Context-Dependent Gait Choice Elicited by EphA4 Mutation in Lbx1 Spinal Interneurons  Daisuke Satoh, Christiane Pudenz, Silvia Arber  Neuron  Volume 89, Issue 5, Pages 1046-1058 (March 2016) DOI: 10.1016/j.neuron.2016.01.033 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 EphA4 Controls Contralateral Premotor Connectivity (A and B) Transverse spinal cord reconstruction of contralateral TA premotor interneurons in WT and EphA4−/− mice shown as scatter and contour plots. (C) Bar plots (∗∗∗p < 0.001, mean + SEM) showing percentage of dorsal and ventral commissural of all premotor interneurons (WT, n = 7, and EphA4−/−, n = 6). (D) Dorso-ventral density plot of averaged contralateral premotor interneuron distribution densities in WT (black, n = 7) and EphA4−/− (turquoise, n = 6) mice. (E) Contralateral dorsal premotor interneuron (magenta) expressing the transcription factor Lbx1 (turquoise) in EphA4−/− mice. (F) Transcriptional codes for spinal progenitor domain territory relevant to this study. (G and H) Ectopic dorsal Lbx1ON commissural premotor interneurons are derived from both dI5, marked by Tlx3 (G) and dI4, marked by Pax2 (H) in Lbx1-Cre; EphA4fl/−; Tau-LSL-INLA mice. Neuron 2016 89, 1046-1058DOI: (10.1016/j.neuron.2016.01.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Cell-Autonomous EphA4 Function in Ectopic Dorsal Premotor Interneurons (A and D) Contralateral transverse spinal cord sections showing TA premotor interneurons (magenta) and Lbx1-nlsLacZ expression (turquoise) in Lbx1-Cre; Tau-LSL-nlsLacZ (A) and Lbx1-Cre; EphA4fl/−; Tau-LSL-nlsLacZ (D) mice. (B and E) Digital reconstructions of contralateral spinal TA premotor interneurons overlaid with contour plots displaying density of premotor interneurons. (C) Bar plots depicting percentage of TA premotor interneurons in contralateral dorsal and ventral spinal cord of WT (n = 7) and Lbx1-Cre; EphA4fl/− (n = 7) mice. (F) Dorso-ventral density plots for contralateral TA premotor interneurons in WT and Lbx1-Cre; EphA4fl/− mice (each trace depicts one mouse analyzed). (G and I) TA premotor interneurons (magenta) and Lbx1 nuclear marker (turquoise) in contralateral Rexed’s lamina V/VI of Lbx1-Cre; Tau-LSL-nlsLacZ (G) and Lbx1-Cre; EphA4fl/−; Tau-LSL-nlsLacZ (I) mice. Inset in (I) shows an example of an Lbx1ON commissural premotor interneuron at high magnification. (H and J) Digital reconstructions (left) and dorso-ventral density plots (right) of Lbx1ON (turquoise) and Lbx1OFF (black) commissural premotor interneurons in Lbx1-Cre; Tau-LSL-nlsLacZ (H) and Lbx1-Cre; EphA4fl/−; Tau-LSL-nlsLacZ (J) mice. Bar plots are ∗∗∗p < 0.001, mean + SEM. See also Figure S1. Neuron 2016 89, 1046-1058DOI: (10.1016/j.neuron.2016.01.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Locomotor Task-Dependent Gait Choice in Lbx1 EphA4 Conditional Mice (A) Behavioral tests employed in this study and scheme displaying predicted relative strength of proprioceptive signaling under respective experimental condition. (B) Histograms of hindlimb phase values in individual mice during locomotion on ground (magenta) and in water (gray). Dashed lines indicate threshold values for gait types (definition, see Figure S2). (C) Bar plots of percentage of synchronous hindlimb movements during locomotion on ground (magenta) and in water (gray) for WT (n = 5), Lbx1-Cre; EphA4fl/− (n = 9), and EphA4−/− (n = 4) mice. (D) Bar plots of percentage of synchronous limb movements during airstepping for WT (filled bars, hindlimbs: n = 6, forelimbs: n = 5) and Lbx1-Cre; EphA4fl/− (open bars, hindlimbs: n = 8; forelimbs: n = 7) mice. Bar plots are ∗∗∗p < 0.001, mean + SEM. See also Figure S2. Neuron 2016 89, 1046-1058DOI: (10.1016/j.neuron.2016.01.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Neuronal Silencing of Local Lbx1 Neurons in Lbx1 EphA4 Conditional Mice Reverts Gait Defects (A) Timeline for pharmacogenetic silencing experiments in Lbx1-Cre; Tau-LSL-FlpO (control) and Lbx1-Cre; EphA4fl/−; Tau-LSL-FlpO (experimental) mice. (B) Expression of PSAM-GlyR (white) by Lbx1-nlsLacZ (turquoise) neurons. (C) Distribution pattern of PSAM-GlyR expressing Lbx1ON interneurons at injection site (single section). (D and E) Bar plots of swimming gait pattern for hindlimbs (D) and forelimbs (E) (left: Lbx1-Cre; Tau-LSL-FlpO with injection of conditional AAV, n = 4; middle: Lbx1-Cre; EphA4fl/−; Tau-LSL-FlpO without injection of AAV, n = 4; right: Lbx1-Cre; EphA4fl/−; Tau-LSL-FlpO with injection of conditional AAV, n = 4). Light gray, turquoise, and dark gray bars indicate pre, 10 min after, and post PSEM308 administration. Bar plots are ∗∗p < 0.01, ∗p < 0.05, mean + SEM. Lines show values from single animals. See also Figure S2. Neuron 2016 89, 1046-1058DOI: (10.1016/j.neuron.2016.01.