Dynamic Functional Pathway Development in Type 1 Spinal Interneurons: Stage-specific roles of retinoic acid activity

Abstract

BackgrounddI1 spinal interneuron development requires precise coordination of proliferation, differentiation, and migration, yet the molecular and cellular properties underlying the temporal coupling of these processes remain unresolved. ResultsSingle-cell RNA sequencing of the E4 quail neural tube under control and retinoic acid (RA)-deprived conditions generated a high-resolution atlas spanning a continuous trajectory from dorsal progenitors to differentiated neurons. Pseudotime and RNA velocity analyses revealed transitions consistent with medial-to-lateral displacement in vivo. Early progenitors expressed cell cycle and BMP-associated programs, followed by induction of cytoskeletal and extracellular matrix remodelling genes and later activation of pan-neuronal and synaptic pathways. BMP signaling declined during differentiation through upregulation of BMP antagonists, whereas apoptosis-related genes were poised during specification without overt cell death. Autophagy-associated genes peaked during differentiation. Disruption of RA signaling by dominant-negative RAR403 uncovered stage-specific functions of RA. In progenitors, RA inhibition increased proliferation. In differentiating neurons, it upregulated BARHL1/2 and enhanced ion channel, synaptic, cytoskeletal, and extracellular matrix gene programs. RA inhibition also impaired ventral displacement of BarHL2-positive dI1 neurons, causing abnormal positioning. ConclusionsThese findings define a dI1-centered single-cell atlas of the progenitor-to-neuron transition and identify stage-specific roles for RA in dorsal hinterneuron maturation. Key findingsO_LIWe provide a high-resolution single-cell atlas of dI1 spinal interneuron development in the E4 quail neural tube, analysis of this single stage reveals a continuous trajectory from proliferative dorsal progenitors to differentiated neurons. C_LIO_LIDevelopment along this trajectory is tightly staged: early cells are dominated by cell-cycle and progenitor-maintenance programs, while later cells progressively activate cytoskeletal, extracellular matrix, axon-guidance, synaptic, and mature neuronal pathways. C_LIO_LIBMP signaling is strongest in early dI1 progenitors and is progressively attenuated during differentiation through the induction of BMP inhibitors, supporting a stage-specific role for BMP in early specification rather than late maturation. C_LIO_LIRetinoic acid acts in a stage-dependent manner: RA inhibition increases progenitor proliferation, alters differentiation-associated gene programs, and upregulates neuronal maturation, ion-channel, and synaptic genes in differentiating neurons. C_LIO_LIRepressing RA signaling causes BarHL2-positive dI1 neurons to fail their normal ventral migration. Instead, they accumulate near their dorsal origin and show ectopic localization, indicating that RA is required for proper migration. C_LI