Serotonin neuron development: shaping molecular and structural identities

Abstract

The continuing fascination with serotonin (5-hydroxytryptamine, 5-HT) as a nervous system chemical messenger began with its discovery in the brains of mammals in 1953. Among the many reasons for this decades-long interest is that the small numbers of neurons that make 5-HT influence the excitability of neural circuits in nearly every region of the brain and spinal cord. A further reason is that 5-HT dysfunction has been linked to a range of psychiatric and neurological disorders many of which have a neurodevelopmental component. This has led to intense interest in understanding 5-HT neuron development with the aim of determining whether early alterations in their generation lead to brain disease susceptibility. Here, we present an overview of the neuroanatomical organization of vertebrate 5-HT neurons, their neurogenesis, and prodigious axonal architectures, which enables the expansive reach of 5-HT neuromodulation in the central nervous system. We review recent findings that have revealed the molecular basis for the tremendous diversity of 5-HT neuron subtypes, the impact of environmental factors on 5-HT neuron development, and how 5-HT axons are topographically organized through disparate signaling pathways. We summarize studies of the gene regulatory networks that control the differentiation, maturation, and maintenance of 5-HT neurons. These studies show that the regulatory factors controlling acquisition of 5-HT-type transmitter identity continue to play critical roles in the functional maturation and the maintenance of 5-HT neurons. New insights are presented into how continuously expressed 5-HT regulatory factors control 5-HT neurons at different stages of life and how the regulatory networks themselves are maintained. WIREs Dev Biol 2018, 7:e301. doi: 10.1002/wdev.301 This article is categorized under: Nervous System Development > Vertebrates: General Principles Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Nervous System Development > Secondary: Vertebrates: Regional Development.

Figure 1. Anatomy of developing 5-HT neurons in the mouse brain

3-D imaging of whole E12.5 mouse embryos. Tissue was fixed, immunostained in toto with 5-HT antisera and imaged with light sheet microscopy after tissue clarification. A) Dorsal view of the embryo, showing the anterior (B4–B9) and posterior (B1–B3) cell clusters. At this stage 5-HT neurons form two continuous parasagittal bands on either side of the midline. B) Higher magnification of the same embryo shows the 3D organization of cell bodies and the 5-HT efferent fiber tracts (arrowheads) that are directed both rostrally or caudally. The tentative position of earlier rhombomeric divisions (r1–r7) is indicated with different colors. Note the absence of 5-HT-labeled cells and axons in the brainstem segment corresponding to r4 at this embryonic stage.

Figure 2. Maturation gradient of serotonin neurons

A) Progressive midline fusion of the newborn 5-HT neurons in rat embryonic brains. The scheme indicates the different anteroposterior levels of sectioning in the hindbrain shown as red lines (1,2,3,4) that are illustrated in the micrographs. The position of the 5-HT neurons is indicated as green dots. Micrographs on the right show 5-HT immunostained sections at E13, E15 and E18 at these different rostral to caudal levels. Note that 5-HT cell groups are initially entirely separated by floor plate cells at E13. The floor plate starts to be invaded by outgrowing neurites at E15 at rostral levels (1, 2), midline fusion of the anterior cluster neurons begins at E18, while the fusion of the cells in the posterior cluster (3,4) is achieved postnatally. Pictures were taken from Wallace and Lauder, 1983. B) The rostral to caudal sequential appearance and maturation of 5-HT neurons is best seen on whole mounts of rat embryonic hindbrains immunostained with 5-HT. The drawings were taken from Aitken and Törk 1988 (41).

Figure 3. Diversity of 5-HT neuron identities

A) The identities of 5-HT neurons comprise common and subtype-specific characteristics. Common characteristics include 5-HT pathway terminal effector proteins for 5-HT synthesis (Tryptophan hydroxylase 2, TPH2; Aromatic amino acid decarboxylase, AADC; Guanosine triphosphate cyclohydrolase, GTPCH; 6-pyruvoyl-tetrahydropterin synthase, PTPS; Sepiapterin reductase, SR), reuptake (Serotonin transporter, SERT, Slc6a4) and vesicular transport (Vesicular monoamine transporter 2, VMAT2, Slc18a2). Activation of the genes encoding these proteins endows newborn neurons with a 5-HT-type transmitter identity. Subtype-specific characteristics are expressed in subsets of 5-HT neurons and thus diversify 5-HT neuron molecular identity. Various peptides are well-established subtype-specific features of 5-HT neurons. A large number of other characteristics such as GPCRs, transporters, ion channels and TFs further diversify 5-HT neurons. Some examples are depicted: Vesicular glutamate transporter 3, VGLUT3 (Slc17a8); preprotachykinin, TKN1 (Substance P); lysophosphatidic acid 1 receptor, LPAR1; engrailed1 (homeodomain TF), EN1. Black squares indicate the numerous other 5-HT subtype specific proteins that diversify 5-HT neurons (see (–60). B) Single cell RNAseq and single molecule FISH revealed that virtually all r2-derived Pet1⁺ precursors of the MRN give rise to 5-HT neurons but with negatively correlated levels of Tph2 and Slc17a8. Thus, two subtypes with either high Tph2/low Slc17a8 or low Tph2/high Slc17a8 expression are present in the r2-derived MRN. In contrast, Pet1⁺ precursors give rise to 5-HT neurons in the DRN with more uniform levels of TPH2; some of these 5-HT neurons express VGLUT3 (Slc17a8). See (58, 126).

