Single Cell Transcriptomic Analysis of Spinal Dmrt3 Neurons in Zebrafish and Mouse Identifies Distinct Subtypes and Reveal Novel Subpopulations Within the dI6 Domain

Abstract

The spinal locomotor network is frequently used for studies into how neuronal circuits are formed and how cellular activity shape behavioral patterns. A population of dI6 interneurons, marked by the Doublesex and mab-3 related transcription factor 3 (Dmrt3), has been shown to participate in the coordination of locomotion and gaits in horses, mice and zebrafish. Analyses of Dmrt3 neurons based on morphology, functionality and the expression of transcription factors have identified different subtypes. Here we analyzed the transcriptomes of individual cells belonging to the Dmrt3 lineage from zebrafish and mice to unravel the molecular code that underlies their subfunctionalization. Indeed, clustering of Dmrt3 neurons based on their gene expression verified known subtypes and revealed novel populations expressing unique markers. Differences in birth order, differential expression of axon guidance genes, neurotransmitters, and their receptors, as well as genes affecting electrophysiological properties, were identified as factors likely underlying diversity. In addition, the comparison between fish and mice populations offers insights into the evolutionary driven subspecialization concomitant with the emergence of limbed locomotion.

Adult spinal Dmrt3 neurons receive direct somatosensory inputs from ipsi‐ and contralateral primary afferents and from brainstem motor nuclei

“…Therefore, interneurons that connect to group Ia and group II afferents are presumably separate populations. Dmrt3 neurons can be subclassified based on their expression of other markers (Andersson et al., 2012 ; Iglesias González et al., 2021 ; Schnerwitzki et al., 2018 ); thus, it will be important to examine how the different types of inputs can be functionally linked to Dmrt3 interneuron subpopulations. Furthermore, it is known that descending tracts connect to Ia interneurons (Hultborn et al., 1976 ), as well as to lamina VIII commissural interneurons (Bannatyne et al., 2003 ).…”

“…Dmrt3 neurons in the dI6 mouse spinal cord population have previously been divided into functional subtypes (Griener et al., 2017 ; Perry et al., 2019 ). A recent single cell sequencing study divided the spinal Dmrt3 neurons from mice into six clusters (M1–6) based on their transcriptomic profile (Iglesias González et al., 2021 ). Most clusters, except M1, express the 5HT–7 receptor, providing some of the molecular set up required to receive descending serotonergic input from the Raphe nuclei, and it has been reported that interneurons in laminae VII and VIII are likely sites for serotonergic modulation of the centrally activated locomotor network (Noga et al., 2009 ).…”

“…Our analysis suggests that also Dmrt3 inhibitory interneurons of the dI6 cardinal class contribute to the further transmission of proprioceptive signaling. It is also clear, however, that the Dmrt3 population is not homogenous; thus, refined techniques to provide a new level of detail regarding the composition of the Dmrt3 population (Iglesias González et al., 2021 ) will be required to tease out the properties of each of these subpopulations of spinal Dmrt3 interneurons.…”

“…In mice, Dmrt3‐expressing spinal interneurons are inhibitory and mainly commissural (Iglesias González et al., 2021 ; Perry et al., 2019 ). The role of these neurons in locomotor circuits, together with previous findings indicating that the population has a direct connection to motor neurons and a fast response to dorsal root stimulation, suggests an involvement in spinal sensorimotor reflex circuits (Perry et al., 2019 ).…”

Spinal cords: Symphonies of interneurons across species

“…However, expression of WT1, together with GABAergic and glycinergic neurotransmitters, define a broader population of dI6 neurons in the mouse ( Figure 6B ; Goulding, 2009 ; Andersson et al, 2012 ; Haque et al, 2018 ). This population can be further divided into subtypes based on morphology, electrophysiology, neurotransmitters, birth order, transcription factors and axon guidance gene expression ( Andersson et al, 2012 ; Griener et al, 2017 ; Schnerwitzki et al, 2018 ; Perry et al, 2019 ; Kishore et al, 2020 ; Iglesias González et al, 2021 ).…”