Mice

Mice were group-housed in ventilated cages, given access to food (PicoLab^® Rodent Diet 20, 5053) and water ad libitum and maintained on a 12-h light/dark cycle (lights off at 8 pm). All experimental procedures were approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee (IACUC) and were performed in strict accordance with National Institutes of Health guidelines for the care and use of laboratory animals. Camk2a-iCre mice (C57BL/6J background) were used for excitatory neurons; mice were generated in the laboratory of G. Schütz [23] and provided by the laboratory of C.-K. J. Shen. Gad2-Cre mice (stock number: 010802, C57BL/6J background) were used for inhibitory neurons and Gfap-Cre mice (stock number: 012886, C57BL/6J background) were used for astrocytes; both were purchased from the Jackson Laboratories, Bar Harbor, Maine. The full strain name of Ai14 Cre reporter mice [24] is B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J (stock number: 007914), which were provided by the laboratory of S.L. Lin. C57BL/6J (B6) mice (stock number: 000664) and CAST/EiJ (CAST) mice (stock number: 000928) were purchased from Jackson Laboratories. To obtain cell-type-specific fluorescent cells, we bred cell-type-specific Cre-expression mice and Cre-reporter mice with a B6 background. Next, we crossed the mutant mouse (B6) with a wild-type mouse in CAST background [F1 initial cross (F1i); CAST wild-type mother x B6 mutant father] (Fig 1a, top). This breeding produced offspring with a B6 and CAST background and red fluorescent signals in specific cells. The B6/CAST mixed background has the advantage of yielding more identifiable single nucleotide polymorphisms (SNPs). To confirm our observations from F1i, we performed a reciprocal cross [F1 reciprocal cross (F1r): B6 mother x CAST father]. Offspring were euthanized at postnatal day 28 (P28) with 1% isoflurane followed by decapitation, and all efforts were made to minimize suffering. Brains were collected and processed according to the specific protocols described below.

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Fig 1. Process for detection and isolation of single cells from layer 2/3 of mouse visual cortex.

(a) Schematic flowchart of the experimental design. Top: mating strategy to obtain B6/CAST mice. Middle: 7 μm cryostat sections from P28 mice were cut and single cells were identified for laser capture microdissection (LCM) as excitatory neurons, inhibitory neurons or astrocytes in layer 2/3 of the visual cortex. Scale bar, 50 μm. Bottom: LCM single cells were collected and RNA was extracted. A cDNA library, was generated for each cell type followed by RNA-Seq, and allele-specific expression in the cellular transcriptome was analyzed. (b) Schematic diagram showing cross of cell-type-specific Cre lines with a Cre/loxP reporter line (Ai14). (c) Confirmation of each specific cell type captured by LCM in Cre reporter cell lines. Left panels (i, v, ix): Representative images of the visual cortex from the three cell-type-specific Cre mouse lines. Cells are visualized with tdTomato (red). Cell-type specific Cre reporter mouse lines are indicated above each image. Squares indicate areas enlarged in left panel. Scale bar, 100 μm. Right panels (ii-iv, vi-viii, x-xii): Enlarged images of cells labeled with antibodies (green) to anti-CAMK2A (excitatory neurons), anti-GAD2 (for inhibitory neurons) or anti-S100B (for astrocytes). Double-labeled cells (overlay) confirmed cell type of red fluorescent cells from the cell-type-specific Cre reporter lines. Sections were counterstained with DAPI (blue). Scale bar, 20 μm.

