phox2a  (Bioss)

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Associated with . ( A ) Schematic of vestibulo-ocular reflex circuitry and the genetic loss-of-function approach used to perturb motor-derived signals. ( B ) Fluorescent in situ hybridization showing phox2a transcript expression in statoacoustic ganglion sensory afferents (left), central projection neurons in the tangential nucleus (middle), or nIII/nIV extraocular motor neurons (right) at 5 days post-fertilization (dpf). Top: probe only, nuclei outlined with dashed lines. Bottom: probe (green) merged with somata, labeled by Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS-E1b:Kaede ) (sensory, central) or Tg(isl1:GFP ) (motor). ( C ) Schematic of CRISPR/Cas9 mutagenesis approach. Top: Red star shows location of guides against phox2a DNA. Bottom: RNA sequence in wildtype and phox2a d22 alleles. Red dashed lines show deleted sequence; ‘STOP’ box shows predicted premature stop codon due to deletion. Right shows predicted protein sequence. ( D ) Transmitted light image of a 5 dpf wildtype (top) and phox2a null mutant (bottom). White arrows point to a normally inflated (top) or absent (bottom) swim bladder. ( E ) Images of nIII/nIV motor neurons in one hemisphere, labeled by Tg(isl1:GFP ), in wildtype siblings (left) and phox2a null mutants (right) at 5 dpf. Scale bar, 20 µm. ( F ) Quantification of the number of Tg(isl1:GFP )+neurons in nIII/nIV from N=6 wildtype siblings and N=10 phox2a null mutants.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Derivative Assay, In Situ Hybridization, Expressing, Labeling, CRISPR, Mutagenesis, Sequencing

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: ( A ) Fluorescent in situ hybridization images of nIII/nIV motor neurons, labeled by Tg(isl1:GFP ), in wildtype larvae at 2 dpf. Panels from left to right show: (1) isl1 + motor neurons, (2) mRNA for vachta , a marker for cholinergic motor neurons, (3) DAPI, and (4) merge. White dashed lines show the extent of nIII/nIV. ( B ) Loss of both isl1 and vachta identity in nIII/nIV motor neurons in phox2a mutants at 2 dpf. All scale bars, 20 µm.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: In Situ Hybridization, Labeling, Marker

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: ( A ) Images of nIII/nIV motor neurons, labeled in Tg(isl1:GFP ), in wildtype siblings (left) and phox2a heterozygotes (middle) at 5 dpf. Wildtype image same as in . One hemisphere shown. White dashed lines outline the dorsal extent of nIII, which contains inferior rectus and medial rectus neurons . Scale bar, 20 µm. ( B ) Location of the earliest-born neurons in nIII/nIV (left, magenta) against all nIII/nIV neurons labeled in Tg(isl1:Kaede ) (right, grey). Larvae birthdated at 34 hpf (Materials and methods). One hemisphere shown. White dashed lines outline the dorsal extent of nIII. Scale bar, 20 µm. ( C ) Quantification of the number of Tg(isl1:GFP )+ neurons in nIII/nIV from N=6 wildtype siblings (grey) and N=8 phox2a heterozygotes (teal). Wildtype data same as . ( D ) Distributions showing probability of nIII/nIV soma location across each spatial axis in wildtype (black) and heterozygous (teal) phox2a larvae. Solid and shaded lines show mean and standard deviation, respectively, from bootstrapped data. Data from same fish quantified in C . ns, not significant; star, significant at the p<0.001 level.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Labeling, Standard Deviation

