A Novel Ultrasensitive In Situ Hybridization Approach to Detect Short Sequences and Splice Variants with Cellular Resolution
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Investigating the expression of RNAs that differ by short or single nucleotide sequences at a single-cell level in tissue has been limited by the sensitivity and specificity of in situ hybridization (ISH) techniques. Detection of short isoform-specific sequences requires RNA isolation for PCR analysis—an approach that loses the regional and cell-type-specific distribution of isoforms. Having the capability to distinguish the differential expression of RNA variants in tissue is critical because alterations in mRNA splicing and editing, as well as coding single nucleotide polymorphisms, have been associated with numerous cancers, neurological and psychiatric disorders. Here we introduce a novel highly sensitive single-probe colorimetric/fluorescent ISH approach that targets short exon/exon RNA splice junctions using single-pair oligonucleotide probes (~ 50 bp). We use this approach to investigate, with single-cell resolution, the expression of four transcripts encoding the neuregulin (NRG) receptor ErbB4 that differ by alternative splicing of exons encoding two juxtamembrane (JMa/JMb) and two cytoplasmic (CYT-1/CYT-2) domains that alter receptor stability and signaling modes, respectively. By comparing ErbB4 hybridization on sections from wild-type and ErbB4 knockout mice (missing exon 2), we initially demonstrate that single-pair probes provide the sensitivity and specificity to visualize and quantify the differential expression of ErbB4 isoforms. Using cell-type-specific GFP reporter mice, we go on to demonstrate that expression of ErbB4 isoforms differs between neurons and oligodendrocytes, and that this differential expression of ErbB4 isoforms is evolutionarily conserved to humans. This single-pair probe ISH approach, known as BaseScope, could serve as an invaluable diagnostic tool to detect alternative spliced isoforms, and potentially single base polymorphisms, associated with disease.
KeywordsSchizophrenia ErbB4 Neuregulin Alternative splicing Oligodendrocytes RNA expression Transcriptome BaseScope
Alternative mRNA splicing increases the functional complexity of the genome, with > 90% of all human multi-exon genes being differentially spliced . In the central nervous system (CNS), alternative splicing is tightly regulated in a spatio-temporal manner, as well as by neuronal activity [2, 3, 4]. Different mRNA isoforms encode for ion channels, neurotransmitter receptors, adhesion molecules, and signaling proteins with distinct functional properties [5, 6, 7, 8]. Splicing abnormalities are observed in different cancers and neurological diseases [9, 10], but are particularly abundant in psychiatric disorders, such as affective and addictive disorders, schizophrenia (Scz) and autism spectrum disorders . In the postmortem brain of Scz patients, splice variant expression of many at-risk alleles is altered ; including those that encode: trophic factors [13, 14, 15, 16, 17, 18, 19], neuronal migration and adhesion proteins [20, 21], structural components of myelin and synapses [22, 23] and isoforms associated with dopaminergic, GABAergic and glutamatergic neurotransmission and signaling [24, 25, 26, 27, 28].
The four ErbB4 isoforms differ functionally. JMa-containing ErbB4 isoforms, but not JMb variants, are susceptible to extracellular metalloprotease-mediated cleavage followed by gamma-secretase intramembranous cleavage that releases a transcriptionally active intracellular domain (ICD) to regulate gene expression [38, 42, 43, 44, 45]. CYT-1-containing isoforms encode a site for phosphatidyl inositol 3-kinase recruitment that increases the downstream signaling capacities of CYT-1 variants [37, 43].
Because of the different functions imparted by distinct splice variants, in this case ErbB4, it is critically important to identify the cells that express distinct isoforms. Whereas quantitative real-time PCR (qRT-PCR) and RNA sequencing (RNAseq) can be designed to detect specific RNA splice variants with high sensitivity in different brain regions, these methodologies require the disruption of dissected tissue to isolate RNA. The technical requirements of RNA isolation come at the expense of losing in vivo cell-type-specific resolution of splice variant expression. Traditionally, in situ hybridization (ISH) using radioactively- and fluorescently-labeled complementary RNA probes have provided the sensitivity to detect abundant transcripts at cellular level, but fail to unambiguously identify cells expressing rare splice variants. Recent advances in ISH using multiple non-radioisotropic oligonucleotide probe pairs targeting a single transcript, combined with chemical signal amplification [46, 47], enable specific and sensitive co-detection of rare transcripts (known as “multiplexing”; ). However, the optimal target lengths of these probes (> 300 bp) exceed the size of most alternative spliced variants. Due to these limitations, in the present study we implement a novel ISH approach based on an ultrasensitive amplification chemistry that allows the specific detection of mRNA exon junctions by a single pair of 18–25 bp anti-sense oligonucleotide probes targeting adjacent mRNA sequences; hereafter denoted as “single-pair probe”.