https://doi.org/10.1371/journal.pone.0163663.g001

Genotyping

PCR was performed using Camk2a-iCre/F (5’-CTC TGA CAG ATG CCA GGA CA-3’), Camk2a-iCre/R (5’-TGA TTT CAG GGA TGG ACA CA-3’), Gad2-Cre-WT/F (5’-CTT CTT CCG CAT GGT CAT CT-3’), Gad2-Cre-Common (5’-CAC CCC ACT GGT TTT GAT TT-3’), Gad2-Cre-MT/F (5’-AAA GCA ATA GCA TCA CAA ATT TCA-3’), Gfap-Cre/F (5’-AAA ATT TGC CTG CAT TAC CG-3’), Gfap-Cre/R (5’-ATG TTT AGC TGG CCC AAA TG-3’), Ai14 WT/F (5’-AAG GGA GCT GCA GTG GAG TA-3’), Ai14 WT/R (5’-CCG AAA ATC TGT GGG AAG TC-3’), Ai14 mutant/F (5’-GGC ATT AAA GCA GCG TAT CC-3’), Ai14 mutant/R (5’-CTG TTC CTG TAC GGC ATG G-3’). The PCR cycling conditions were as follows: for Camk2a-iCre primers; initial denaturation at 94°C for 3 min followed by 35 cycles of 94°C for 30 s, 56°C for 30 s and 70°C for 60 s and a final extension at 72°C for 2 min; for Gad2-Cre primers; initial denaturation at 94°C for 3 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s and a final extension at 72°C for 2 min; for Gfap-Cre primers; initial denaturation at 94°C for 2 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 20 s and a final extension at 72°C for 5 min; for Ai14 WT and mutant primers; initial denaturation at 94°C for 3 min followed by 35 cycles of 94°C for 20 s, 61°C for 30 s and 72°C for 30 s and a final extension at 72°C for 2 min. The size of PCR product is 381 bp for Camk2a-iCre primers, 250 bp for Gad2/WT primers, 352 bp for Gad2/MT primers, 350 bp for Gfap-Cre primers, 297 bp for Ai14 WT primers, 196 bp for Ai14 MT primers. Amplification was performed on a C1000 Touch Thermal Cycler (Bio-Rad).

Immunofluorescence staining

For immunofluorescence staining, P28 brains were immersion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) for 12 h and then cryoprotected with 30% sucrose in 0.1 M PB at 4°C overnight. Fixed brains were embedded in O.C.T. compound (Surgipath, FSC 22). Cryostat sections (7 μm) were cut with a cryostat (Leica, CM3050 S) collected on glass slides, permeabilized with 0.3% Triton X-100 in 1X phosphate buffer saline (PBS) for 30 min at room temperature, and blocked in 1X PBS containing 5% goat serum and 0.3% Triton X-100 for 1 h. Sections were further incubated with anti-CAMK2A antibody (1:500, Acris Antibodies, AM 12015PU-S) at 4°C overnight or anti-GAD67 antibody (1:800, Millipore, MAB5406) at 4°C overnight, or anti-S100 beta antibody (1:500, Abcam, ab52642) at room temperature for 1 h, and then incubated with Alexa Fluor 488 goat anti-mouse IgG1 (γ1) antibody (1:500, Invitrogen, A21121 for CAMK2A) or Alexa Fluor 488 goat anti-mouse IgG2a antibody (1:500, Invitrogen, A21131 for GAD67) or Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody (1:500, Invitrogen, A11008 for GFAP) and DAPI (1:10,000, Invitrogen, D-1306) for 2 h at room temperature. Sections were washed in 1X PBS containing 0.1% Triton X-100 three times (5 min/wash) following incubations with primary and secondary antibodies. Sections were coverslipped with Fluoromount aqueous mounting medium (Sigma, F4680) and images were acquired using a Zeiss LSM 780 confocal microscope. Montage images were acquired with a 20X/0.8 NA objective and enlarged images were acquired with a 63X/1.4 NA objective.

