Fig. 1: Spatiotemporal analysis of mDA neuron development.

a Experimental design of the study. Following analysis of the developmental trajectory of mDA neurons, transcriptomic profiling was performed to (I) compare mDA neurons (Pitx3-GFP+) and cells in their environment (GFP-) at E14, and (II) to determine RNA expression at several key developmental stages. Based on these observations circRNA expression and function were studied in more detail. b Immunohistochemistry of E14, E16 and P0 Pitx3-ITC:ACTB-Flp coronal vibratome sections. At P0, mDA dendrites reach into SNr. A few mDA neurons are present in the SNr (TH⁺/ITC^- = orange arrow, TH⁺/ITC⁺ = white arrow). Dashed line indicates SNc/SNr boundary. Scale bars: column 1 = 100 µm, columns 2 and 3 = 20 µm. Representative images from n = 1. c Whole-mount immunostaining for TH followed by 3DISCO tissue clearing and FLSM of E12, E13, E15 and E18 WT brains. White arrows indicate TH⁺ axons growing towards the Hb. Scale bars: E12 and E13 = 100 µm, E15 = 150 µm, E18 = 200 µm. Representative images from n = 1. d Schematic overview of key developmental events in mDA neuron development based on published literature and results from the present study. Key events include floorplate division¹⁰⁶, mDA neuron specification into SNc and VTA subsets⁶⁴, axon extension towards the forebrain¹⁰⁷, re-orientation of mDA neuron axis during migration, formation of the SNr, first Ca²⁺ events, mDA neurons in SNr (visible until adolescence), fasciculation of mDA dendrites in SNr and formation of ‘dendrons’³⁸, first Na⁺ spikes⁴⁵, first DA release into the striatum⁴⁵ and apoptotic waves (around P0 and between P15 and P20)¹⁰⁸. Ctx Cortex, dMb dorsal Midbrain, E embryonic day, mDA midbrain dopamine, MFB medial forebrain bundle, P postnatal day, SNc Substantia nigra pars compacta, SNr Substantia nigra pars reticulata, Str Striatum; TH Tyrosine hydroxylase, vMB ventral Midbrain, VTA Ventral tegmental area. Embryo and cell dish icons were from BioRender.com.