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Associated with . ( A ) Schematic of pitch vestibulo-ocular reflex circuitry. Dashed lines outline projection neurons as calcium imaging target. Nose-down/eyes-up channel represented with blue; orange, nose-up/eyes-down. ( B ) Schematic of tonic pitch-tilt stimulus delivered with Tilt-In-Place Microscopy (TIPM). Shaded regions show calcium imaging windows when fish were oriented horizontally immediately following tilts. Inset shows timecourse of the rapid step to restore horizontal position after tilts. Imaging experiments used larvae from the Tg(isl1:GFP);Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:GCaMP6s ) line. ( C ) Proportion of subtypes observed in sibling controls and phox2a null mutants. Blue: nose-down. Orange: nose-up. Grey: Neurons without directional tuning (criteria in Materials and methods). ( D, E ) Soma position of nose-down (blue) and nose-up (orange) neurons in sibling controls (left) and phox2a null mutants (right). Soma size scaled by strength of directional selectivity (min = 0; max = 1; see Materials and methods). ( F/I ) Heatmaps showing example tilt responses from nose-down ( F ) or nose-up ( I ) neurons in sibling controls (top) and phox2a null mutants (bottom). n=10 neurons with strongest ΔF/F responses to tilts shown. Each row shows an individual neuron. Shaded bars show calcium imaging window immediately following restoration from eccentric position. Black arrow points to first second of tilt response used for analyses. ( G/J ) Distributions of maximum ΔF/F responses to tilts for nose-down ( G ) or nose-up ( J ) neurons in sibling controls (black) and phox2a null mutants (red). Solid and shaded lines show mean and standard deviation, respectively, of bootstrapped data (Materials and methods). ( H/K ) Distributions of directional tuning score to tilts for nose-down ( H ) or nose-up ( K ) neurons in sibling controls (black) and phox2a null mutants (red). Tuning score ranges from 0 (equal responses to both tilt directions, no tuning) to 1 (responses to one tilt direction only); criteria detailed in Materials and methods. Solid and shaded lines show mean and standard deviation, respectively, of bootstrapped data.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Imaging, Microscopy, Standard Deviation

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Statistical comparisons of tilt responses across genotypes. WT (sampled) refers to an n=125 neuron subset, sampled with replacement from a reference dataset of wildtype projection neurons. Data shown is mean/standard deviation unless otherwise noted. p val generated from a one-way ANOVA with multiple comparisons. Associated with Figure 2 and Figure 3 .

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Generated

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Statistical comparisons of projection neuron topography across genotypes. WT (sampled) refers to an n=125 neuron subset, sampled with replacement from a reference dataset of wildtype projection neurons. Data shown is the median/standard deviation distance from the ventro-lateral and rostral edges of the tangential nucleus (total size: 40 µm across each spatial axis). p val from one-way ANOVA (individual spatial axes) or multivariate ANOVA (global organization), respectively.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques:

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Associated with . ( A ) Schematic of impulse tilt experiments. Yellow dashed lines outline projection neurons as calcium imaging target. Impulse-responsive neurons (ventrally-localized) shown with purple; unresponsive neurons, grey. ( B ) Schematic of impulse stimuli delivered with TIPM. Shaded regions show calcium imaging windows at horizontal immediately following impulses. Inset shows timecourse of impulse stimulus. Imaging experiments used larvae from the Tg(isl1:GFP);Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:GCaMP6s ) line. ( C ) Proportion of impulse-responsive (purple) and unresponsive (grey) neurons observed in sibling controls and phox2a null mutants. ( D ) Soma position of impulse-responsive neurons in sibling controls (left) and phox2a null mutants (right). Soma size scaled by strength of calcium response (ΔF/F), normalized by max observed ΔF/F. ( E ) Heatmaps showing example impulse responses from neurons in sibling controls (left) and phox2a null mutants (right). n=10 example neurons shown. Each row shows an individual neuron. Shaded bars show calcium imaging window immediately following impulse delivery. Black arrow points to first second of tilt response used for analyses. Note smaller scale (0–0.75) of impulse responses relative to . ( F ) Distributions of maximum ΔF/F responses to impulses in sibling controls (black) and phox2a null mutants (red). Solid and shaded lines show mean and standard deviation, respectively, from bootstrapped data. ( G ) Distributions of directional tuning score to impulses in sibling controls (black) and phox2a null mutants (red). Tuning score ranges from 0 (equal responses to both tilt directions, no tuning) to 1 (responses to one tilt direction only); criteria detailed in Materials and methods. Solid and shaded lines show mean and standard deviation, respectively, from bootstrapped data.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Imaging, Standard Deviation