Fluorescence-based laser capture microdissection

P28 mouse brains were cut in half coronally and immersed in RNAlater stabilization solution (Ambion, AM7021) at 4°C for 1 h. A minimum of 6 h fixation is required to maintain a fluorescent signal during laser capture with 7 μm-thick cryostat sections of brain tissues. Therefore, brains were fixed in 4% paraformaldehyde (Sigma, SI-P6148) in 0.1 M PB at 4°C for 6 h followed by cryoprotection with 30% sucrose (J.T.-Baker, JT-4097-04) in 0.1 M PB at 4°C overnight. Brains were placed into a cryomold, embedded with O.C.T. compound (Surgipath, FSC 22) and frozen in a pre-chilled bath of isopentane (Sigma, AL-M32631) cooled with dry ice. To avoid obtaining more than one cell per capture, frozen brains were sectioned at a thickness of 7 μm with a cryostat (Leica, CM3050 S) and collected on membrane slides (Zeiss, 415190-7041-000). Sections were air-dried and dehydrated by performing one 30 second-dip in 50% ethanol (J.T. Baker, JT-8006-05) in DEPC (Sigma, D5758)-treated water, one 10 second-dip in 95% ethanol in DEPC-treated water, two 30 second-dips in 100% ethanol and two 2 minute-dips in 100% xylene (J.T. Baker, JT-9490-03). Red fluorescent positive cells in layer 2/3 of primary visual cortex were captured using a laser capture microdissection system (Zeiss, PALM MicroBeam) and collected into AdhesiveCap 500 opaque PCR tubes (Zeiss, 415190-9201-000). Layer 2/3 was defined by its relative location to all six layers of primary visual cortex [25, 26]. Tubes were replaced every hour. A total of 6000 cells per type were captured for further processing (Fig 1a, middle). In preliminary studies, we determined a minimum of 6000 cells per type was required for RNA-Seq analysis. A flowchart of the overall process of obtaining the LCM-captured cells is shown in Fig 1a. We also determined a minimum of 2000 cells per type was required for the confirmation of at least two imprinted genes with Sanger sequencing (S1 Fig).

RNA isolation, library preparation and sequencing

Total RNA was extracted from the pooled LCM-captured cells with the NucleoSpin totalRNA FFPE XS kit (Macherey-Nagel, REF740969) with minor modifications. In short, cells in the cap were covered with lysis buffer, incubated at 56°C for 15 min and then spun down. Extracted RNA was bound onto the column with binding buffer and genomic DNA was digested for 15 min in rDNase incubation solution. After two washes, RNA was eluted with 10 μl of RNase-free water. rRNA was removed from the purified RNA with the Ribo-Zero Magnetic Gold Kit (Epicentre, MRZG126). The purified RNA was amplified with the SMARTer Universal Low Input RNA Kit for Sequencing (Clontech, 634938) and libraries were generated with the Low Input Library Prep Kit (Clontech, 634947). Libraries were sequenced on an Illumina HiSeq2000 (100 PE bp) at the Welgene Biotech Company, which generated 6 Gb of data per sample. RNA was quantified and qualitated with the Bioanalyzer 2100 (Agilent Technologies) using a RNA 6000 Pico LabChip (Agilent, 5065–4401). DNA was quantified and qualitated with an Agilent 2200 TapeStation system using High Sensitivity D1000 ScreenTape (Agilent, 5067–5584) and High Sensitivity D1000 Reagents (Agilent, 5067–5585).

RNA-sequencing (RNA-Seq) analysis

To analyze the RNA-Seq data from the three cell types, qualified sequencing reads were obtained by filtering the generated sequences (S2 Fig and S1 Table). Per base sequence quality and per sequence quality scores from RNA-Seq data were analyzed by FastQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Subsequently, Trimmomatics was implemented to trim or remove the reads according to the quality score [27]. Qualified reads after filtering low-quality data were analyzed using TopHat/Cufflinks for gene expression estimation [28]. The gene expression level was calculated as FPKM (Fragments Per Kilobase of transcript per million mapped reads). The reference genome and gene annotations were retrieved from the Ensembl database. The Genome Analysis Toolkit (GATK) was used for SNP finding and genotype determination. MMSEQ was applied for Allelic-Specific-Expression (ASE) analysis. Monoallelic gene expression was defined as log(Ma/Pa) larger than 0.5 or less than -0.5. The identities of monoallelically-expressed genes in each cell type are shown in S2 Table.

