Distinct transcriptomes define rostral and caudal serotonin neurons

Abstract

The molecular architecture of developing serotonin (5HT) neurons is poorly understood, yet its determination is likely to be essential for elucidating functional heterogeneity of these cells and the contribution of serotonergic dysfunction to disease pathogenesis. Here, we describe the purification of postmitotic embryonic 5HT neurons by flow cytometry for whole-genome microarray expression profiling of this unitary monoaminergic neuron type. Our studies identified significantly enriched expression of hundreds of unique genes in 5HT neurons, thus providing an abundance of new serotonergic markers. Furthermore, we identified several hundred transcripts encoding homeodomain, axon guidance, cell adhesion, intracellular signaling, ion transport, and imprinted genes associated with various neurodevelopmental disorders that were differentially enriched in developing rostral and caudal 5HT neurons. These findings suggested a homeodomain code that distinguishes rostral and caudal 5HT neurons. Indeed, verification studies demonstrated that Hmx homeodomain and Hox gene expression defined an Hmx(+) rostral subtype and Hox(+) caudal subtype. Expression of engrailed genes in a subset of 5HT neurons in the rostral domain further distinguished two subtypes defined as Hmx(+)En(+) and Hmx(+)En(-). The differential enrichment of gene sets for different canonical pathways and gene ontology categories provided additional evidence for heterogeneity between rostral and caudal 5HT neurons. These findings demonstrate a deep transcriptome and biological pathway duality for neurons that give rise to the ascending and descending serotonergic subsystems. Our databases provide a rich, clinically relevant resource for definition of 5HT neuron subtypes and elucidation of the genetic networks required for serotonergic function.

Figure 1.

ePet-EYFP expression marks rostral and caudal 5HT neurons in the embryonic ventral hindbrain. A, Whole-mount view of an ePet-EYFP transgenic brain at E14.5. Both rostral and caudal 5HT neuronal cell bodies and axonal projections were brightly marked by EYFP fluorescence. A′, High-magnification view (8×) of caudal 5HT axonal projections to the spinal cord. B–D, Tiled confocal z-stack images of 5HT immunoreactivity (red) and EYFP fluorescence at E12.5 (B, sagittal view) and at E14.5 in the caudal (C, sagittal view) and rostral (D, coronal view) domains. Scale bars: B, 100 μm; C, D, 200 μm.

Figure 2.

Purification and expression profiling of rostral and caudal 5HT neurons. A, E12.5 ePet-EYFP neural tubes were dissected and incisions made at the mesencephalic flexure, pontine flexure, and spinal cord to separate the rostral and caudal domains. B, Single-cell suspensions of rostral and caudal neural tubes were subjected to FACS. EYFP ⁺ cells were collected in gate P7 (top) and nonfluorescent cells were collected in gate P8 (bottom). The fluorescent and nonfluorescent fractions were separated by one log unit to ensure pure populations for gene expression profiling. C, Sorting efficiency was verified by examining purified cells under fluorescence (top) and phase-contrast (bottom) microscopy. D, RNA was isolated from rostral YFP⁺ (R⁺), rostral YFP⁻ (R⁻), caudal YFP⁺ (C⁺), and caudal YFP⁻ (C⁻) sorted cells and then assayed by Taqman RT-PCR for two known 5HT neuron-specific genes, Fev and Tph2. E, Total RNA from 200,000 sorted cells each for rostral 5HT YFP⁺ (R⁺), rostral non-5HT (R⁻), caudal 5HT YFP⁺ (C⁺), and caudal non-5HT (C⁻) was used for one round of amplification to generate cRNA probes. F, G, Enriched expression of well established highly expressed (F) and more moderately expressed (G) markers of 5HT neurons were detected in R⁺ and C⁺ neurons by microarray hybridization. The y-axis equals the gene expression level determined by MAS 5.0. Error bars represent SEM. Scale bars: A, C, 100 μm.

Figure 3.

