1. Volume 147, Issue 3, Pages 653-665 (October 2011)
Patterns of Spinal Sensory-Motor Connectivity Prescribed by a Dorsoventral Positional Template 
Gülşen Sürmeli, Turgay Akay, Gregory C. Ippolito, Philip W. Tucker, Thomas M. Jessell 
Cell 
Volume 147, Issue 3, Pages (October 2011)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Motor Neuron Columelar Organization
(A) Motor columels in cat lumbar spinal cord. Color code: dark blue/proximal hip (PH), gray/iliopsoas (IP), light green/adductors (A), pink/quadriceps (Q), orange/hamstring (H), red/anterior crural (AC), dark green/posterior crural (PC), purple/foot (F). Lumbar (L) and sacral (S) segmental levels are indicated. Derived from data in Vanderhorst and Holstege (1997).
(B) The proximodistal organization of muscles in cat hindlimb.
(C) The dorsoventral (DV, μm) positions of motor pools in cat lumbar spinal cord and the proximodistal (PD, cm) positions of muscles in cat hindlimb. Color code as in (A). Colored fields represent columelar/synergy groups, and individual points mark specific motor pools and limb muscles. Muscle and motor neuron key provided in the Extended Experimental Procedures. Motor pool position from Vanderhorst and Holstege (1997), muscle position from Burkholder and Nichols (2004).
(D) Columelar organization along the dorsoventral axis, after rostrocaudal compression into two dimensions. Columels are assigned to dorsoventral tiers that correspond to muscles at individual joints. The approximate positions of relevant motor pools within columelar groups are marked.
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4. Figure 2 Motor Impairment and Pool Disruption in FoxP1 Mutants
(A) Innervation of TA muscle in P2 FoxP1fl and FoxP1MNΔ mice. Motor nerves visualized by neurofilament (NF), and acetylcholine receptors by alpha-bungarotoxin (α-BTX) labeling.
(B) Upper panels: EMG recodings from right and left TA and right GS muscles from FoxP1fl and FoxP1MNΔ mice during swimming. Lower panels: autocorrelograms of muscle burst patterns of right and left TA and right GS in FoxP1fl and FoxP1MNΔ mice.
(C) Motor pools in ∼P20 FoxPfl mice. Top row: columelar positions at L3 to L5, based on our observations and McHanwell and Biscoe (1981). MMC: median motor column, S: sacral motor neurons. Second row: ChAT+ motor neurons at L3 to L5. Third row: organization of motor pools after CTB injection into specific muscles. Arrow in GS panel indicates BF and/or ST motor neurons, labeled through tracer leakage from GS muscle. Bottom row: rostrocaudal distribution of motor pools.
(D) Motor neuron positions in FoxP1MNΔ mice. Top row: motor neuron positions at L3 to L5. Green/gray: zone 1; blue/gray: zone 2. Second row: ChAT+ motor neuron positions at L3 to L5. Third row: distribution of motor neuron pools after CTB injection into individual muscles. Bottom row: rostrocaudal distribution of labeled motor neurons.
See also Figures S3 and S4.
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5. Figure 3 Specificity of Sensory Connections in Wild-Type Mice
(A) vGluT1+ sensory boutons on ChAT+ motor neurons in P18 mice. Bassoon marks sensory terminals and shank1a, motor neuron membrane aligned with sensory boutons.
(B) vGluT1+ bouton density on TA and GS motor neurons in FoxP1fl and FoxP1MNΔ mice.
(C) Experimental design: after CTB and Rh-Dex tracer injection into different muscles, vGluT1+ sensory boutons contact CTB-labled self but not Rh-Dex-labeled nonself motor neurons in P21 wild-type mice (see Figure S6).
(D) CTB-labeled vGluT1+ TA sensory boutons on TA motor neurons.
(E) CTB-labeled vGluT1+ GL sensory boutons are not found on TA motor neurons.
(F) CTB-labeled vGluT1+ GL sensory boutons on GL motor neurons.
