Motor neurons are dispensable for the assembly of a sensorimotor circuit for gaze stabilization

Sensorimotor reflex circuits engage distinct neuronal subtypes, defined by precise connectivity, to transform sensation into compensatory behavior. Whether and how motor neuron populations specify the subtype fate and/or sensory connectivity of their pre-motor partners remains controversial. Here, we discovered that motor neurons are dispensable for proper connectivity in the vestibular reflex circuit that stabilizes gaze. We first measured activity following vestibular sensation in pre-motor projection neurons after constitutive loss of their extraocular motor neuron partners. We observed normal responses and topography indicative of unchanged functional connectivity between sensory neurons and projection neurons. Next, we show that projection neurons remain anatomically and molecularly poised to connect appropriately with their downstream partners. Lastly, we show that the transcriptional signatures that typify projection neurons develop independently of motor partners. Our findings comprehensively overturn a long-standing model: that connectivity in the circuit for gaze stabilization is retrogradely determined by motor partner-derived signals. By defining the contribution of motor neurons to specification of an archetypal sensorimotor circuit, our work speaks to comparable processes in the spinal cord and advances our understanding of general principles of neural development.

;Tg(UAS-E1b:Kaede);Tg(isl1:GFP).Gate A excluded presumptive debris (small cells).Gate B isolated single cells and excluded large cells and doublets.Gate C excluded DAPI+ (dead or unhealthy) neurons.Gate D isolated fluorescent (GFP or Kaede+) neurons; neurons in this gate were sorted.Gates were set using negative controls (not shown; Methods).Gates shown for one of four experimental repeats.(A) Volcano plot showing differentially expressed genes across an unfiltered bulk RNA sequencing dataset.Dashed lines represent significance cutoffs: horizontal line, p adjusted>0.05; vertical line, Log 2 Fold Change > 2.0.Each circle is a gene.Genes to the left and right of 0 on the horizontal axis show downregulated and upregulated genes, respectively.Red color shows genes that are differentially expressed in a filtered subset of projection neurons (Figure 5).Grey-colored genes are below both significance thresholds.(B) Same data as Figure S5A, now highlighting candidate genes evaluated by fluorescent in situ (Figure 5) with red.One candidate (yellow) that did not meet projection neuron filter criteria (Methods) is shown in Figure S5D; remaining candidates (included in filtered data) shown in Figure 5F.(C) Same data as Figure S5A-Figure S5B, showing the number of differentially expressed genes at progressive significance thresholds (p adjusted).Red and blue lines show the number of significantly upregulated and downregulated genes, respectively.(D) Fluorescent in situ hybridization against a candidate gene, slc22a7a (log 2 fold change=10.2,p adj.=1.6x10 -4 ), that did not meet projection neuron filter criteria.Percentage refers to fraction of projection neurons from a single-cell sequencing dataset with expression (Methods).Left columns show RNA (green); right columns, merge with projection neurons labeled with Tg(-6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS-E1b:Kaede) (grey).Dashed lines outline the projection nucleus.Cell and transcript expression outside the projection nucleus is removed for visual clarity.All scale bars, 20 µm.

Figure S2 :
Figure S2: Flow cytometry gating strategy to sort fluorescently-labeled neurons for bulk RNA sequencing.Associated with Figure 5.(A) Sequential gates used to sort fluorescent neurons labeled with Tg(-6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS-E1b:Kaede);Tg(isl1:GFP).Gate A excluded presumptive debris (small cells).Gate B isolated single cells and excluded large cells and doublets.Gate C excluded DAPI+ (dead or unhealthy) neurons.Gate D isolated fluorescent (GFP or Kaede+) neurons; neurons in this gate were sorted.Gates were set using negative controls (not shown; Methods).Gates shown for one of four experimental repeats.