Unsupervised hierarchical clustering of serotonergic gene expression. A, Dendrogram showing unsupervised hierarchical clustering of R⁺, C⁺, R⁻, and C⁻ gene expression profiles from the 12 arrays. Biological replicates show a high degree of reproducibility. B, Heat map displays eight distinct clusters of enriched gene expression in R⁺, C⁺, R⁻, and C⁻ cell populations at E12.5. Each row represents the relative levels of expression for a single gene. The red or green color indicates high or low expression, respectively. Each column shows the expression profile for a single biological sample. Cluster I, Enriched gene expression in rostral 5HT and caudal 5HT neurons (R⁺C⁺); cluster II, enriched gene expression in rostral 5HT neurons (R⁺); cluster III, enriched gene expression in rostral 5HT and rostral non-5HT neurons (R⁺R⁻); cluster IV, enriched gene expression in caudal 5HT neurons (C⁺); cluster V, enriched gene expression in caudal 5HT and caudal non-5HT neurons (C⁺C⁻); cluster VI, enriched gene expression in rostral non-5HT neurons (R⁻); cluster VII, enriched gene expression in caudal non-5HT neurons (C⁻); and cluster VIII, enriched gene expression in rostral and caudal non-5HT neurons (R⁻C⁻).

Figure 4.

Verification of 5HT neuron-enriched gene expression. A, B, Taqman RT-PCR verified 5HT neuron-enriched expression of transcription factors Rnf112, Zcchc12, Tox and RIKEN genes 2010204K13Rik, AW551984, and 1700042O10Rik. C, Double-label immunohistochemistry for EYFP (brown) and in situ hybridization for AW551984 transcript (purple) verified the enriched expression of est AW551984 in 5HT neurons. D–F, Coimmunohistochemical staining of E12.5 neural tube confirmed FoxP1 (E) expression in YFP⁺ (D) 5HT neurons (F, overlay). Scale bars: C, 40 μm; D–F, 100 μm.

Figure 5.

Verification of rostral 5HT neuron-enriched HD gene expression. A, Taqman RT-PCR confirmed enriched expression of Hmx2, Hmx3, and Pou3f1 homeodomain genes in rostral (R⁺) but not caudal (C⁺) 5HT neurons. B–D, Double-label in situ hybridization (blue) and immunohistochemistry for EYFP (brown) confirmed expression of Hmx2 (B) and Hmx3 (C) in E12.5 rostral 5HT neurons. D, Hmx2 expression was maintained in rostral 5HT neurons at E16.5. E, Taqman RT-PCR verification of enriched En1 and En2 gene expression in E12.5 rostral 5HT neurons (R⁺) as well as non-5HT cells (R⁻ and C⁻) but not caudal 5HT neurons (C⁺). F, In situ hybridization for En2 at E12.5 shows a broad domain of expression on either side of the MHB. Immunohistochemical staining with a pan-engrailed antibody (red) detected En1 and En2 proteins in EYFP⁺ (green) 5HT neurons in rhombomere 1 at E12.5. G, En1/2 protein expression persisted in rostral 5HT neurons at E14.5. Scale bars: B, C, G, 50 μm; D, H, 100 μm; F, 500 μm.

Figure 6.

Verification of Hox gene expression in caudal 5HT neurons. A–C, Taqman RT-PCR confirmed the expression of Hox3 (A), Hox4 (B), and Hox5 (C) paralogues in caudal (C⁺) but not rostral (R⁺) 5HT neurons. D–L, Immunohistochemical staining for the Hox cofactors, Meis 1 and Meis 2, and Hox proteins. D–F, Meis1 and Meis2 (E) were detected in caudal 5HT neurons (D) with a pan-anti-Meis antibody (F, overlay). G–I, HoxC4 protein (H) was detected in caudal 5HT neurons (G, I, overlay). J–L, HoxA3 protein (K) was detected in most caudal 5HT neurons (J) at E12.5 (L, overlay). Scale bars: D–L, 50 μm.