(G) CTB-labeled vGluT1+ TA sensory boutons are not detected on GL motor neurons.
(H) Incidence of sensory connections with self motor neurons. GS∗∗ indicates contamination of GS by BF and ST sensory afferents.
(I) Incidence of sensory connections with nonself motor neurons.
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6. Figure 4
Breakdown of Dorsoventral Sensory-Motor Specificity in FoxP1 Mutants
(A) Incidence of TA sensory input to TA and GL motor neurons in FoxP1fl and FoxP1MNΔ mice. Statistical analysis for these and subsequent histograms is presented in Figure S7.
(B) Incidence of GL sensory input to GL and TA motor neurons in FoxP1fl and FoxP1MNΔ mice.
(C) CTB-labeled vGluT1+ TA sensory boutons contact GL∗ motor neurons in FoxP1MNΔ mutants.
(D) CTB-labeled vGluT1+ GL sensory boutons contact TA∗ motor neurons in FoxP1MNΔ mutants.
(E) Plotting the dorsoventral positions of CTB-labeled motor neurons (in this case TA and TA∗ neurons) in FoxP1fl and FoxP1MNΔ mice.
(F) Sensory input status as a function of motor neuron dorsoventral position. Dark gray circles: wild-type TA motor neurons. TA∗ and GL∗ motor neurons with CTB-labeled sensory bouton input are shown in light gray circles. TA∗ and GL∗ motor neurons lacking CTB-labeled sensory input are shown in open circles.
(G) Incidence of sensory input to TA∗ and GL∗ motor neurons in FoxP1MNΔ mutants, gated to zonal position.
See also Figure S7.
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7. Figure 5
Trajectory of Sensory Afferents to Motor Neuron-free Domains in FoxP1MNΔ Mice
(A) CTB-labeled IF sensory afferents and ChAT+ IF motor neurons in P21 control mice. Position of high-power images in (B) denoted by box.
(B) CTB-labeled vGluT1+ IF sensory boutons on dendrites and cell bodies of IF motor neurons in control mice.
(C) CTB-labeled IF sensory afferents and ChAT+ IF motor neurons in P21 FoxP1MNΔ mice. Position of high-power images in (D) denoted by box.
(D) Absence of CTB-labeled vGlut1+ IF sensory boutons on the dendrites or cell bodies of IF∗ motor neurons in P21 FoxP1MNΔ mice.
(E) Incidence of IF and GL sensory bouton inputs to IF motor neurons in FoxP1fl and FoxP1MNΔ mice.
(F) CTB-labeled vGluT1+ GL sensory boutons contact ventrally displaced IF∗ motor neurons.
(G) Spatial distribution of CTB-labeled vGluT1+ GL sensory boutons in P21 FoxP1fl and FoxP1MNΔ mice.
(H) Spatial distribution of CTB-labeled vGluT1+ IF sensory boutons in P21 FoxP1fl mice. In (H) and (I), representative stacks of three (left) and ten (right, color plot) sections are shown.
(I) Spatial distribution of CTB-labeled vGluT1+ IF sensory boutons in P21 FoxP1MNΔ mice.
(J) In FoxPMNΔ mice, CTB-labeled vGluT1+ IF sensory boutons contact neurotrace (NT)-labeled interneurons (blue) in a dorsomedial domain.
(K) Left panel: trajectory of sensory afferents at L6 after Rh-Dex labeling of L5 dorsal roots in E18 FoxP1fl embryos. Middle panel: Isl1/2+ IF motor neurons. Right panel: positional prevalence of Rh-Dex-labeled sensory axons. Ratio of mean fluorescence intensity (f.i.) in dorsal and ventral domains determined from eight sections from three mice.
(L) Left panel: trajectory of sensory afferents at L6 after Rh-Dex labeling of L5 dorsal roots in E18 in FoxP1MNΔ embryos. Middle panel: Isl1/2+ IF motor neurons. Right panel: positional prevalence of Rh-Dex-labeled sensory axons. Ratio of mean f.i. in dorsal and ventral domains determined from eight sections from three mice.