Materials and Methods
For further details see Supplemental Information.
Animals and Human Brain Samples
Homozygous ErbB4 knock-out (KO) mice lacking exon 2  will be hereafter designated as ErbB4-Δ2 KO mice. CNP-GFP , NG2-GFP  and wild-type (WT) C57BL/6J mice were obtained from the Jackson Laboratory. GAD67-GFP mice , were a kind gift from Yuchio Yanagawa (Gunma University, Japan). All procedures were approved by the NIH Animal Care and Use Committee. Ground frozen human brain samples from four male adult control individuals were obtained from the Human Brain Collection Core (National Institute of Mental Health, NIMH).
Exon junction-specific single-pair probes for the detection of distinct ErbB4 isoforms
Target sequence (5′ → 3′)
Post hoc immunohistochemistry immediately following ISH was performed as previously published  using 1 μg/mL mouse monoclonal anti-GFP (isotype IgG2a, clone N86/8; NeuroMab, Davis CA).
RNA was isolated from micro-dissected ROI of 10-week-old male WT mice or ground human brain tissue using TRIReagent Kit (ThermoFisher, Waltham MA). cDNA was synthesized with random hexamers from 1μg RNA using SuperScriptIV Reverse Transcriptase (ThermoFisher). Quantification of ErbB4 isoforms was performed as described  using TaqMan assays (ThermoFisher).
Imaging and Quantification
FastRED fluorescent signal was excited at 530 nm and analyzed at 20x magnification. Unbiased automated signal detection and quantification was performed using CellProfiler . Intensity threshold was determined based on background intensity in ErbB4-Δ2 KO sections and dot diameter threshold (≥ 3 pixels) based on mean dot diameter in WT sections. Dots/area, percentage of positive cells and average number of dots/cell were calculated.
All data represent the mean ± SEM and statistical significance was set at p < 0.05. Statistical analyses were performed using one-way ANOVA and Tukey’s multiple comparison test. Statistical analyses are tabulated in Supplemental Tables.
Sensitivity and Specificity of the Novel Single-Pair Probe ISH Approach
To validate the specificity of the single-pair probes, we used as negative controls sections from ErbB4-Δ2 KO mice that lack exon 2 , and targeted the upstream and downstream junctions of exon 2 with probes pan 1/2 and pan 2/3, respectively. In contrast to the high cellular ErbB4 expression in hippocampal interneurons of WT mice (Fig. 2a–e), the signal was absent in the ErbB4-Δ2 KO (Fig. 2f–j; Fig. S1). In summary, these results show the sensitivity and specificity of single-pair probes to visualize exon junctions.
Semi-Quantitative Analysis of Junction-Specific Single-Pair Probe ISH
Differential Expression of ErbB4 Isoforms in Distinct Regions of the Adult Brain
Expression of the Cleavable JMa Isoform in Cells of the Oligodendrocyte Lineage
A present limitation of the novel single-pair probe ISH approach described here, in contrast to the multiplex system, is that its amplification chemistry is limited to one fluorescent/colorimetric channel per section and, does not allow for the simultaneous detection of independent probes with distinct fluorophores (e.g. ErbB4 exon-specific single-pair probe and cell marker probe such as MAG). To circumvent this limitation, first we had to develop a post hoc immunohistochemical protocol because most of antibody cell markers tested were not compatible with the fixation and latter permeabilization protocol (i.e., protease treatment) necessary for ISH—even on fresh frozen sections that allow for milder pretreatment conditions than formalin-fixed paraffin sections. However, we identified a GFP antibody that is compatible with this ISH procedure and has the advantage that it is of broad use for other studies. Next, to unambiguously determine the cell-type expressing JMa transcripts, we used transgenic mice expressing GFP under specific promoters for GABAergic neurons (GAD) or for precursor (NG2) and mature (CNP) oligodendrocytes (details see Materials and Methods). Interestingly, we found that ErbB4 JMa isoforms are expressed in NG2+ oligodendrocyte precursor cells (OPCs) in the corpus callosum and cortex (Fig. 6d, f), as well as in CNP-GFP+ oligodendrocytes in the cortex (Fig. 6h); JMb isoforms were not detected in neither of these cell-types (Fig. 6e, g, i). Consistent with our hypothesis, GABAergic neurons in the corpus callosum and neocortex expressed high levels of JMb (Fig. 6k, m), but low amounts of JMa isoforms (Fig. 6j, l). Taken together, these findings confirm that the cleavable juxtamembrane isoform JMa is the major, if not the sole, juxtamembrane isoform expressed in cells of the oligodendrocyte lineage, whereas JMb transcripts are predominant in GABAergic neurons.