Figure S3 :
Figure S3: Molecular identification of projection neurons using a reference single-cell RNA sequencing atlas.Associated with Figure 5. (A) UMAP visualization of a single-cell RNA sequencing atlas of n=1,468 neurons labeled in Tg(-6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS-E1b:Kaede), generated with 10x Genomics (Methods).Each circle is a single neuron.Neurons are clustered (colors) according to their transcriptional identity.Annotations are based on validated marker genes (data not shown).TAN, tangential nucleus; MVN, medial vestibular nucleus; r, rhombomere; MNs, motor neurons; inhib, inhibitory neurons.(B) Heatmap showing genes unique to each annotated cluster.Each row is a gene; names unlisted for clarity.Columns show distinct clusters.Color bar on top reflects clusters in Figure S3A.Yellow and purple reflect stronger or weaker gene expression, respectively.(C) Fluorescent in situ hybridization against three markers (pou4f1, penkb, chrna2b) that are negative for tangential nucleus projection neurons and positive for medial vestibular nucleus neurons.Top row shows RNA expression (green); bottom row, merge with neurons labeled in Tg(-6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS-E1b:Kaede).Dashed lines outline the tangential nucleus (TAN) and medial vestibular nucleus (MVN).Data from 72 hpf larvae.Images shown in an axial view.(D) Fluorescent in situ hybridization against a positive marker (evx2) for both tangential nucleus and medial vestibular nucleus neurons.All scale bars, 20 µm.(E) Heatmap showing genes unique to tangential and medial vestibular neurons.Clusters identified using positive and negative fluorescent in situ data from Figure S3C-Figure S3D and unpublished data.

Figure S5 :
Figure S5: Differential gene expression in an unfiltered bulk sequencing dataset of siblings and phox2a mutants.Associated with Figure 5.(A) Volcano plot showing differentially expressed genes across an unfiltered bulk RNA sequencing dataset.Dashed lines represent significance cutoffs: horizontal line, p adjusted>0.05; vertical line, Log 2 Fold Change > 2.0.Each circle is a gene.Genes to the left and right of 0 on the horizontal axis show downregulated and upregulated genes, respectively.Red color shows genes that are differentially expressed in a filtered subset of projection neurons (Figure5).Grey-colored genes are below both significance thresholds.(B) Same data as FigureS5A, now highlighting candidate genes evaluated by fluorescent in situ (Figure5) with red.One candidate (yellow) that did not meet projection neuron filter criteria (Methods) is shown in FigureS5D; remaining candidates (included in filtered data) shown in Figure5F.(C) Same data as FigureS5A-FigureS5B, showing the number of differentially expressed genes at progressive significance thresholds (p adjusted).Red and blue lines show the number of significantly upregulated and downregulated genes, respectively.(D) Fluorescent in situ hybridization against a candidate gene, slc22a7a (log 2 fold change=10.2,p adj.=1.6x10 -4 ), that did not meet projection neuron filter criteria.Percentage refers to fraction of projection neurons from a single-cell sequencing dataset with expression (Methods).Left columns show RNA (green); right columns, merge with projection neurons labeled with Tg(-6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS-E1b:Kaede) (grey).Dashed lines outline the projection nucleus.Cell and transcript expression outside the projection nucleus is removed for visual clarity.All scale bars, 20 µm.

Figure S6 :
Figure S6: phox2a expression in the medial vestibular nucleus may underscore differential gene expression phenotypes in bulk data.Associated with Figure 5. (A) Fluorescent in situ hybridization against phox2a in a 5 dpf larvae (axial view).Top panel shows phox2a RNA (green); bottom panel, merge with neurons visualized with Tg(isl1:GFP);Tg(-6.7Tru.Hcrtr2:GAL4-VP16(;Tg(UAS-E1b:Kaede) (grey).White dashed lines outline three nuclei of interest: projection neurons in the tangential nucleus (TAN), the medial vestibular nucleus (MVN), and the facial nucleus (nVII).All scale bars, 20 µm.(B) Volanco plot showing differentially expressed genes in medial vestibular nucleus neurons between control and phox2a null larvae at 3 dpf.Dashed lines represent significance cutoffs: horizontal line, p>0.05; vertical line, Log 2 Fold Change > 2.0.Each circle is a gene.Genes to the left and right of 0 on the horizontal axis show downregulated and upregulated genes, respectively.Colors indicate percent of reference medial vestibular neurons (Methods) that express a given gene.Grey-colored genes are below both significance thresholds.(C) Same data as Figure S6B.Color shows genes that are differentially expressed in both medial vestibular nucleus neurons and projection neurons.