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8. Figure 6
Breakdown of Specificity in Antagonist Reflex Arcs in FoxP1 Mutants
(A) CTB-labeled vGluT1+ GS∗∗ sensory boutons contact TA∗ motor neurons in P21 FoxP1MNΔ mice. Plots show connectivity of GS∗∗ sensory boutons with TA and GS motor neurons in FoxP1fl and FoxP1MNΔ mice.
(B) CTB-labeled vGluT1+ TA sensory boutons do not contact GS∗∗ motor neurons in P21 FoxP1MNΔ mice. Plots show connectivity of TA sensory boutons with TA and GS motor neurons in FoxP1fl and FoxP1MNΔ mice.
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9. Figure 7
Tier Targeting Provides a Template for Sensory-Motor Connectivity
(Ai) Motor columels in wild-type mice.
(Aii) Tier termination of group Ia sensory afferents supplying individual muscles.
(Bi) Tier targeting of TA (red) and GL (blue) sensory afferents in FoxP1MNΔ mice, despite scrambling of TA∗ and GL∗ motor neuron position.
(Bii) Tier targeting of sensory afferents at perinatal stages in FoxP1MNΔ mice, despite the absence of tier 1 motor neurons. At later stages, sensory afferents withdraw to a dorsomedial position.
(Biii) Breakdown of TA and GS antagonist exclusion in FoxP1MNΔ mice.
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10. Figure S1
Spatial Relationship between Motor Pool and Limb Muscle Position in Mouse, Related to Figure 1
The relationship between the dorsoventral (DV, μm) position of specific motor pools in adult mouse lumbar spinal cord and the proximodistal (PD, expressed as a fraction of total limb length) position of the target muscle groups in embryonic mouse hindlimb. Individual points mark specific motor pools or clusters of motor pools that are marked through peripheral nerve tracing. Motor neuron key: GLUT: gluteal muscles, PEC: pectineus, ADD: adductors, GRAC: gracilis, RF: rectus femoris, V: vastus lateralis, BF: biceps femoris, SM: semimembranosus ST: semitendinosus, GS: gastrocnemius, SOL: soleus, TA: tibialis anterior, EDL: extensor digitorum longus, PER: peroneus longus. Motor pool identity and dorsoventral position obtained from analysis of adult spinal cord, in McHanwell and Biscoe (1981); and proximodistal muscle position obtained from analysis of embryonic limb, in Delaurier et al. (2008).
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11. Figure S2
Molecular Phenotype of Conditional FoxP1 Mutants, Related to Figure 2
(A) Design for generation of motor neuron FoxP1-deficient mice: an Olig2::Cre [O2:Cre] driver line used to direct recombination of a conditional FoxP1 allele.
(B) Preservation of generic (Isl1/2) motor neuron character, but loss of LMC columnar (RALDH2) and pool (Nkx6.1) characters in motor neuron FoxP1 mutants. Pool-specific expression of Pea3, Nkx6.2, and Sema3e is also lost (data not shown; see Dasen et al., 2008). The ∼15% reduction in total number of Isl1/2+ motor neurons observed at lumbar levels in FoxP1MNΔ embryos may reflect the loss of RALDH2-dependent proliferative signals for motor neuron progenitors (Sockanathan and Jessell, 1998).
(C) Patterns of motor innervation of GL, IF and GS muscles in P2 control FoxP1fl and mutant FoxP1MNΔ mice. Motor nerves visualized by neurofilament (NF) immunolabeling and acetylcholine receptors visualized by alpha-bungarotoxin (α-BTX) labeling.
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12. Figure S3
Mapping the Position of Biceps Femoris and Semitendinosus Motor Neurons, Related to Figure 2
(A) Retrograde labeling of BF-ST (CTB-Alexa555) and GS∗ (CTB-Alexa488, green) motor neurons. Inset shows colabeled motor neurons after GS and BF/ST injections, indicating that GS muscle injections label contaminating BF and/or ST motor neurons.
