. The anterior and posterior subplates as well as anterior and posterior layer 6 clustered tightly and separated into closely related groups (Supplementary Fig. 2). The differences between layer 6 and subplate were larger than the differences between corresponding layers in the 2 cortical areas.

Pathways and Gradients

Analyzing the pathways that are consistently differentially expressed between subplate and layer 6 at P8 did not highlight any pathways or signaling cascades contributing to apoptosis, suggesting that by P8 the period of major cell death reported by Price et al. (1997) is over. On the other hand, cell maturation pathways (cell development, cell fate determination and commitment, and cellular morphogenesis) were consistently highlighted in MAPPFinder as containing a significant proportion of genes expressed at a higher level in the subplate compared with layer 6.

Identification of Putative Subplate Markers in Wild-Type

Owing to the broadness of the categories highlighted by MAPPFinder, we decided to focus on verifying the expression pattern of genes with a very high fold difference in expression levels that are also expressed at a high level, irrespective of their proposed function. We conducted either in situ hybridization or immunohistochemistry against 4 transcripts and 3 proteins of interest to verify their location and selectivity of expression in wild-type mouse brains (Fig. 1). Brains from the reeler, scrambler, and p35-KO strains, all of which have an altered location of subplate cells as a result of cell migration defects, were also examined (Figs 2, 3, and 6 and Supplementary Fig. 3).

Figure 1.

Subplate-selective gene expression in P8 mouse cortex. The panels of this figure show six genes that are each expressed in the subplate but not in adjacent layer 6. Cplx3 and CTGF show subplate-specific mRNA localization in mouse P8 cortex. Tmem163 (2610024A01Rik) and MoxD1 mRNA are present in the subplate and at a lower level also in cortical layer 5 at P8. In dorsal mouse cortex at P8, the DDC protein is localized to cells in the subplate and white matter, whereas the transcription factor Nurr1/Nr4a2 is localized to the subplate only in dorsal cortex (shown here). Cortical layers are indicated by the abbreviations: MZ, marginal zone; L2/3, layer 2/3; L4, layer 4; L5, layer 5; L6, layer 6; SP, subplate; WM, white matter. Scale bars 100 and 50 μm (inset).

Figure 1.

Subplate-selective gene expression in P8 mouse cortex. The panels of this figure show six genes that are each expressed in the subplate but not in adjacent layer 6. Cplx3 and CTGF show subplate-specific mRNA localization in mouse P8 cortex. Tmem163 (2610024A01Rik) and MoxD1 mRNA are present in the subplate and at a lower level also in cortical layer 5 at P8. In dorsal mouse cortex at P8, the DDC protein is localized to cells in the subplate and white matter, whereas the transcription factor Nurr1/Nr4a2 is localized to the subplate only in dorsal cortex (shown here). Cortical layers are indicated by the abbreviations: MZ, marginal zone; L2/3, layer 2/3; L4, layer 4; L5, layer 5; L6, layer 6; SP, subplate; WM, white matter. Scale bars 100 and 50 μm (inset).

Figure 2.

mRNA localization of subplate gene expression in mutants affecting cortical plate formation. The panels of this figure show in situ hybridization signal in P8 reeler (AD) and p35-KO (EH) cortices for 4 genes selectively expressed in the subplate of wild-type mice. In reeler brains, the cortical preplate fails to split into marginal zone and subplate such that subplate cells are relocated to the superplate, that is, the outermost layer of cortex. All CTGF-, MoxD1-, and Tmem163-expressing “subplate” cells are located in the superplate of reeler brains (B, C, D), but only some Cplx3-expressing subplate cells are shifted to the superplate (A). The remainder of the Cplx3-expressing subplate cells is shifted downward into the white matter in reeler brains (A). In p35-KO brains, subplate cells are relocated to a thin layer of cells in the middle of cortex. All subplate cells expressing Cplx3, CTGF, MoxD1, or Tmem163 are relocated to the middle of the cortical plate in p35-KO brains (EH), whereas Tmem163- and MoxD1-labeled layer 5 cells are shifted outward to just underneath the marginal zone. Scale bars = 100 μm.

Figure 2.

mRNA localization of subplate gene expression in mutants affecting cortical plate formation. The panels of this figure show in situ hybridization signal in P8 reeler (AD) and p35-KO (EH) cortices for 4 genes selectively expressed in the subplate of wild-type mice. In reeler brains, the cortical preplate fails to split into marginal zone and subplate such that subplate cells are relocated to the superplate, that is, the outermost layer of cortex. All CTGF-, MoxD1-, and Tmem163-expressing “subplate” cells are located in the superplate of reeler brains (B, C, D), but only some Cplx3-expressing subplate cells are shifted to the superplate (A). The remainder of the Cplx3-expressing subplate cells is shifted downward into the white matter in reeler brains (A). In p35-KO brains, subplate cells are relocated to a thin layer of cells in the middle of cortex. All subplate cells expressing Cplx3, CTGF, MoxD1, or Tmem163 are relocated to the middle of the cortical plate in p35-KO brains (EH), whereas Tmem163- and MoxD1-labeled layer 5 cells are shifted outward to just underneath the marginal zone. Scale bars = 100 μm.

