Abstract
Midbrain dopamine (DA) neurons exist as several subtypes and are found in a heterogeneous environment including GABAergic and glutamatergic neurons as well as various types of co-releasing neurons. Developmental programs underlying this heterogeneity have remained elusive. In this study, combinatorial mRNA analysis was performed at stages when neuronal phenotypes are first specified. Vesicular transporters for dopamine and other monoamines (VMAT2), GABA (VIAAT), and glutamate (VGLUT2) were assessed by systematically applying fluorescent in situ hybridization through the mes-di-encephalon of the mouse embryo at embryonal days (E) 9.5–14.5. The results show that early differentiating dopamine neurons express the gene encoding VGLUT2 before onset of any dopaminergic markers. Prior to its down-regulation in maturing dopamine neurons, Vglut2 mRNA co-localizes extensively with Tyrosine hydroxylase (Th) and Nurr1, commonly used as markers for DA neurons. Further, Vglut2 and Nurr1 mRNAs are shown to overlap substantially in diencephalic neurons that maintain a glutamatergic phenotype. The results suggest that Vglut2/Nurr1-double positive cells give rise both to dopaminergic and glutamatergic neurons within the mes-di-encephalic area. Finally, analysis of markers representing subtypes of dopamine neurons, including the newly described NeuroD6 subtype, shows that certain subtype specifications arise early. Histological findings are outlined in the context of neuroanatomical concepts and the prosomeric model of brain development. The study contributes to the current decoding of the recently discovered heterogeneity among neurons residing along the cephalic flexure.
Highlights
The midbrain dopamine (DA) system, originally described in the 1960’s, is a key brain substrate at the intersection between emotional, cognitive, and motor functions
Schematic illustrations of the histological findings described at E14.5 in mes-di-encephalic brain area, with each mRNA analyzed in the current study compared to Tyrosine hydroxylase (Th) and how they relate to Vglut2 and Th within the prosomeric model, show how gene expression patterns overlap within differentiating mes-di-encephalic neurons as they adopt a dopaminergic and/or glutamatergic phenotype that together represent distinct subregions or distinct prosomeres
By addressing combinations of some newly described and some more established gene expression profiles implementing high spatial resolution analysis at the embryonal stages when DA neurons subtypes are first formed and specified, some unexpected insights into the development of midbrain DA (mDA) neurons and their neighboring cells were reached: Firstly, we identify Vglut2 expression in the absolute majority of neurons destined to become dopaminergic prior to their expression of Nurr1 and Th, suggesting that Vglut2 is a marker of early, non-differentiated DA neurons, the function of which remains to be explored
Summary
The midbrain dopamine (DA) system, originally described in the 1960’s, is a key brain substrate at the intersection between emotional, cognitive, and motor functions. Through technical advancements allowing for mRNA sequencing from single cells (scRNAseq), analysis of stem cell, murine and human DA neurons has led to the identification of molecularly defined subtypes of both developing and mature DA neurons (Poulin et al, 2014; La Manno et al, 2016; Kee et al, 2017; Tiklová et al, 2019). These findings will likely form the foundation for a new generation of knowledge of how to generate and to distinguish between mDA neurons. Findings are outlined and discussed in the context of common anatomical concepts and the more recently described prosomeric, segmental, model of brain development
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