(B) Retrograde labeling of GS∗ (CTB-Alexa488, green) motor neurons. Note the ventral position of contaminating BF-ST ChAT+ motor neurons.
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13. Figure S4
Spatial Analysis of Motor Neuron Positioning in Motor Neuron FoxP1 Mutants, Related to Figure 2
(A) Scattering of CTB-labeled TA∗ neurons in P18 FoxP1MNΔ mice within the general ChAT+ motor neuron population.
(B) TA motor neuron distribution in FoxP1fl mice. The summed pairwise distance between all CTB-labeled motor neurons present in a single section (n = 5) (red dot). 200-fold iterative shuffling of the location of CTB-labeled neurons—to compute summed pairwise distances, to yield a distribution histogram. Mean (black circle) and standard deviations (black line) are shown. The distribution of actual and randomly shuffled motor neuron positions differ by 2.86 standard deviations.
(C) TA∗ motor neuron distribution in FoxP1MNΔ mice. The pairwise distance value derived from experimentally observed positions differs from the mean of the randomly derived distribution by 0.25 standard deviations.
(D) Analysis of FoxP1fl and FoxP1MNΔ sections. For controls, the mean pairwise distance (Z score) between CTB-labeled neurons was 4.06 standard deviations separated from random prediction. The mean Z score in FoxP1MNΔ mutant sections was 0.06 standard deviations separated from random. Red dots indicate analysis derived from sections shown in (B) and (C).
(E) Scattering of CTB-labeled GS∗∗ neurons in P18 FoxP1MNΔ mice. Motor neurons revealed by ChAT expression.
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14. Figure S5
Motor Neuron Dendritic Arborization and γ-Motor Neuron Character Preserved in FoxP1 Mutant Mice, Related to Figure 3
(A) Dendritic arborization of motor neurons revealed by Rh-Dex retrograde labeling from L4 ventral root in P0 FoxP1fl mice.
(B) Dendritic arborization of motor neurons revealed by Rh-Dex retrograde labeling from L4 ventral root in P0 FoxP1MNΔ mice.
(C and D) Err3+, ChAT+ γ-motor neurons detected in P21 FoxP1fl (C) and FoxP1MNΔ (D) mice. Note the small size of γ-motor neurons.
(E and F) Paucity of vGluT1+ sensory bouton on small ChAT+, presumed γ-motor neurons in P21 FoxP1fl (E) and FoxP1MNΔ (F) mice.
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15. Figure S6
Specificity of Sensory-Motor Connections Revealed by Transganglionic Tracing, Related to Figure 3
(A) Tracer injection of rhodamine-dextran (Rh-Dex, blue) into individual hindlimb muscles of P18 wild-type mice labels motor neurons but not sensory terminals. Images show absence of Rh-Dex tracer accumulation in vGluT1+ (red) sensory terminals that contact motor neurons.
(B) Tracer CTB injection into individual hind limb muscles of P18 wild-type mice labels both motor neurons and many sensory terminal boutons. Images show CTB tracer accumulation in vGluT1+ sensory terminals that contact motor neuron cell bodies and dendrites.
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16. Figure S7
Quantitation of Sensory-Motor Connections in Control and FoxP1 Mutant Mice, Related to Figures 3, 4, 5, and 6
(A) Incidence of innervation of motor neurons by sensory afferents in control and FoxP1 mutant mice. Individual motor neurons were marked as innervated if the number of CTB-labeled sensory boutons exceeded 10% of the total number of vGluT1+ bouton contacts on that motor neuron. The number of motor neurons analyzed is indicated.
(B) Fraction of vGluT1+ sensory bouton contacts with motor neurons that accumulated CTB in control and FoxP1 mutant mice. The number of sensory boutons analyzed is indicated.