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: ( A ) Schematic of retrograde photofill experiments. Projection neuron axons expressing the photolabile protein Kaede are targeted at the midbrain-hindbrain boundary with ultraviolet light. Converted protein (magenta) retrogradely diffuses to the soma, while the unconverted nucleus remains green. ( B ) Projection neuron somata in sibling controls (left) and phox2a null mutants (right) after retrograde photolabeling. Experiments performed at 5 dpf. Neurons visualized in Tg(isl1:GFP);Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:E1b-Kaede ). ( C ) Top two panels: Projection neuron axons at the hindbrain (inset) and midbrain-hindbrain boundary in sibling controls (top) and phox2a null mutants (bottom). Axons visualized using Tg(isl1:GFP);Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:E1b-Kaede ). White dashed outline (circle) shows arborization fields in nIII/nIV. Dashed box over axons shows location of two bottom panels. MHB and yellow dashed line, midbrain-hindbrain boundary. nucMLF: nucleus of the longitudinal fasciculus. Bottom two panels: Zoom of axons (dashed rectangle above). Spatial segregation between early-born (magenta +green) and late-born (green only) axons. White dashed line reflects separation between dorsal (nose-up, early-born) and ventral (nose-down, late-born) axon bundles. Image at 5 dpf in sagittal view. ( D ) Projection neuron axon bundle in a phox2a null mutant at 3 dpf. White arrows point to single collateral to two nIII/nIV neurons that were not eliminated following phox2a knockout. ( E ) Fluorescent in situ hybridization against RNA for three pre-synaptic markers: synaptophysin a ( sypa ; left), synaptic vesicle glycoprotein 2 ( sv2 , middle), and synapsin I ( syn1 , right). Top row, sibling controls. Bottom row, phox2a null mutants. For each panel set, left images show in situ probe expression (green) and right images show merge with projection neurons labeled in Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:E1b-Kaede ). Dashed lines outline the projection nucleus. Cell and transcript expression outside the projection nucleus is removed for visual clarity. Images taken at 5 dpf in sagittal mount. All scale bars, 20 µm.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Expressing, Mutagenesis, Knock-Out, In Situ Hybridization, In Situ, Labeling

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Associated with , , . ( A ) Schematic of sequencing approach. Central projection neurons ( Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:E1b-Kaede )) are harvested from 3 dpf larvae. Flow cytometry is used to exclude neurons not labelled by Tg(–6.7Tru.Hcrtr2:GAL4-VP16 ). Bulk RNA sequencing is performed to compare the profiles of projection neurons in siblings and phox2a null mutants. ( B ) Example of projection neurons before (left) and after (right) harvesting. Neurons visualized with Tg(isl1:GFP);Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:E1b-Kaede ). Dashed lines outline projection neurons in the tangential nucleus; dotted lines, medial vestibular nucleus. Yellow region shows margin of harvesting error: non-projection neurons that may be included in bulk sequencing dataset. ( C ) Number of differentially expressed genes in projection neurons at 3 dpf after applying progressive filters based on gene expression in a reference single-cell dataset. Data shown on logarithmic scale. Solid, dashed, and dotted lines represent differentially-expressed gene with p adjusted<0.5, p adjusted<0.01, or p adjusted<0.001 significance, respectively. ( D ) Volcano plot showing differentially expressed genes in projection 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 cells that express a given gene. Grey-colored genes are below both significance thresholds. ( E ) Same data as Figure 5D. Colored genes show eight candidates evaluated with fluorescent in situ hybridization: red, upregulated; blue, downregulated; yellow, highly-expressed controls ( evx2) . ( F ) Fluorescent in situ hybridization against candidate genes that met projection neuron filter criteria. Top row shows sibling controls; bottom row, phox2a null mutants. For each gene, left panels show RNA probe (green) and right panels show merge with projection neurons labeled by Tg(–6.7Tru.Hcrtr2:GAL4-VP16 ) (grey). Dashed lines outline the projection nucleus. Cell and transcript expression outside the projection nucleus is masked for visual clarity. Arrows denote whether genes are upregulated (red), downregulated (blue), or not significantly changed (yellow). Percentage refers to fraction of cells in a single-cell RNA sequencing reference atlas (Materials and methods) with detected transcript. Candidates: itga9 (log 2 fold change = 23.0, p adj.=3.9 × 10 –6 ), twf1b (log 2 fold change = 5.9, p adj.=0.024), p4hb (log 2 fold change = 5.1, p adj.=0.04), mapk6 (log 2 fold change = 5.1, p adj.=0.06), rxfp2a (log 2 fold change = −8.5, p adj.=1.1 × 10 –5 ), satb1a (log 2 fold change = −3.0, p adj.=0.001), evx2 (log 2 fold change = 0.46, p adj.=0.99), myt1la (log 2 fold change = 2.6, p adj.=0.44). All scale bars, 20 µm.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Sequencing, Flow Cytometry, RNA Sequencing Assay, Expressing, Control, In Situ Hybridization, Labeling