Figure 3.

Protein localization of putative subplate cell markers in mutants affecting cortical plate formation. The Cplx3 and Nurr1 protein localization shifts from the normal position in the subplate in wild-type brains to the superplate in reeler brains (A, C) and to a thin band in the middle of the cortical plate in p35-KO brains at P8 (D, F). DDC protein, which is normally localized to cells in the subplate, white matter, and lower layer 6, is only rarely detectable in cells in the superplate in reeler mutants, but DDC-positive cells are distributed across the lower half of the cortex (B), in a band broader than in the wild type (H). DDC-positive cells can also be found in the middle of cortex in p35-KO brains (E). However, the majority of DDC-positive cells remains in the white matter and subplate in both mutants. Scale bars = 100 μm.

Figure 3.

Protein localization of putative subplate cell markers in mutants affecting cortical plate formation. The Cplx3 and Nurr1 protein localization shifts from the normal position in the subplate in wild-type brains to the superplate in reeler brains (A, C) and to a thin band in the middle of the cortical plate in p35-KO brains at P8 (D, F). DDC protein, which is normally localized to cells in the subplate, white matter, and lower layer 6, is only rarely detectable in cells in the superplate in reeler mutants, but DDC-positive cells are distributed across the lower half of the cortex (B), in a band broader than in the wild type (H). DDC-positive cells can also be found in the middle of cortex in p35-KO brains (E). However, the majority of DDC-positive cells remains in the white matter and subplate in both mutants. Scale bars = 100 μm.

The two known subplate markers CTGF (Heuer et al. 2003) and the orphan nuclear receptor Nurr1/Nr4a2 (Liu and Baker 1999; Arimatsu et al. 2003) were both confirmed in our screen with high differential expression levels and/or multiple probe sets. The gene encoding CTGF was expressed at 11.9- and 14.9-fold higher levels in the anterior and posterior subplates, respectively, and is the most differentially expressed gene both in anterior and posterior comparisons in the present comparison. The Nurr1/Nr4a2 gene was reported as differentially expressed by 3 probe sets with an up to 5-fold higher expression in subplate compared with layer 6 (see Supplementary Table 1).

Four Novel Subplate Markers Were Identified and Confirmed

The most differentially expressed novel gene in our screen is MoxD1, also referred to as monooxygenase X (unknown substrate) or dopamine β-hydroxylase related with a 9.5- and 5.6-fold higher expression level in the anterior and posterior subplates, respectively. Mouse MoxD1 (mMOX) is expressed in a wide variety of tissues including olfactory bulb, cerebellum, brain stem, and parietal cortex (Xin et al. 2004). Here we demonstrate by in situ hybridization that MoxD1 gives a strong label in the subplate of the wild-type P8 cerebral cortex (n = 3; Figs 1and 2K). This subplate label is uniform across the entire anterior–posterior expanse of the cortex. In some wild-type brains, a fainter cortical label is also visible in layer 5.

Cplx3 showed 6.7- and 8.2-fold different levels of expression. Cplx3 belongs to a family of 4 genes (Cplx1–4). Cplx3 and Cplx4 are highly homologous but share only little identity with the well-characterized Cplx1 and 2 (Reim et al. 2005). Cplx3 was previously demonstrated by in situ hybridization to be expressed in the adult mouse hippocampus and the cerebellum (Reim et al. 2005), but so far no expression has been reported in the cerebral cortex. Here we demonstrate by in situ hybridization that Cplx3 antisense mRNA gives a subplate-specific label in wild-type brains (n = 3; Figs 1and 2I). We also assessed the protein distribution of Cplx3 with immunohistochemistry. In P8 wild-type mice, the strongest cortical label is in the subplate (Fig. 3G) and spans the entire anterior–posterior width of the cortex. In contrast to the mRNA, the protein is not exclusively localized to the subplate as a less intense stain can also be seen in some layer 5 cells.

The most highly differentially expressed gene of unknown function that was also expressed at high levels was 2610024A01Rik, also known as Tmem163 (NM_028135). Expression levels were 5.1- and 3.1-fold higher in anterior or posterior subplate to adjacent layer 6, respectively. In situ hybridization revealed that gene expression in the cortex is restricted to the subplate and layer 5 (Figs 1and 2L; n = 3). The intensity of labeling and the density of labeled subplate cells both appear uniform across the anterior–posterior expanse of the cortex. Subplate cells are more strongly labeled than layer 5 cells.

Lastly, we also investigated the localization of the DDC protein. DDC was expressed at 4.4- and 2.9-fold higher levels in the anterior and posterior subplates compared with adjacent layer 6, respectively. Cortical DDC labeling was observed in the subplate, white matter, and occasional cells in layer 6 (Figs 1and 3H; n = 5).