Below is the quantitative analysis of synaptic connectivity data:
Figure 3B: TA to TA∗, wild-type (WT): mean: 4.2% standard error of the mean (SEM): 0.1; mutant (mt): 3.6% SEM: 0.3. GS to GS∗, WT: mean: 3.7% SEM: 0.8; mt: mean: 3.9% SEM: 0.1.
Figure 3H: TA to TA∗, WT: mean: 45.69% SEM: 7.1, n = 3 mice, 13 cells, 197 synapses; GL to GL∗, WT: mean: 33.52% SEM: 2.94, n = 3 mice, 42 cells, 719 synapses; IF to IF∗, WT: mean: 52.9% SEM.: 8.8, n = 3 mice, 9 cells, 170 synapses; GS to GS∗, WT: mean: 42.0% SEM: 3.9, n = 3 mice, 9 cells, 317 synapses.
Figure 4A: TA to TA∗, WT: 84%, 13 cells; mt: 56% 45 cells; TA to GL∗, WT: 0%, 9 cells; mt: 50%, 22 cells. TA to TA∗, WT: mean: 45.69% SEM: 7.1, n = 3 mice, 13 cells, 197 synapses; TA to GL∗, mean 0% SEM: 0, n = 3 mice, 9 cells, 206 synapses; mt: TA to TA∗, mean: 33.2% SEM: 4.6, n = 5 mice, 45 cells, 754 synapses. TA to GL∗, mean: 29.5% SEM: 6.2, n = 3 mice, 22 cells, 492 synapses.
Figure 4B: GL to GL∗, WT: 90%, 42 cells; mt: 45%, 51 cells; GL to TA∗, WT: 0%, 13 cells; mt: 56%, 23 cells. GL to GL∗, WT: mean: 33.52% SEM: 2.94, n = 3 mice, 42 cells, 719 synapses; GL to TA∗, WT: mean 0% SEM: 0, n = 3 mice, 13 cells, 314 synapses; GL to GL∗, mt: mean: 16.39% SEM: 2.43, n = 3 mice, 51 cells, 1129 synapses; GL to TA∗: 19.17% SEM: 3.67, n = 3 mice, 23 cells, 480 synapses.
Figure 4G: TA to TA∗ zone 1, mean: 54.30% SEM: 5.1, 26 cells, 453 synapses; zone 2, mean: 1.6% SEM: 1.4, 16 cells, 301 synapses; TA to GL∗ zone 1, mean: 55.8% SEM: 4.6%, 11 cells, 256 synapses; zone 2, mean: 0% SEM: 0.3, 11 cells, 236 synapses ; GL to GL∗ zone 1, mean: 1.46% SEM: 0.67, 26 cells, 548 synapses; zone 2, mean: 30.5% SEM: 2.77, 25 cells, 581 synapses; GL to TA∗ zone 1, mean: 0% SEM: 0, 9 cells, 154 synapses; zone 2, mean: 28.2% SEM: 3.6, 13 cells, 326 synapses.
Figure 5E: IF to IF∗, WT: mean: 52.9% SEM: 8.8, n = 3 mice, 9 cells, 170 synapses; mt: mean: 0% SEM: 0, n = 2 mice, 6 cells, 110 synapses. GL to IF∗, WT: mean: 0% SEM: 0%, n = 3 mice, 8 cells, 143 synapses; mt: mean: 28.8% SEM: 3.8, n = 3 mice, 12 cells, 144 synapses.
Figure 6A: GS to TA∗, WT: 0%, 17 cells; mt: 54% 33 cells. GS to TA∗, WT: GS to TA∗, WT: mean: 0% SEM: 0, n = 4 mice, 17 cells, 587 synapses; mt: mean: 14.84% SEM: 2.3, n = 5 mice, 33 cells, 728 synapses.
Figure 6B: TA to GS∗, WT: 0%, 20 cells; mt: 0% 12 cells. TA to GS∗, WT: mean: 0% SEM: 0, n = 4 mice, 20 cells, 302 synapses; mt: mean: 3.3% SEM: 1.8, n = 2 mice, 9 cells, 156 synapses.
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