Quantitative real-time PCR (Q-RT-PCR)

Total RNA was extracted from LCM-captured excitatory neurons and astrocytes with the NucleoSpin miRNA kit (Macherey-Nagel, 740971). Q-RT-PCR was performed with a StepOnePlus Real Time PCR System (Applied Biosystem) using the SuperScript III Platinum SYBR Green One-Step qPCR Kit w/ROX (Invitrogen, 11746). The PCR cycling condition was as follows: initial denaturation at 95°C for 5 min followed by 50 cycles of 95°C for 15 s and 60°C for 30 s and a final extension at 40°C for 1 min. Ct values were generated by StepOne Software version 2.2.2. The expression level of each gene was normalized to B2m. All primer sequences are provided in S3 Table. The PCR product was purified with NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, 740609) and analysed by Sanger sequencing.

A multi-stage approach analysed parent-of-origin-specific monoallelic expression in individual cell types of the mouse visual cortex

This study employed a multi-stage approach to identify parent-of-origin-specific monoallelic expression in different cell types of the mouse visual cortex on a genome-wide scale. We targeted three major cell types: excitatory neurons, inhibitory neurons and astrocytes within distinct regions of the mouse visual cortex and resolved parent-of-origin-specific monoallelic expression patterns. To identify the individual cell types, we took advantage of the Cre-LoxP system. Cell-type-specific Cre mice were mated with td-Tomato Cre-reporter Ai14 mice (Fig 1a, top). Single cells with red fluorescent signals from layers 2/3 of the visual cortex were isolated with laser capture microdissection (LCM) (Fig 1a, middle). We focused on these two layers because this is where plasticity of the visual cortex occurs [21, 22]. Next, to distinguish parent-of-origin-specific monoallelic expression, we took advantage of single nucleotide polymorphisms (SNPs), which were identified from the genomic DNA of C57BL/6J (B6) and CAST/EiJ (CAST) strain mice. Both have very distant sub-strains; therefore, it is much easier to distinguish the two sub-strain SNPs. Finally, RNAs were extracted from LCM-captured cells, which were then depleted of ribosomal ribonucleic acids (rRNAs). A cDNA library was constructed from the rRNA-depleted RNAs and further processed with genome-wide RNA-sequencing (RNA-Seq) followed by allele-specific expression analysis (Fig 1a, bottom).

Cell-type-specific CRE expression is robust in the mouse visual cortex

Our multi-stage approach employed cell-type-specific Cre mice. We used Camk2a-iCre mice for excitatory neurons, Gad2-Cre mice for inhibitory neurons, and GFAP-Cre mice for astrocytes. To obtain red fluorescent signals in the cells, the cell-type-specific Cre mice were mated with Cre-reporter mice (Ai14), offspring were bred with CAST/EiJ mice, and the resulting offspring were used for LCM (Fig 1a and 1b). Red fluorescent cells in the visual cortex confirmed the presence of the three distinct cell types (Fig 1ci, 1cv and 1cix). Each cell type was further validated by immunofluorescence with cell-type-specific antibodies: α-CAMK2A for excitatory neurons; α-GAD2 for inhibitory neurons; and α-S100B for astrocytes (Fig 1ciii, 1cvii and 1cxi). Double-labeled cells confirmed gene and protein expression in Camk2a-iCre::Ai14, Gad2-Cre::Ai14 and Gfap-Cre::Ai14 mice (Fig 1civ, 1cviii and 1cxii). Our results demonstrate that cell-type-specific expression can be detected in the mouse visual cortex using this Cre-LoxP system, and the level of expression is robust.

LCM-captured cells maintain their cellular identities in distinct layers of visual cortex

We focused on three major cell types in the mouse visual cortex for implementing fluorescence-based LCM: excitatory neurons (E), inhibitory neurons (I), and astrocytes (A) (Fig 2a). We extracted RNAs from the LCM-captured cells and then depleted rRNAs. We converted the rRNA-depleted RNAs into a cDNA library and the resulting concentration and cDNA fragment size (Fig 2b) met the criteria for further analysis with RNA-Seq. RNA-Seq analysis demonstrated the LCM-captured cells exhibited high expression levels of cell-type-specific genes for excitatory neurons, inhibitory neurons, and astrocytes (Fig 2c). The appropriate genetic properties displayed by each cell type confirmed our methodology of obtaining cells from the visual cortex by LCM did not disrupt their cellular identities.