Temporal Expression Profile of Putative Subplate Markers in Wild-Type Mouse

The temporal expression pattern for CTGF in mouse has been previously reported, with expression first detectable around E16 and adult levels being reached by P14 (Heuer et al. 2003). Similarly, in our study, we found many faintly CTGF-labeled cells in the subplate at E18 (see Fig. 4). The intensity of labeling increases at P3 and further by P8 (as well as increasing in numbers) but then appears constant until adulthood.

Figure 4.

Time course of subplate gene expression in wild-type. CTGF expression is already detectable in the subplate region at E18 and increases in the number of cells and the intensity of labeling at P3 and P8. Expression levels remain similar at P21 and in adult brains. Tmem163 expression follows a similar time course with very weak expression seen at E18 and higher levels at P3, P8, and P21. In contrast to CTGF expression, Tmem163 is only expressed in few cells in the adult subplate. Layer 5 labeling is strongest at P8 and barely visible at most other ages. MoxD1 expression is not detected at E18. Some cells are labeled in the P3 subplate, and labeling continues with similar intensity and cell numbers at P8 and P21, but fewer cells are labeled in the adult subplate. None of the brains used for the time series showed MoxD1 expression in layer 5. Scale bars = 100 μm (applies to all large panels in the same column) and 20 μm (for all insets).

Figure 4.

Time course of subplate gene expression in wild-type. CTGF expression is already detectable in the subplate region at E18 and increases in the number of cells and the intensity of labeling at P3 and P8. Expression levels remain similar at P21 and in adult brains. Tmem163 expression follows a similar time course with very weak expression seen at E18 and higher levels at P3, P8, and P21. In contrast to CTGF expression, Tmem163 is only expressed in few cells in the adult subplate. Layer 5 labeling is strongest at P8 and barely visible at most other ages. MoxD1 expression is not detected at E18. Some cells are labeled in the P3 subplate, and labeling continues with similar intensity and cell numbers at P8 and P21, but fewer cells are labeled in the adult subplate. None of the brains used for the time series showed MoxD1 expression in layer 5. Scale bars = 100 μm (applies to all large panels in the same column) and 20 μm (for all insets).

The temporal expression pattern of Nurr1 has previously been described in rats (Arimatsu et al. 2003). Here we report that strongly labeled nuclei are visible at E18 in the mouse subplate (Fig. 5). Across all mouse ages from E18 to adulthood, the label is exclusive to the subplate in very dorsal regions but spread across most layers in lateral regions. However, at E18, the region of subplate-specific labeling extends further laterally than it does at later ages. The region of subplate-selective labeling is progressively more restricted to dorsal areas in older stages.

Figure 5.

Time course of subplate marker protein localization in wild-type. Cplx3 is not detectable at E18, but by P2, cells including proximal processes are labeled in the subplate. Number of labeled cells and processes increases by P8 with a dense fiber network in the area of the subplate. Labeled cells continue to be visible at P21 and in the adult subplate region. DDC is not seen in cells at E18, but labeled fibers span the lower half of cortex and are particularly dense in the subplate area. At P2, neither fibers nor cells are labeled. Labeled cells and proximal processes are visible in the subplate, white matter, and layer 6 at P8 and P21 but only very rarely in the adult cortex. Nurr1 is detectable at E18 and continues to be present at similar levels throughout all postnatal ages tested. Scale bars = 100 μm (applies to all large panels in the same column) and 20 μm (for all insets).

Figure 5.

Time course of subplate marker protein localization in wild-type. Cplx3 is not detectable at E18, but by P2, cells including proximal processes are labeled in the subplate. Number of labeled cells and processes increases by P8 with a dense fiber network in the area of the subplate. Labeled cells continue to be visible at P21 and in the adult subplate region. DDC is not seen in cells at E18, but labeled fibers span the lower half of cortex and are particularly dense in the subplate area. At P2, neither fibers nor cells are labeled. Labeled cells and proximal processes are visible in the subplate, white matter, and layer 6 at P8 and P21 but only very rarely in the adult cortex. Nurr1 is detectable at E18 and continues to be present at similar levels throughout all postnatal ages tested. Scale bars = 100 μm (applies to all large panels in the same column) and 20 μm (for all insets).

The expression pattern of MoxD1 is of later onset and not as consistent across ages as the expression of CTGF and Nurr1. MoxD1 mRNA was first detected using in situ hybridization at P3 with most cells in the subplate being faintly labeled (Fig. 4). The intensity further increased at P8 and remained similar in intensity and number of cells at P21, whereas it appeared that fewer cells were labeled in the adult subplate.

Cplx3 has a similar onset of expression as MoxD1 with no labeled cells or processes visible in the subplate at E18 when using the Cplx3 antibody (Fig. 5). The mRNA probe also does not label individual cell bodies in the E18 cortex (data not shown). By P2, strongly labeled cells are present in the subplate and the intensity of labeling remains similar until adulthood, but the density of labeled fibers within the subplate increases from P3 to P8 at which point it peaks.