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Differentially expressed genes in projection neurons. Star indicates a gene was evaluated using fluorescent in situ hybridization. # symbol indicates a gene was also differentially expressed in adjacent phox2a -expressing medial vestibular neurons (see Figure 5—figure supplement 5 ). ”% of projection neurons with expression” refers to detection in a filtered subset of projection neurons from a single-cell reference atlas of neurons labeled in Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS-E1b:Kaede ) (Materials and methods, Figure 5—figure supplement 2 ). Genes sorted by p adjusted value. Data associated with Figure 5 .

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: In Situ Hybridization, Expressing, Labeling, Control

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: ( A-A’ ) Fluorescent in situ hybridization against itga9 for three sibling ( A ) or phox2a null mutant ( A’ ) larvae (72 hpf), imaged with identical conditions. Left column shows RNA (green); right column, merge with projection neurons visualized 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. Percentage (1.9%) refers to fraction of cells in a single-cell RNA sequencing reference atlas (Materials and methods) with detected transcript. All scale bars, 20 µm. ( B-B’ ) Fluorescent in situ hybridization against htt for three sibling ( B ) and phox2a mutant ( B’ ) larvae (72 hpf). ( C-C’ ) Fluorescent in situ hybridization against evx2 for three sibling ( C ) and phox2a mutant ( C’ ) larvae (72 hpf).

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: In Situ Hybridization, Mutagenesis, Expressing, RNA Sequencing Assay

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Top 50 expressed genes in an unfiltered bulk RNA sequencing dataset of phox2a siblings. ‘% of unfiltered 10 x neurons’ refers to gene detection in a single-cell atlas of neurons labeled in Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS-E1b:Kaede ) (n=1,468 neurons). ‘% of projection neurons’ refers to gene detection in a subset of the single-cell atlas containing projection neurons in the tangential nucleus (n=159 neurons). Data associated with Figure 5 .

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: RNA Sequencing Assay, Labeling, Expressing, Control

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Top 50 differentially expressed genes in an unfiltered bulk RNA sequencing dataset of phox2a siblings and null mutants. One star indicates a gene was retained in a filtered subset of projection neurons; %, evaluated using fluorescent in situ hybridization. ‘% of unfiltered 10 x neurons’ refers to gene detection in an unfiltered single-cell reference atlas of neurons labeled in Tg(–6.7Tru.Hcrtr2:GAL4-VP16 ); Tg(UAS-E1b:Kaede ) (n=1,468 neurons). Putative origin inferred from gene expression in the annotated 10 x dataset (Materials and methods, Figure 5—figure supplement 2 ). Genes sorted by p adjusted value. Data associated with Figure 5 .

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: RNA Sequencing Assay, In Situ Hybridization, Labeling, Expressing, Inhibition, Control

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: ( 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 phox2a -expressing 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 phox2a -expressing medial vestibular neurons (Materials and methods, ) that express a given gene. Grey-colored genes are below both significance thresholds. Data for all differentially expressed candidates in medial vestibular neurons shown in . ( C ) Same data as B . Color shows genes that are differentially expressed in both medial vestibular nucleus neurons and projection neurons.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: In Situ Hybridization, Expressing, Control

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Differentially expressed genes in phox2a -expressing medial vestibular neurons. Star indicates a gene was evaluated in projection neurons using fluorescent in situ hybridization. # indicates a gene was significantly differentially expressed in projection neurons. ‘% of medial vestibular neurons’ refers to detection in a subset of phox2a -expressing medial vestibular neurons in a single-cell reference atlas (Materials and methods, Figure 5—figure supplement 2 ). ‘% of projection neurons with expression’ refers to detection in a filtered subset of projection neurons. Gene sorted by p adjusted value. Data associated with Figure 5 .