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Fig 2. Cellular identity and genomic imprinting status were maintained in LCM-captured cells of mouse visual cortex.

(a) Schematic diagram of the mouse visual cortex and the three cell types captured with LCM: excitatory neurons (E), inhibitory neurons (I) and astrocytes (A). (b) Quality of the cDNA library was evaluated by the fragment peak size and concentration of cDNA using the TS HS D1000 (Agilent) assay. Top: excitatory neurons; fragment peak size = 284 bp; concentration = 7 nM. Middle: inhibitory neurons; fragment peak size = 293 bp; concentration = 180 nM. Bottom: astrocytes; fragment peak size = 327 bp; concentration = 42 nM. A minimum fragment peak size of 240 bp and concentration of 4 nM was required for performing RNA-Seq. (c) Heat map showing expression of cell-type-specific genes, which confirmed the identities of the LCM captured cells were excitatory neurons, inhibitory neurons, and astrocytes. (d) Left panel: Excitatory neurons (top), inhibitory neurons (middle) and astrocytes (bottom) exhibited similar patterns of monoallelic gene expression. Right panel: Known maternal and paternal imprinted genes in the three cell types. The imprinting status of known imprinted genes was maintained in all cell types. Excitatory neurons were obtained from offspring of a female CAST x male B6 cross. Inhibitory neurons and astrocytes were obtained from offspring of a female B6 x male CAST cross.

https://doi.org/10.1371/journal.pone.0163663.g002

LCM-captured cells maintain their genomic imprinting status associated with distinct layers of the visual cortex

To determine whether LCM-captured cells exhibited a parent-of-origin-specific expression pattern, we profiled monoallelic expression in the LCM captured cells. The three major cell types of the mouse visual cortex exhibited similar patterns of monoallelic gene expression: one diagonal line, which indicated equal expression of genes from paternal and maternal alleles, one cohort above the diagonal line, representing expression of genes from maternal alleles, and one cohort below the diagonal line, representing expression of genes from paternal alleles, which was seen in initial and reciprocal crosses (Fig 2d, left and S3 Fig). Unfortunately, the low density of genes in the inhibitory neurons and astrocytes of the initial crosses prevented any further analysis of initial crosses.

To further investigate whether monoallelically expressed genes are predominantly expressed in three major cell types of mouse visual cortex and whether there is any overlappability of monoallelically expressed genes between the three cell types in mouse visual cortex, we calculated the percent of monoallelically expressed genes in the three cell types and the overlappability of monoallelically expressed genes among them. Our results showed that, although the percentages of monoallelically expressed genes differed between excitatory neurons, inhibitory neurons and astrocytes, each cell type had a similar percentage from paternal and maternal alleles (S4 Table). The percent of monoallelically expressed genes was less than 12% regardless of allele source. Examination of the overlappability of these monoallelically expressed genes in these three cell types indicated the percent of overlap was less than 20% regardless of allele source (S4 Fig and S5 Table). Monoallelically expressed genes from S4 Fig and overlapped genes from S5 Table for each cell type are shown in S2 Table. Finally, known imprinted genes including maternally expressed Meg3 and paternally expressed Peg3 were detected in our LCM-captured cells regardless of cell type (Fig 2d, right), which indicated LCM-capturing did not alter the genomic imprinting status. We also confirmed the presence of a known imprinted gene, Meg3 and a known neuron-specific imprinted gene, Ube3a, in our LCM-captured cells with Sanger Sequencing (S1 Fig), suggesting that RNA obtained from our method of LCM-capture can be validated by a second approach. These data suggest that monoallelic gene expression is prevalent in the brain transcriptome regardless of cell type.