Tmem163 mRNA is detectable as a very faint label in very few subplate cells at E18 (Fig. 4). The labeling becomes more prominent in the P3 subplate and first becomes visible in layer 5. The intensity and number of labeled cells in the subplate remains similar until P21, but fewer cells are labeled in the adult subplate.

DDC immunoreactivity does not label cells in the subplate region at E18, but many labeled fibers are visible in the white matter and lower half of the cortical plate (Fig. 5). Neither cellular nor fiber label is visible in the subplate region at P2. Labeled white matter, subplate, and layer 6 cells and proximal processes are abundant at P8 and P21 but become almost absent in the adult cortex.

Reeler/scrambler and p35-KO Mutants

We analyzed the gene expression or protein localization for each of these potential markers in the cortices of 3 mutant mouse strains with disrupted cortical layering: reeler or scrambler mice and p35-KO mice. In reeler and scrambler mice, the preplate fails to split and the cells normally contributing to the subplate are located in the outermost cortical layer. This layer is referred to as superplate, and thalamic fibers navigate to it as they normally would to the subplate (Molnár et al., 1998). In p35-KO mice, the neocortex does not show the clear 6-layered cortical structure, whereas entorhinal and piriform cortices are reported as fairly unaffected by the mutation (Chae et al. 1997). Layer 1 is present and also forms the outermost layer in the p35-KO cortex, but the large pyramidal cells of the normal layer 5 occupy the layer underneath (Chae et al. 1997; see Supplementary Fig. 3B). All cells usually forming the remaining layers can be identified morphologically in the p35-KO cortex, but their distribution does not follow any clear layering (Chae et al. 1997). Using the marker FoxP2, layer 6 cells can be shown to be distributed throughout the upper two-thirds of the cortex with the exception of the cell-free marginal zone (Supplementary Fig. 3E).

CTGF is expressed exclusively in the superplate in reeler mice (n = 2; Fig. 2B), except for the junction between putative auditory cortex and piriform cortex where the labeled cells are distributed throughout all cortical layers. In the dorsal cortex of reeler brains, Nurr1+ cells are either absent (toward the midline) or located in the superplate (n = 2 brains; Fig. 3C). In the lateral aspects of cortex, some labeled cells are detected in the superplate and the cell layer underneath, but the majority remains spread throughout the lower cortical layers. A similar distribution of Nurr1+ cells is also observed in P8 cortices of scrambler mice (n = 2), but again fewer labeled cells are detected in the marginal zone (Supplementary Fig. 4C). The in situ hybridization signal for MoxD1 is distributed similarly to that of CTGF. The strong signal representing subplate cells is located in the superplate (n = 2; Fig. 2C) except at the midline and in the piriform cortex where it is spread throughout all cortical layers. The weaker label representing layer 5 was not detectable in the reeler brains. Cplx3-positive cells are differentially distributed in the reeler and scrambler mutants based on whether the mRNA localization or the protein localization is analyzed. In situ hybridization for Cplx3 revealed cells both in the white matter as well as the superplate in reeler brains (n = 2; see Fig. 2A), demonstrating an unusual split. Immunohistochemistry for Cplx3 revealed that the strong immunoreactivity representing the subplate cells is entirely in the superplate in dorsal aspects of reeler (n = 2; Fig. 3A) or scrambler (n = 1; Supplementary Fig. 4A) brains. The weaker label representing layer 5 cells is detectable underneath the dorsal superplate. In the lateral cortex of these 2 mutants, however, the strongly Cplx3-immunoreactive cells are not exclusively localized to the superplate. Instead, many cells remain at the white matter/gray matter boundary, and some strongly labeled cells, including inverted pyramidal cells, are found at all depths of the cortex (data not shown). The in situ hybridization signal for Tmem163 also revealed some labeled cells in the white matter but many more in the superplate of the reeler dorsal cortex (n = 2; Fig. 2D). No cells were found at the white matter/gray matter boundary. In more lateral regions, fewer Tmem163-labeled cells shift to the marginal zone/superplate and instead some cells remain at the boundary of white and gray matters (see Fig. 6C). The faint labeling representing layer 5 shifted outward as an uninterrupted band and is located throughout the upper half of the cortex. The distribution of DDC+ cells also differs by dorsolateral aspect, and furthermore, DDC+ cells are also differentially affected by the scrambler versus reelin gene mutations. Very few DDC+ cells are located in the superplate in reeler brains (n = 2), and these are only in the lateral cortex. The majority of DDC+ cells remain in the white matter and the lowest cortical layer, but a few DDC+ cells could be found in all cortical layers (Fig. 3B). In scrambler brains (n = 2), some DDC+ cells are located in the lower third of the cortex, but no labeled cells were found in the superplate (Supplementary Fig. 4B).

Figure 6.