Figure 4. Development of 5-HT axons

A-1, Scheme of E16 rat embryo, (adapted from 24). Ascending 5-HT axons grow rapidly between E12 and E16, following different preexisting axonal tracts such as the medial forebrain bundle (mfb), the mamillotegmental tract (mtg), the supracallosal striae (scs), external capsule (ec) and the stria medullaris (sm). Subsequent axon terminal branching and invasion of targets is a slower protracted process that continues late into early postnatal life. Axon guidance molecules such as WNT and SLIT1/SLIT2 have been implicated in directing this growth. A-2, The onset of 5-HT terminal Innervation in different brain structures shows marked regional differences. This is schematized as triangles with different shadings along the developmental stage E16 to P21. Light green = early target invasion; dark green = late target invasion. 5-HT fiber ingrowth has been found to be modulated by several factors, namely GAP43, STOP, 5-HT, protocadherin alpha, and ephrinAs.

Figure 5. 5-HT neuron gene regulatory networks

Depicted regulatory interactions (arrows) at the indicated stages of life are based on germ line or conditional loss of function data for each indicated TF. Terminal effector genes are depicted in rectangles and transcription factors in ovals. 5-HT pathway genes encoding 5-HT synthesis (Tph2, Ddc, Gch1, Qdpr), reuptake, (Slc6a4, Slc22a3), vesicular transport (Slc18a2) and metabolism (Maoa, Maob) are shown in rectangles with black letters. Terminal effector genes depicted in blue letters (Gria4, Htr1a, Htr1b, Adra1b, Lpar1, Hcrtr1) encode AMPA receptor subunit GLUR4 and GPCRs required for responses to diverse synaptic inputs. Arrows indicate that deficiency of a specific TF results in complete or partial loss of gene expression of a particular target gene in some or nearly all 5-HT neurons in which the TF expressed. Red blunted line from Pet1 to Hcrtr1 indicates that loss of Pet1 leads to upregulation of Hcrtr1. Slc17a8 (VGLUT3) is a subtype-specific terminal effector of 5-HT neurons. Homeobox gene, Engrailed1, (En1) encodes a 5-HT subtype specific TF as it is expressed only in r1-derived 5-HT and non-5-HT neurons but not at more posterior hindbrain levels. Dashed line indicates that Lmx1b controls maintenance of Pet1 at fetal stage. Solid circles indicate that evidence in support of direct regulation of a TF or terminal effector gene by a particular TF was obtained by chromatin immunoprecipitation (ChIP)–PCR or ChIP sequencing. In the case of Htr1a, evidence in support of direct regulation by Pet1 was obtained with cell line reporter assays and in vitro mobility shift DNA binding assays (187). Further ChIP studies may reveal PET1 occupancy at the other PET1 regulated effector genes shown in the scheme. Chromatin immunoprecipitation has not been reported for LMX1B in 5-HT neurons. Red curved arrow indicates positive autoregulation by Pet1.

Figure 6. Temporally dynamic Pet1 target dependencies in postmitotic 5-HT neurons

Stage-specific conditional targeting with AAV-Cre and tamoxifen-inducible approaches reveal changing sensitivities of target genes to loss of Pet1 in postmitotic 5-HT neurons. Target sensitivities to Pet1 loss (y-axis) are presented in arbitrary scale based on RT-qPCR and in situ hybridization assays of target gene expression. Dashed line segments indicate that no data is available for early postnatal targeting of Pet1. Slc6a4, high affinity, low capacity 5-HT plasma membrane transporter; Slc22a3, low affinity, high capacity 5-HT plasma membrane transporter; Slc18a2, vesicular monoamine transporter 2; Tph2, tryptophan hydroxylase 2; Gch1, GTP cyclohydrolase 1; Htr1a, 5-HT1a receptor; Maob, monoamine oxidase b.

Figure 7

Schematic representation of the ascending 5-HT raphe axons from the DRN (B7) and the MRN (B5–B8). Different patterns of altered 5-HT terminal innervation are noted in Gap43^−/− (164), Tph2^−/− (90), ephrin A5^−/− (179), and Pcdhα ^ΔCR/ΔCR (176), reflecting the requirement of trophic (5-HT) or guidance molecules for the establishment of the 5-HT raphe circuits.