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Developmental Analysis of Swimming Gaits across Genotypes (A–C) Histograms of phase values for hindlimbs and forelimbs of single P5 WT (A), Lbx1-Cre; EphA4fl/− (B), EphA4−/− (C) mice during locomotion in water. Dashed lines indicate threshold values for gait types (as defined in Figure S2). (D and E) Percentages of alternating and synchronous gait for hindlimbs (D) and forelimbs (E) (black: WT, n = 6; magenta: Lbx1-Cre; EphA4fl/− [abbreviated EphA4 cond.], n = 13; turquoise: EphA4−/−, n = 5 for hindlimbs, n = 4 for forelimbs). Percentages for Asy hindlimb strokes are as follows: WT (3.5%), Lbx1 EphA4 conditional mice (26.7%), and full EphA4 mutants (1.7%) (∗∗∗p < 0.001 for WT or full EphA4 mutants versus Lbx1 EphA4 conditional mice). (F–H) Developmental time course of swimming gait, displaying synchrony in WT (n = 5; frequency P9: 3.1 Hz; P15: 5.5 Hz), Lbx1-Cre; EphA4fl/− (n = 9; frequency P9: 3.0 Hz; P15: 4.9 Hz), and EphA4−/− (n = 4) mice. (I) Summary table listing gait phenotypes in different environments and at different developmental stages. Bar plots are ∗∗∗p < 0.001, ∗p < 0.05, mean + SEM. See also Figures S2 and S3. Neuron 2016 89, 1046-1058DOI: (10.1016/j.neuron.2016.01.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Aberrant Commissural Connectivity in Adult Lbx1 EphA4 Conditional Mice (A) Scheme for anterograde tracing using AAV-FRT-membrane-tdTomato, AAV-FRT-synaptophysin-GFP, and AAV-FRT-nlsGFP coinjection, as markers for axons, presynaptic terminals, and injection sites, respectively. Unilateral and dorsally restricted injections are performed into mice expressing Flp under the control of Lbx1-Cre. (B and C) Spinal cord sections from Lbx1-Cre; Tau-LSL-FlpO and Lbx1-Cre; EphA4fl/−; Tau-LSL-FlpO mice showing membrane-tdTomato (black) and nlsGFP (red). (D) Reconstructed surfaces of motor neurons (purple) and contacting synaptic terminals (yellow) on ipsilateral and contralateral spinal sides in Lbx1-Cre; Tau-LSL-FlpO and Lbx1-Cre; EphA4fl/−; Tau-LSL-FlpO mice. (E) Synaptic terminal analysis of Lbx1ON interneurons opposed to motor neurons on ipsilateral and contralateral sides at segmental level of injection. Each dot in graph represents single motor neuron (Lbx1-Cre; Tau-LSL-FlpO ipsi, n = 50, Lbx1-Cre; Tau-LSL-FlpO contra, n = 42, Lbx1-Cre; EphA4fl/−; Tau-LSL-FlpO ipsi, n = 58, Lbx1-Cre; EphA4fl/−; Tau-LSL-FlpO contra, n = 41; ∗∗∗p < 0.001). (F) Connectivity scheme for WT and Lbx1 EphA4 conditional mice. See also Figure S4. Neuron 2016 89, 1046-1058DOI: (10.1016/j.neuron.2016.01.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Input-Output Circuitry of Ectopic Commissural Premotor Interneurons in Lbx1 EphA4 Conditional Mice (A) Injection scheme to visualize muscle sensory afferents (CTb) and left-right double connected premotor interneurons (monosynaptically restricted transsynaptic rabies). (B) CTb (green) and vGlut1 (red) labeled proprioceptive terminals and ectopic commissural premotor interneurons in P13 dorsal spinal cord (blue). (C) Reconstructed ectopic commissural premotor interneuron in dorsal spinal cord. (D) Bar plot of quantification of CTbON proprioceptive inputs per total proprioceptive input to ectopic commissural premotor interneurons in the dorsal spinal cord (TAprop, n = 20; GSprop, n = 22; ∗∗∗p < 0.001). (E) Exemplary image for rabies-GFP and rabies-RFP double-positive premotor interneuron. (F) Distribution pattern of double positive premotor interneurons in Lbx1 EphA4 conditional mice. (G) Summary of wiring diagram for findings illustrated in this figure. See also Figure S5. Neuron 2016 89, 1046-1058DOI: (10.1016/j.neuron.2016.01.033) Copyright © 2016 Elsevier Inc. Terms and Conditions