Schematic summary of gene expression or protein localization for putative subplate markers in wild type, reeler, and p35-KO brains. The distribution of cells labeled with putative subplate markers is affected differently according to dorsolateral position in some of the mutant brains. The colored bands represent the area in which labeled cells are found but do not correspond to any measure of cell density or intensity of cell label. Some areas contain labeled cells in both mutant and wild-type brains and are therefore labeled in the respective color mixtures of red, blue, and green. (A) Distribution of Cplx3-expressing cells, (B) distribution of CTGF-expressing cells, (C) distribution of Tmem163-expressing cells, (D) distribution of DDC+ cells, (E) distribution of MoxD1-expressing cells, (F) distribution of Nurr1+ cells.

Figure 6.

Schematic summary of gene expression or protein localization for putative subplate markers in wild type, reeler, and p35-KO brains. The distribution of cells labeled with putative subplate markers is affected differently according to dorsolateral position in some of the mutant brains. The colored bands represent the area in which labeled cells are found but do not correspond to any measure of cell density or intensity of cell label. Some areas contain labeled cells in both mutant and wild-type brains and are therefore labeled in the respective color mixtures of red, blue, and green. (A) Distribution of Cplx3-expressing cells, (B) distribution of CTGF-expressing cells, (C) distribution of Tmem163-expressing cells, (D) distribution of DDC+ cells, (E) distribution of MoxD1-expressing cells, (F) distribution of Nurr1+ cells.

We also analyzed the distribution of each of these markers in the p35-KO mouse cortex. CTGF-labeled cells are confined to a very narrow band in the middle of the p35-KO cortex (n = 3; Fig. 2F), whereas Nurr1+ cells are in the middle of the p35-KO cortex (n = 3; Fig. 3F) only in dorsal locations. In the lateral aspects, Nurr1+ cells remain in the normal pattern spread throughout the lower cortical layers (Fig. 6F). The in situ hybridization signal for MoxD1 is also located in a thin band roughly in the middle of the p35-KO cortex in dorsal aspects (n = 3; Fig. 2G), whereas more laterally, it is spread throughout the lower half of the cortex. In 1 p35-KO cortex, the previously mentioned faint labeling of layer 5 cells is visible and is shifted outward to just underneath the marginal zone (Fig. 2G). The Cplx3 mRNA is exclusively localized to a very narrow band of cells in the middle of the cortex, similar to the label observed for CTGF (n = 3; Fig. 2E). The protein localization reveals the same band of strongly labeled Cplx3+ cells representing the subplate (n = 2; Fig. 3D), but in addition, a few weakly labeled cells are underneath the marginal zone in the position of layer 5 in the p35-KO cortex. A similar distribution was observed for Tmem163. The strongly labeled cells representing the subplate are relocated to the middle of the dorsal cortex in p35-KO brain sections (n = 3; Fig. 2H). The fainter labeling representing layer 5 cells could not be detected in all sections of p35-KO brains, but when present was relocated to just underneath the marginal zone (the position of layer 5 cells in the p35-KO cortex; Chae et al. 1997). In more lateral aspects of these brains, the narrow band of subplate labeling turns downward toward the white matter and is eventually located just above the white matter (Fig. 6C). The distribution of DDC+ cells in the p35-KO cortex is again dependent upon the dorsolateral aspect (n = 2 brains). In very dorsal aspects, no DDC+ cells are present in the cortex. At slightly more lateral positions, the distribution of DDC+ cells is broader than in wild type including the white matter and the lower two-thirds of the cortical plate (Fig. 3E). In lateral positions above the striatum, the DDC+ cell distribution is very similar to wild-type brains with cells confined to the white matter, subplate, and lower layer 6. Overall, more DDC+ cells are outside of their wild-type distribution in p35-KO than in scrambler mice.

For a summary of the distribution of all types of labeled cells in the reeler mutant and p35-KO brain, see Figure 6.

Colocalization with GABA and with Each Other

Lastly, for those potential markers where antibodies were obtainable, we also investigated the colocalization with the known subplate marker Nurr1 as well as with GABA. Approximately half of all GABA-immunoreactive cells in the postnatal cortex are also GFP positive in the Gad65-GFP mouse (López-Bendito et al. 2004).

Nurr1 was previously reported to label glutamatergic but not GABAergic cells in the rat cortex (Arimatsu et al. 2003). We can confirm this for the mouse cortex at P8 where less than 1% of Nurr1+ cell were found to be also GABA+ (n = 1 double-labeled cell of 420 Nurr1+ cells, n = 3 brains; data not shown). Furthermore, no Nurr1+ cell was also Gad65-GFP+ in the Gad65-GFP mouse (n = 3 brains, 396 cells; see Fig. 7A). Neither Cplx3+ cells (n = 3 brains, 593 cells; Fig. 7B) nor DDC+ cells (n = 3 brains, 408 cells; Fig. 7D) were ever GABA+. No colocalization of DDC or Cplx3 with GFP+ cells was found in the Gad65-GFP mouse either (586 Cplx3+ cells and 276 DDC+ cells in n = 3 brains). We assessed whether the Cplx3 protein colocalizes with Nurr1 in the cortex. Just less than half of the Cplx3+ cells in the subplate were also Nurr1+ (n = 3 brains, 567 cells, 46 ± 12% [mean ± standard deviation {SD}] colocalization; see Fig. 7C for an example of double- and single-labeled cells). Conversely, approximately two-thirds of Nurr1+ cells are also Cplx3+ (n = 3 brains, 317 Nurr1+ cells, 69 ± 8% [mean ± SD] colocalization). DDC+ cells on the other hand do not colocalize with Nurr1+ cells (n = 2 brains, 564 cells; Fig. 7E).