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Expressing, In Situ Hybridization

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet:

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Electron Microscopy, Recombinant, In Situ Hybridization, Isolation, Purification, Sequencing, Software, CRISPR, Flow Cytometry

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Associated with . ( A ) Schematic of vestibulo-ocular reflex circuitry and the genetic loss-of-function approach used to perturb motor-derived signals. ( B ) Fluorescent in situ hybridization showing phox2a transcript expression in statoacoustic ganglion sensory afferents (left), central projection neurons in the tangential nucleus (middle), or nIII/nIV extraocular motor neurons (right) at 5 days post-fertilization (dpf). Top: probe only, nuclei outlined with dashed lines. Bottom: probe (green) merged with somata, labeled by Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS-E1b:Kaede ) (sensory, central) or Tg(isl1:GFP ) (motor). ( C ) Schematic of CRISPR/Cas9 mutagenesis approach. Top: Red star shows location of guides against phox2a DNA. Bottom: RNA sequence in wildtype and phox2a d22 alleles. Red dashed lines show deleted sequence; ‘STOP’ box shows predicted premature stop codon due to deletion. Right shows predicted protein sequence. ( D ) Transmitted light image of a 5 dpf wildtype (top) and phox2a null mutant (bottom). White arrows point to a normally inflated (top) or absent (bottom) swim bladder. ( E ) Images of nIII/nIV motor neurons in one hemisphere, labeled by Tg(isl1:GFP ), in wildtype siblings (left) and phox2a null mutants (right) at 5 dpf. Scale bar, 20 µm. ( F ) Quantification of the number of Tg(isl1:GFP )+neurons in nIII/nIV from N=6 wildtype siblings and N=10 phox2a null mutants.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Derivative Assay, In Situ Hybridization, Expressing, Labeling, CRISPR, Mutagenesis, Sequencing

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: ( A ) Fluorescent in situ hybridization images of nIII/nIV motor neurons, labeled by Tg(isl1:GFP ), in wildtype larvae at 2 dpf. Panels from left to right show: (1) isl1 + motor neurons, (2) mRNA for vachta , a marker for cholinergic motor neurons, (3) DAPI, and (4) merge. White dashed lines show the extent of nIII/nIV. ( B ) Loss of both isl1 and vachta identity in nIII/nIV motor neurons in phox2a mutants at 2 dpf. All scale bars, 20 µm.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: In Situ Hybridization, Labeling, Marker

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: ( A ) Images of nIII/nIV motor neurons, labeled in Tg(isl1:GFP ), in wildtype siblings (left) and phox2a heterozygotes (middle) at 5 dpf. Wildtype image same as in . One hemisphere shown. White dashed lines outline the dorsal extent of nIII, which contains inferior rectus and medial rectus neurons . Scale bar, 20 µm. ( B ) Location of the earliest-born neurons in nIII/nIV (left, magenta) against all nIII/nIV neurons labeled in Tg(isl1:Kaede ) (right, grey). Larvae birthdated at 34 hpf (Materials and methods). One hemisphere shown. White dashed lines outline the dorsal extent of nIII. Scale bar, 20 µm. ( C ) Quantification of the number of Tg(isl1:GFP )+ neurons in nIII/nIV from N=6 wildtype siblings (grey) and N=8 phox2a heterozygotes (teal). Wildtype data same as . ( D ) Distributions showing probability of nIII/nIV soma location across each spatial axis in wildtype (black) and heterozygous (teal) phox2a larvae. Solid and shaded lines show mean and standard deviation, respectively, from bootstrapped data. Data from same fish quantified in C . ns, not significant; star, significant at the p<0.001 level.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Labeling, Standard Deviation

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Associated with . ( A ) Schematic of pitch vestibulo-ocular reflex circuitry. Dashed lines outline projection neurons as calcium imaging target. Nose-down/eyes-up channel represented with blue; orange, nose-up/eyes-down. ( B ) Schematic of tonic pitch-tilt stimulus delivered with Tilt-In-Place Microscopy (TIPM). Shaded regions show calcium imaging windows when fish were oriented horizontally immediately following tilts. Inset shows timecourse of the rapid step to restore horizontal position after tilts. Imaging experiments used larvae from the Tg(isl1:GFP);Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:GCaMP6s ) line. ( C ) Proportion of subtypes observed in sibling controls and phox2a null mutants. Blue: nose-down. Orange: nose-up. Grey: Neurons without directional tuning (criteria in Materials and methods). ( D, E ) Soma position of nose-down (blue) and nose-up (orange) neurons in sibling controls (left) and phox2a null mutants (right). Soma size scaled by strength of directional selectivity (min = 0; max = 1; see Materials and methods). ( F/I ) Heatmaps showing example tilt responses from nose-down ( F ) or nose-up ( I ) neurons in sibling controls (top) and phox2a null mutants (bottom). n=10 neurons with strongest ΔF/F responses to tilts shown. Each row shows an individual neuron. Shaded bars show calcium imaging window immediately following restoration from eccentric position. Black arrow points to first second of tilt response used for analyses. ( G/J ) Distributions of maximum ΔF/F responses to tilts for nose-down ( G ) or nose-up ( J ) neurons in sibling controls (black) and phox2a null mutants (red). Solid and shaded lines show mean and standard deviation, respectively, of bootstrapped data (Materials and methods). ( H/K ) Distributions of directional tuning score to tilts for nose-down ( H ) or nose-up ( K ) neurons in sibling controls (black) and phox2a null mutants (red). Tuning score ranges from 0 (equal responses to both tilt directions, no tuning) to 1 (responses to one tilt direction only); criteria detailed in Materials and methods. Solid and shaded lines show mean and standard deviation, respectively, of bootstrapped data.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Imaging, Microscopy, Standard Deviation