Conservation of Differential ErbB4 Isoform Expression in Human Cortex and Corpus Callosum
Here, we demonstrate the use of a novel sensitive non-radioisotropic ISH approach, called BaseScope, to analyze exon junctions in tissue sections at a single-cell level that has universal applicability to study short RNA sequences—including splice variants in the brain and other tissues. We carefully validate the sensitivity and specificity of junction-specific probes used for this ISH approach, and show that single-pair probes are generally comparable. Moreover, the semi-quantitative results obtained are consistent with established isoform analyses using TaqMan qRT-PCR. By using this novel ISH approach that provides cellular resolution, we identified differential regional ErbB4 isoform expression in the adult mouse brain that is conserved in humans, and that results from the predominant cell-type-specific expression of juxtamembrane isoforms in neurons (JMb) and cells of the oligodendrocyte lineage (JMa).
Differential and Cell-Type-Specific Expression of ErbB4 Isoforms in the Adult CNS
Our analyses identified ErbB4 transcripts harboring the JMb and CYT-2 exons as the two major isoforms in most adult mouse brain areas (e.g. hippocampus, cortex, reticular thalamic nucleus); in line with other studies analyzing ErbB4 isoform expression in the different brain areas across species—including humans [37, 38, 39, 60, 61, 62, 63]; but see . Taking advantage of the expression overview of ErbB4 isoforms by single-pair probe ISH, we identified brain regions where—although generally low—ErbB4 JMa and CYT-1 isoforms comprise most ErbB4 expressed, namely the corpus callosum and thalamus. Of note, the exclusive detection of JMa ErbB4 isoforms in the oligodendrocyte lineage (Fig. 6) is entirely consistent with a recent study that found this distribution of ErbB4 by using RNAseq from cell-sorted brain cells . The fact that JMa, but not JMb, isoforms are cleaved by metalloproteases, which is a requirement for intramembranous gamma-secretase cleavage that releases a transcriptionally active ICD [38, 43, 44], raises the possibility that NRG/ErbB4 signaling uniquely regulates oligodendrocyte maturation through ErbB4-dependent transcriptional mechanisms. Consistent with the expression of ErbB4 in oligodendrocytes, previous studies have reported a role of NRG/ErbB signaling in glial development and myelination [64, 65, 66, 67].