Figure 7.

Cellular colocalization of putative subplate markers with each other and with Gad65-GFP or GABA. Half of GABAergic cells in the adult cortex of Gad65-GFP mice are GFP positive, and this is used here as a marker for putative GABAergic interneurons. (A) Confocal image of the protein colocalization of Nurr1 (red) with Gad65-GFP (green). No colocalization was observed. (B) Confocal image of Cplx3 (green) protein colocalization with GABA (red). No colocalization was observed. (C) Confocal image of Nurr1 protein (green) colocalization with Cplx3 (red). Cplx3+/Nurr1+ double-labeled cells are indicated by a white arrowhead, Cplx3-only cells are indicated by a red arrow and Nurr1-only cells are indicated by a green arrow. Approximately two-thirds of all Nurr1+ cells were also Cplx3+. Conversely, approximately half of all Cplx3+ cells were also Nurr1+. (D) Confocal image of the protein colocalization of DDC (green) with GABA (red). No colocalization was observed. (E) Confocal image of Nurr1 protein (green) colocalization with DDC (red). No colocalization was observed. The blue label in panels (AE) is the nuclear counterstain DAPI, and narrow white lines delineate the subplate. (F) Schematic distribution of putative subplate cell markers and GABA across different cell layers in the dorsal mouse cortex at P8, and preliminary subdivision of the subplate into different groups of cells. Scale bar = 50 μm for A - C, E and 100μm for GP.

Figure 7.

Cellular colocalization of putative subplate markers with each other and with Gad65-GFP or GABA. Half of GABAergic cells in the adult cortex of Gad65-GFP mice are GFP positive, and this is used here as a marker for putative GABAergic interneurons. (A) Confocal image of the protein colocalization of Nurr1 (red) with Gad65-GFP (green). No colocalization was observed. (B) Confocal image of Cplx3 (green) protein colocalization with GABA (red). No colocalization was observed. (C) Confocal image of Nurr1 protein (green) colocalization with Cplx3 (red). Cplx3+/Nurr1+ double-labeled cells are indicated by a white arrowhead, Cplx3-only cells are indicated by a red arrow and Nurr1-only cells are indicated by a green arrow. Approximately two-thirds of all Nurr1+ cells were also Cplx3+. Conversely, approximately half of all Cplx3+ cells were also Nurr1+. (D) Confocal image of the protein colocalization of DDC (green) with GABA (red). No colocalization was observed. (E) Confocal image of Nurr1 protein (green) colocalization with DDC (red). No colocalization was observed. The blue label in panels (AE) is the nuclear counterstain DAPI, and narrow white lines delineate the subplate. (F) Schematic distribution of putative subplate cell markers and GABA across different cell layers in the dorsal mouse cortex at P8, and preliminary subdivision of the subplate into different groups of cells. Scale bar = 50 μm for A - C, E and 100μm for GP.

DDC is an essential enzyme in the synthesis pathway for the neurotransmitter dopamine, so we assessed whether the rate-limiting enzyme in this pathway, TH, is present in the DDC+ cells in the subplate. Only one TH+ cell was found in the subplate (n = 3 brains) despite abundant labeling in other known dopaminergic regions of the brain (data not shown). No colocalization with DDC was found (data not shown).

Because of its presence in the white matter, we decided to assess whether DDC expression is confined to neurons. At P8, DDC+ cells never colocalized with the neuronal marker NeuN (n = 5 brains, 358 cells; data not shown). Thus, although DDC+ cells have the morphology of neurons including the spherically symmetric nucleus typical of neurons in DAPI-counterstained material, we cannot conclude for certain that DDC expression is confined to neurons in the cerebral cortex.

Discussion

Subplate neurons integrate into the cortical circuits in different manners (Antonini and Shatz 1990; Arimatsu et al. 2003), and cells within the subplate show different electrophysiological properties (Friauf and Shatz 1991; Hanganu et al. 2001). The classification of subplate cells according to their projection sites, neurochemical properties, and physiological characterization could have considerable importance in basic research of cortical circuit formation, in neuropathological diagnosis, and in comparative neurobiology. Antonini and Shatz (1990) have already tried to relate subplate connectivity with markers, but the repertoire of available markers was limited in those times and has not been extended by much since then. Here we have identified 3 novel markers that can distinguish between subplate and the adjacent layer 6, but where expression is also seen in layer 5, at least in some brains (Cplx3, MoxD1, and Tmem163). Our data suggest that Cplx3 in situ hybridization can be used to extend the repertoire of available subplate-selective markers. DDC immunoreactivity revealed a distinct subplate population. The current analysis was restricted to a few selected genes; our microarray data contain further candidates, which will be studied in the future.