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Statistical comparisons of tilt responses across genotypes. WT (sampled) refers to an n=125 neuron subset, sampled with replacement from a reference dataset of wildtype projection neurons. Data shown is mean/standard deviation unless otherwise noted. p val generated from a one-way ANOVA with multiple comparisons. Associated with Figure 2 and Figure 3 .

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Generated

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Statistical comparisons of projection neuron topography across genotypes. WT (sampled) refers to an n=125 neuron subset, sampled with replacement from a reference dataset of wildtype projection neurons. Data shown is the median/standard deviation distance from the ventro-lateral and rostral edges of the tangential nucleus (total size: 40 µm across each spatial axis). p val from one-way ANOVA (individual spatial axes) or multivariate ANOVA (global organization), respectively.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques:

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Associated with . ( A ) Schematic of impulse tilt experiments. Yellow dashed lines outline projection neurons as calcium imaging target. Impulse-responsive neurons (ventrally-localized) shown with purple; unresponsive neurons, grey. ( B ) Schematic of impulse stimuli delivered with TIPM. Shaded regions show calcium imaging windows at horizontal immediately following impulses. Inset shows timecourse of impulse stimulus. Imaging experiments used larvae from the Tg(isl1:GFP);Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:GCaMP6s ) line. ( C ) Proportion of impulse-responsive (purple) and unresponsive (grey) neurons observed in sibling controls and phox2a null mutants. ( D ) Soma position of impulse-responsive neurons in sibling controls (left) and phox2a null mutants (right). Soma size scaled by strength of calcium response (ΔF/F), normalized by max observed ΔF/F. ( E ) Heatmaps showing example impulse responses from neurons in sibling controls (left) and phox2a null mutants (right). n=10 example neurons shown. Each row shows an individual neuron. Shaded bars show calcium imaging window immediately following impulse delivery. Black arrow points to first second of tilt response used for analyses. Note smaller scale (0–0.75) of impulse responses relative to . ( F ) Distributions of maximum ΔF/F responses to impulses in sibling controls (black) and phox2a null mutants (red). Solid and shaded lines show mean and standard deviation, respectively, from bootstrapped data. ( G ) Distributions of directional tuning score to impulses in sibling controls (black) and phox2a null mutants (red). Tuning score ranges from 0 (equal responses to both tilt directions, no tuning) to 1 (responses to one tilt direction only); criteria detailed in Materials and methods. Solid and shaded lines show mean and standard deviation, respectively, from bootstrapped data.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Imaging, Standard Deviation