Alterations of ErbB4 Isoform Expression in Scz
Whereas JMa and CYT-1 are the minor ErbB4 isoforms in the adult brain (this study; [37, 38]), they have been repeatedly reported to play an important role during neurodevelopment [68, 69, 70] and higher expression of JMa and CYT-1 ErbB4 isoforms has been reported in postmortem DLPFC of Scz patients independently by several groups [29, 39, 40, 41]. This is interesting considering the increased expression of disease-associated genes in neurodevelopmental disorders during fetal development [71, 72] and high NRG1 expression at ages with highest risk for Scz onset [73, 74]. Further it raises the question whether the increased expression of JMa and CYT-1 isoforms in the DLPFC of Scz results from alterations in the expression or number of cells from the oligodendrocyte lineage and/or a switch in ErbB4 isoform expression in GABAergic neurons. A proposed role of oligodendrocytes and myelination deficits associated with Scz has been emerging (see ). An ErbB4 SNP was shown to affect brain white matter integrity , subcortical white matter is lost in Scz patients [77, 78], and genes related to oligodendrocyte function have been associated with Scz [79, 80]. These observations are interesting in the context of our novel finding that OPCs and oligodendrocytes express predominantly or exclusively the ErbB4 JMa isoform. On the other hand numerous postmortem studies implicate alterations in GABAergic neurons in the DLPFC and hippocampus of persons with Scz [81, 82], where a reduction of GABAergic neuron markers  in particular those associated with fast-spiking interneurons [84, 85], has been frequently reported. Interestingly, the changes have been proposed to occur in specific subtypes of interneurons [39, 40]. Future studies, using ErbB4 isoform-specific single-pair probes reported here, will be important to investigate ErbB4 JMa/JMb and CYT-1/CYT-2 ratios in postmortem human brains of Scz patients and controls to precisely identify the cell-type(s) that underlie the changes in ErbB4 isoforms. Because in addition to ErbB4 the alternative splice variants of many at-risk genes are frequently aberrant in Scz  and affective, addictive and autism spectrum disorders , single-pair probe ISH at a cellular level could generally advance our understanding of isoform changes in psychiatric disorders.
General Considerations for the Broad Application of the Single-Pair Probe ISH Approach
This study is the first to analyze exon junctions using a fluorescent ISH assay. This approach is not limited to splice variants studies, but could be generally used to analyze short mRNA sequences (e.g. pre-miRNAs and snoRNAs), highly homologous transcripts and circular RNAs, as well as point mutations. In addition, the freely-available automated analytic tool developed here renders this ISH approach a valuable semi-quantitative tool to analyze expression at a single-cell level, which complements other quantitative methodologies such as qRT-PCR and RNAseq analysis to study splice variants. However, single-probe ISH (BaseScope) has the added benefit of post-assay analyses in morphological conserved tissue. Using post-hoc immunohistochemical analysis following hybridization of single-pair probes on sections of transgenic mice, we show how to overcome the current single-plex platform limitation to identify the cell-types expressing specific splice variants. Of note, the anti-GFP antibody used herewith is one of few antibodies (< 10%) compatible with protease permeabilization.
Altogether, the advances of this novel ISH approach in analyzing short sequences and isoforms at cellular resolution in the tissue environment by far outweigh a few limitations or difficulties of this technology that merit to be mentioned. Probes targeting highly abundant transcripts tend to produce signal accumulations (clumps) during the enzymatic conversion of FastRED (see Fig. 2b–e; Fig. S1). As shown earlier (Figs. 3 and 4), in our experience hybridization efficiencies between unrelated single-pair probes are in general extremely similar but on occasion, as was the case of CYT probes, can give weaker signals relative to the panErbB4 or juxtamembrane single-pair probes (compare Figs. 3a and 4h); the differences observed could have resulted from intrinsic differences of the targeted mRNA sequences (i.e., looping). Therefore, quantification using this novel single-pair ISH should be considered carefully. Nevertheless, the relative signals for CYT-1/CYT-2 isoforms were conserved as confirmed by qRT-PCR analysis (Fig. 4j, S3A), supporting the semi-quantitative nature of this approach.
Taken together, our study underscores the important and unique utility of this novel single-pair probe ISH technique to investigate, with cellular resolution in tissues, the expression of short and highly homologous RNA sequences. As discussed above, whilst BaseScope should be considered as semi-quantitative approach, it can be used to complement other traditionally used methodologies like qRT-PCR and RNAseq. Its numerous applications renders the single-pair probe ISH as an indispensable tool to advance studies on mRNA regulation and complexity, and their association with numerous neurological and psychiatric diseases.
This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD; ZIA-HD000711). We thank the Human Brain Core Collection, NIMH for providing human brain tissue samples and Dr. Pavan Auluck for micro-dissecting human brain tissue and critical review of the manuscript. We are grateful to Vincent Schram from the NICHD microscopy and imaging core (MIC) for expert assistance with confocal and bright-field microscopy. The authors thank Prof. Dr. Andreas Zimmer (University Bonn) for his insightful comments and suggestions.
Compliance with Ethical Standards
Conflict of Interest
L. E. and A.B. declare no competing financial interests. MX.H., A.L. and E.P. are employed by Advanced Cell Diagnostics.
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