The markers described here provide evidence of distinct subgroups of subplate cells, some of which further subdivide the known class of glutamatergic cells. Furthermore, we have demonstrated that these subgroups are not only expressing different markers but also differentially affected by mutations causing cortical plate migration defects in the reeler and p35-KO mouse strains. Lastly, the novel markers extend the available repertoire in the postnatal period as all genes continue to be expressed in a thin band of cells in between layer 6 and the white matter in adult tissue.

Experimental Considerations

The recent advances in cell separation and gene expression analysis methods provide us with the possibility of establishing a detailed molecular taxonomy of a selected cell population of the cerebral cortex (Markram et al. 2004; Molnár and Cheung 2006; Sugino et al. 2006; Molyneaux et al. 2007; Luo et al. 2008). Here we contribute to the growing field of molecular taxonomy with some further potentially valuable markers. It is very encouraging as an internal validation step that our screen identified 2 known subplate markers, Nurr 1 (Liu and Baker 1999; Arimatsu et al. 2003) and CTGF (Heuer et al. 2003; Watakabe et al. 2007).

In the current study, we restricted our investigation to 2 layers (subplate and layer 6) and the time point P8, which was chosen to facilitate identification of markers that persist into adulthood. Indeed, we found that Cplx3, CTGF, MoxD1, Nurr1 and Tmem163 are all expressed in adult subplate cells. Accordingly, the pathways highlighted by Ingenuity as consistently differentially expressed between subplate and layer 6 did not include any apoptotic signaling cascades, which is expected since the majority of early-born subplate cells have already disappeared by P8 (Price et al. 1997). Selecting P8 as a starting point for marker identification also had the advantage that all cortical neurons have completed their migration, and we can thus avoid contamination from migratory cells located transiently in the subplate. We also extended our expression analysis to earlier and later stages confirming the presence of subplate expression. However, subplate is a dynamic population of neurons with different functions at different stages of development (Kostović and Rakic 1990; Allendoerfer and Shatz 1994; Kanold 2004), and further studies at earlier stages are required. A detailed characterization of developmental expression in combination with birthdating studies might identify differential survival of subplate neurons.

Utility of Subplate Markers in the Understanding of Cortical Circuits

Integration of the subplate cells into the intra- and extracortical circuitry has been previously studied with tracing, cell filling, or in reporter gene–expressing mouse lines (McConnell et al. 1989; Antonini and Shatz 1990; Friauf et al. 1990; Kolk et al. 2005; Jacobs et al. 2007). It has been suggested that some subplate neurons project to the cortical plate (marginal zone and layer 4; Friauf et al. 1990) and could participate in the establishment of cortical modules in the visual cortex, that is, ocular dominance and orientation columns (Kanold et al. 2003; Kanold 2004; Kanold and Shatz 2006).

Interestingly, the anti-DDC antibody also gives a periphery-related patterning in barrel cortex of wild-type P8 mice (Figs 1and 3H). This patterning is still evident in the brains of scrambler mice but absent from the cortices of p35-KO mice (data not shown). This observation suggests that DDC-immunoreactive neurites, potentially derived from the subplate, integrate into the overlying cortex. The age-specific integration of GFP-positive neurites deriving from subplate or layer 6 cells into the barrel cortex has been recently described in the Golli-tau-eGFP mouse (Aye et al. 2006; Jacobs et al. 2007; Jethwa et al. 2007; Piñon et al. 2008). In view of the gap between the disappearance of the early DDC+ fibers from the subplate region and the onset of cellular labeling in that region, we propose that the earlier fiber labeling is derived from noncortical sources and may be involved in earlier patterning events. The periphery-related patterning observed at P8 is most likely derived from labeled subplate processes.

The identification of novel subplate markers reported here will allow us to establish more selective subplate reporter gene lines or genetic models to selectively control the activity (input or output) of subplate subpopulations. It may even be possible to identify different subgroups of subplate cells based on their integration into the cortical circuitry.

Early-born subplate cells are not believed to persist in the postnatal mouse brain (Price et al. 1997); yet, cells remain visible in a distinct subplate layer in juvenile and adult mouse brains, well after the death of the early-born cells. MoxD1, Cplx3, Tmem163, Nurr1, and CTGF continue to label a band of cells in the location of the subplate in the adult and may therefore help to elucidate the function and developmental origin of the cells contributing to the subplate in the postnatal and more mature cortex.