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: ( A ) Schematic of retrograde photofill experiments. Projection neuron axons expressing the photolabile protein Kaede are targeted at the midbrain-hindbrain boundary with ultraviolet light. Converted protein (magenta) retrogradely diffuses to the soma, while the unconverted nucleus remains green. ( B ) Projection neuron somata in sibling controls (left) and phox2a null mutants (right) after retrograde photolabeling. Experiments performed at 5 dpf. Neurons visualized in Tg(isl1:GFP);Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:E1b-Kaede ). ( C ) Top two panels: Projection neuron axons at the hindbrain (inset) and midbrain-hindbrain boundary in sibling controls (top) and phox2a null mutants (bottom). Axons visualized using Tg(isl1:GFP);Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:E1b-Kaede ). White dashed outline (circle) shows arborization fields in nIII/nIV. Dashed box over axons shows location of two bottom panels. MHB and yellow dashed line, midbrain-hindbrain boundary. nucMLF: nucleus of the longitudinal fasciculus. Bottom two panels: Zoom of axons (dashed rectangle above). Spatial segregation between early-born (magenta +green) and late-born (green only) axons. White dashed line reflects separation between dorsal (nose-up, early-born) and ventral (nose-down, late-born) axon bundles. Image at 5 dpf in sagittal view. ( D ) Projection neuron axon bundle in a phox2a null mutant at 3 dpf. White arrows point to single collateral to two nIII/nIV neurons that were not eliminated following phox2a knockout. ( E ) Fluorescent in situ hybridization against RNA for three pre-synaptic markers: synaptophysin a ( sypa ; left), synaptic vesicle glycoprotein 2 ( sv2 , middle), and synapsin I ( syn1 , right). Top row, sibling controls. Bottom row, phox2a null mutants. For each panel set, left images show in situ probe expression (green) and right images show merge with projection neurons labeled in Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:E1b-Kaede ). Dashed lines outline the projection nucleus. Cell and transcript expression outside the projection nucleus is removed for visual clarity. Images taken at 5 dpf in sagittal mount. All scale bars, 20 µm.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Expressing, Mutagenesis, Knock-Out, In Situ Hybridization, In Situ, Labeling

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Associated with , , . ( A ) Schematic of sequencing approach. Central projection neurons ( Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:E1b-Kaede )) are harvested from 3 dpf larvae. Flow cytometry is used to exclude neurons not labelled by Tg(–6.7Tru.Hcrtr2:GAL4-VP16 ). Bulk RNA sequencing is performed to compare the profiles of projection neurons in siblings and phox2a null mutants. ( B ) Example of projection neurons before (left) and after (right) harvesting. Neurons visualized with Tg(isl1:GFP);Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS:E1b-Kaede ). Dashed lines outline projection neurons in the tangential nucleus; dotted lines, medial vestibular nucleus. Yellow region shows margin of harvesting error: non-projection neurons that may be included in bulk sequencing dataset. ( C ) Number of differentially expressed genes in projection neurons at 3 dpf after applying progressive filters based on gene expression in a reference single-cell dataset. Data shown on logarithmic scale. Solid, dashed, and dotted lines represent differentially-expressed gene with p adjusted<0.5, p adjusted<0.01, or p adjusted<0.001 significance, respectively. ( D ) Volcano plot showing differentially expressed genes in projection 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 cells that express a given gene. Grey-colored genes are below both significance thresholds. ( E ) Same data as Figure 5D. Colored genes show eight candidates evaluated with fluorescent in situ hybridization: red, upregulated; blue, downregulated; yellow, highly-expressed controls ( evx2) . ( F ) Fluorescent in situ hybridization against candidate genes that met projection neuron filter criteria. Top row shows sibling controls; bottom row, phox2a null mutants. For each gene, left panels show RNA probe (green) and right panels show merge with projection neurons labeled by Tg(–6.7Tru.Hcrtr2:GAL4-VP16 ) (grey). Dashed lines outline the projection nucleus. Cell and transcript expression outside the projection nucleus is masked for visual clarity. Arrows denote whether genes are upregulated (red), downregulated (blue), or not significantly changed (yellow). Percentage refers to fraction of cells in a single-cell RNA sequencing reference atlas (Materials and methods) with detected transcript. Candidates: itga9 (log 2 fold change = 23.0, p adj.=3.9 × 10 –6 ), twf1b (log 2 fold change = 5.9, p adj.=0.024), p4hb (log 2 fold change = 5.1, p adj.=0.04), mapk6 (log 2 fold change = 5.1, p adj.=0.06), rxfp2a (log 2 fold change = −8.5, p adj.=1.1 × 10 –5 ), satb1a (log 2 fold change = −3.0, p adj.=0.001), evx2 (log 2 fold change = 0.46, p adj.=0.99), myt1la (log 2 fold change = 2.6, p adj.=0.44). All scale bars, 20 µm.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Sequencing, Flow Cytometry, RNA Sequencing Assay, Expressing, Control, In Situ Hybridization, Labeling

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Differentially expressed genes in projection neurons. Star indicates a gene was evaluated using fluorescent in situ hybridization. # symbol indicates a gene was also differentially expressed in adjacent phox2a -expressing medial vestibular neurons (see Figure 5—figure supplement 5 ). ”% of projection neurons with expression” refers to detection in a filtered subset of projection neurons from a single-cell reference atlas of neurons labeled in Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS-E1b:Kaede ) (Materials and methods, Figure 5—figure supplement 2 ). Genes sorted by p adjusted value. Data associated with Figure 5 .

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: In Situ Hybridization, Expressing, Labeling, Control

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: ( A-A’ ) Fluorescent in situ hybridization against itga9 for three sibling ( A ) or phox2a null mutant ( A’ ) larvae (72 hpf), imaged with identical conditions. Left column shows RNA (green); right column, merge with projection neurons visualized 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. Percentage (1.9%) refers to fraction of cells in a single-cell RNA sequencing reference atlas (Materials and methods) with detected transcript. All scale bars, 20 µm. ( B-B’ ) Fluorescent in situ hybridization against htt for three sibling ( B ) and phox2a mutant ( B’ ) larvae (72 hpf). ( C-C’ ) Fluorescent in situ hybridization against evx2 for three sibling ( C ) and phox2a mutant ( C’ ) larvae (72 hpf).

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: In Situ Hybridization, Mutagenesis, Expressing, RNA Sequencing Assay

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Top 50 expressed genes in an unfiltered bulk RNA sequencing dataset of phox2a siblings. ‘% of unfiltered 10 x neurons’ refers to gene detection in a single-cell atlas of neurons labeled in Tg(–6.7Tru.Hcrtr2:GAL4-VP16);Tg(UAS-E1b:Kaede ) (n=1,468 neurons). ‘% of projection neurons’ refers to gene detection in a subset of the single-cell atlas containing projection neurons in the tangential nucleus (n=159 neurons). Data associated with Figure 5 .

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: RNA Sequencing Assay, Labeling, Expressing, Control

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Top 50 differentially expressed genes in an unfiltered bulk RNA sequencing dataset of phox2a siblings and null mutants. One star indicates a gene was retained in a filtered subset of projection neurons; %, evaluated using fluorescent in situ hybridization. ‘% of unfiltered 10 x neurons’ refers to gene detection in an unfiltered single-cell reference atlas of neurons labeled in Tg(–6.7Tru.Hcrtr2:GAL4-VP16 ); Tg(UAS-E1b:Kaede ) (n=1,468 neurons). Putative origin inferred from gene expression in the annotated 10 x dataset (Materials and methods, Figure 5—figure supplement 2 ). Genes sorted by p adjusted value. Data associated with Figure 5 .

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: RNA Sequencing Assay, In Situ Hybridization, Labeling, Expressing, Inhibition, Control

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: ( 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 phox2a -expressing 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 phox2a -expressing medial vestibular neurons (Materials and methods, ) that express a given gene. Grey-colored genes are below both significance thresholds. Data for all differentially expressed candidates in medial vestibular neurons shown in . ( C ) Same data as B . Color shows genes that are differentially expressed in both medial vestibular nucleus neurons and projection neurons.

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: In Situ Hybridization, Expressing, Control

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet: Differentially expressed genes in phox2a -expressing medial vestibular neurons. Star indicates a gene was evaluated in projection neurons using fluorescent in situ hybridization. # indicates a gene was significantly differentially expressed in projection neurons. ‘% of medial vestibular neurons’ refers to detection in a subset of phox2a -expressing medial vestibular neurons in a single-cell reference atlas (Materials and methods, Figure 5—figure supplement 2 ). ‘% of projection neurons with expression’ refers to detection in a filtered subset of projection neurons. Gene sorted by p adjusted value. Data associated with Figure 5 .

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Expressing, In Situ Hybridization

Journal: eLife

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

doi: 10.7554/eLife.96893

Figure Lengend Snippet:

Article Snippet: PCR generates a 169 bp product (wildtype), 147 bp product ( phox2a d22 ), 150 bp product ( phox2a d19 ), or 171 bp product ( phox2a i2 ). phox2a d22 and phox2a d19 DNA was evaluated using gel electrophoresis; phox2a i2 was assessed via sequencing with the reverse primer (Genewiz, Azenta Life Sciences, South Plainfield, New Jersey).

Techniques: Electron Microscopy, Recombinant, In Situ Hybridization, Isolation, Purification, Sequencing, Software, CRISPR, Flow Cytometry