Suggested Subplate Subgroups

Subplate cells are a diverse group of cells based on projections, neurochemical properties, and electrophysiological criteria (Kostović and Rakic 1990; Allendoerfer and Shatz 1994). Although none of the novel markers reported here could be demonstrated to be useful in identifying GABAergic cells in the subplate, some of them are potential candidates to subdivide and further characterize the large group of glutamatergic subplate cells.

One of the potential subplate markers found in this study is Cplx3. Cplx3 is a component of both glutamatergic and glycinergic synapses in the mouse retina but is not associated with GABAergic synapses (Reim et al. 2005). Interestingly, only 3 of the 4 identified isoforms of Cplxs (Cplxs1–3) are expressed in the mouse brain (Reim et al. 2005), with expression levels of Cplx3 being very low in the neocortex. Electrophysiological studies on cultured hippocampal glutamatergic and striatal GABAergic neurons revealed that the neurotransmitter release of Cplx1/2 double knockout and Cplx1/2/3 triple knockout mice showed almost identical phenotypes, and it was concluded that Cplx1 and 2 are the predominant isoforms at the tested synapses (Xue et al. 2008). Here we demonstrated that Cplx3-positive cortical cells are located in the subplate with a few cells also present in layer 5. It may prove interesting to correlate Cplx3 expression with electrophysiological cell properties. It has been hypothesized (Reim et al. 2005) that Cplx3 presence at the presynaptic terminal permits particularly fast synaptic vesicle release, which raises interesting questions about the functional role of postnatal subplate neurons if some of them are capable of very fast synaptic release.

Nurr1 has been previously reported as a marker of subplate cells in dorsal mouse cortex, and Nurr1+ cells in the mouse subplate also express CTGF (Watakabe et al. 2007). In the rat and macaque, Nurr1+ cells possess corticocortical projections (Arimatsu et al. 2003; Watakabe et al. 2007) and are glutamatergic in the macaque (Watakabe et al. 2007). We observed that approximately one-third of Cplx3+ cells are positive for the transcription factor Nurr1, whereas conversely, about two-thirds of Nurr1+ cells are also Cplx3+. Combining these findings, we can now further extend the classification of murine subplate neurons (Fig. 7F). First, there are Nurr1+/Cplx3+ cells, which we also expect to be CTGF+. Second, there are Nurr1+/Cplx3− cells, which we expect to be CTGF+. Lastly, there are CTGF+/Nurr1− cells (Watakabe et al. 2007), and we have identified Cplx3+/Nurr1− cells. Whether these last 2 groups are distinct is currently not clear but could be resolved with double in situ hybridization or in situ hybridization combined with immunohistochemistry.

Furthermore, some subplate cells are DDC+, and in contrast to Cplx3+ cells, the vast majority of DDC+ cells remain in the lower half of cortex in the reeler mouse, suggesting that DDC+ and Cplx3+ cells are developmentally distinct. The finding that no DDC+ cells are Nurr1+ strengthens this notion. Colocalization of DDC and Cplx3 was not performed for technical reasons. Lastly, we have identified MoxD1-expressing cells and Tmem163-expressing cells, but it is not yet clear how the expression of these two markers is correlated with Cplx3, Nurr1, DDC, and CTGF.

Lastly, we are very intrigued by the finding that 3 novel subplate markers are all expressed in layer 5 pyramidal cells, but no other cortical cells. So far, the only known similarity between these two cell types is that both pioneer some subcortical axon pathways, albeit at much earlier times than the genes reported here begin to be expressed (Molnár et al. 2007).

Markers for Neuropathological Diagnosis

The newly identified subplate markers and our improved understanding of subgroups will enable us to study the pathological changes of subplate in animal models and in human disease in more detail (Hevner 2007). We have already demonstrated in this study that in two relatively well-characterized mutant models, the subplate-specific gene expression changed location according to the abnormal position of the subplate but not all subplate-associated genes changed their expression pattern in the same way. The identification of markers for different subgroups of subplate cells will enable us to study the differential sensitivity of subpopulations in developmental abnormalities, in response to pre- or perinatal insults (Volpe 2001; McQuillen and Ferriero 2005) or in more complex cognitive disorders such as schizophrenia (Eastwood and Harrison 2003, 2005).

Supplementary Material

Supplementary material can be found at http://www.cercor.oxfordjournals.org/.

Funding

Medical Research Council to ZM (G0300200, G0700377); Wellcome Trust 4-year PhD studentship (to AH-S); Wellcome Trust (OXION initiative) to SL; University of Oxford John Fell Fund to ZM and WZW.

Thanks goes to Jamin De Proto for his expert help with the confocal microscopy, Amanda Cheung for her invaluable help in perfusion and tissue collection, and to Franziska Oeschger for her thoughtful comments on the manuscript. We are grateful to Thomas Jessell for his gift of Er81 antibody and Margareta Nikolić for her help with the p35-KO mice. Conflict of Interest: None declared.

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Author notes

Anna Hoerder-Suabedissen and Wei Zhi Wang are joint first authors, contributed equally to this work