Article Figures & Data
Figures
- Figure 1.
Abnormal gait in hind limbs of hop mice. A, Gait analysis painting forelimbs (red) and hind limbs (blue) show that control mice move their forelimbs and hindlimbs in an alternating pattern while hop mice move their hind limbs in a synchronous manner. Horizontal arrow L is the distance between the left and right paw and R the distance covered by the same right paw. B, Graph showing quantification of walking pattern with the ratio L/R and significant genotype differences for hindlimb (p = 0.021, r = −0.81) but not forelimb (p = 1, r = 0) coordination (n = 4 mice per group over a distance of 30 cm). C–E, Hop mice show a characteristic synchronous left-right air-stepping pattern in vivo. C, Schematic drawing of the experimental setup for recording from neonatal mice. E, G, Raw EMG recordings from the left and right tibialis muscles of hop (G) and control (E) mice following a tail pinch stimulus illustrating representative synchronous and alternating traces, respectively (n = 2 mice per group). The circular plots in E, G represent the phase of onset of the left and right tibialis anterior bursts with respect to each other. Each dot represents an individual burst, while the arrow indicates the tendency for the bursts to be coupled to each other. A phase value close to zero indicates synchrony in the case of the hop while a value close to 180° suggests an alternating pattern in the case of the control animals (p < 0.01). D, Schematic illustration of ventral root recording setup measuring fictive locomotion. F, H, The hop deficit can be reproduced in vitro using isolated spinal cord preparations. Control mice exhibit an alternating (F) and hop mice a synchronous (H) left-right pattern (n = 20 mice per group). Circular plots (F, H) indicate phase values across experiments. Each point equals one 60-s section of data (5 per animal). The length of the arrow provides an index of the strength of the coupling between the rhythms and the direction equals phase. I–K, Bath application of GABA and glycinergic reuptake blockers affect the synchronous rhythm’s stability in the hop mutant mouse. Control rhythm in hop mice (I) evoked by bath application of NMDA, 5-HT, and DA. Twenty minutes after adding nipecotic acid (J), one L2 root would double burst occasionally and at random. After application of nipecotic acid and sarcosine, occasional alternating segmental activity could be observed (K), although the ipsilateral L2–L5 rhythm was disrupted. For burst pattern following hemisection, please refer to Extended Data Figure 1-1. *Significant effect of genotype p < 0.05.
- Figure 2.
Altered spinal cord morphology in postnatal hop mice. A, Photomicrographs (ventral view) and (B) graph of measured sizes of early postnatal (p0–p5) cervical and lumbar spinal cords. At cervical levels from control and hop mice, no difference is apparent (p = 1, r = 0; n = 4 animals per group). At lumbar levels, the lumbar enlargement of the spinal cord is less prominent in hop mice (black bar) compared with control mice (white bar, p = 0.009, r = −0.83; n = 5 animals per group). The visible ventral spinal artery in control wild-type mice (arrow) is absent from the lumbar part of the hop spinal cord (position noted with star). C, Photomicrographs of immunohistochemistry staining with NeuN in red on spinal cord open-book preparations demonstrated lumbar specific defects in hop mice. White boxes indicate areas of higher magnification displayed in C'. C', Arrows point to the border between white matter and NeuN-positive cells, and stars denote areas where aberrant NeuN-positive cells are found. D, Fluorescence intensity analysis of NeuN signal within ROI as indicated (dotted lines in C') reflects aberrant NeuN positioning in hop mice (p = 0.009, r = −0.83; n = 8 per group). E–H, Immunohistochemistry staining with MBP in green and NeuN in red, coupled to density plots in G, H, shows misplaced neurons in the ventral spinal cord and a dorsally shifted central canal in hop mice at P5 (F, H) compared with control (E, G). I, Peroxidase staining on 60-μm transverse sections of adult spinal cords showed abnormal morphology in the lumbar spinal cord of hop mice. The central canal is dislocated dorsally and is smaller in size (arrowhead), the ventral commissure is missing, and the ventral white matter is disorganized or absent (black arrow). J–L, Close up and quantification of central canal phenotype. Dorsal shift (p ≤ 0.001, d = 3.88; K) and smaller size (p ≤ 0.001, d = 6.10; L) of central canal in hop mice (n = 12) compared with controls (n = 10). M, N, Immunohistochemistry staining with NeuN in red and MBP in green confirms misplaced neurons in the gray matter of ventral spinal cord in hop mice at adult age (N) compared with controls (M). O, Quantification of number NeuN-positive cells/section (n = 10 per genotype) in the lumbar white matter in the hop adult mice compared with control (p ≤ 0.001, d = 7.21). For brain morphology, refer to Extended Data Figure 2-1. The dotted line in E indicates the midline, and in M, N, the border between white and gray matter. Scale bars: 300 μm (A), 150 μm (I), 100 μm (J), and 50 μm (C–E, M, N). Significant effect of genotype **p < 0.01, ***p ≤ 0.001.
- Figure 3.
Neurotransmitter phenotype in the ventral spinal cord of postnatal hop mice. A–H, In situ hybridization on adult 60-μm floating sections using probes against Viaat (A, E), Vglut2 (B, F), and VAChT (C, D, G, H). In control mice, neurons marked with either Viaat (A) or Vglut2 (B) respect the border between white and gray matter, whereas in hop mice, Viaat (E) or Vglut2 (F) mRNA-expressing neurons intermingle with the white matter. ROI as indicated with dotted black line. Number of displaced Viaat+ cells (p < 0.001, d = −2.31; I) and Vglut2+ (p < 0.001, d = −3.16; J) neurons in ROI are significant higher in lumbar but not cervical spinal cord (p ≥ 0.05). VAChT stained cholinergic cells are found around the central canal in control (C) but are ventrally displaced relative to the central canal in hop mice (G). D, H, K, Motor neuron position (LMC-MMC) and number are similar (K) in hop mice (D) and controls (H). n = 11–13 sections (L1 to L3) from n = 3 mice per genotype; Black box in A indicates area of enlargements. Neurons in the white matter are marked with black arrows. Arrowheads indicate central canal. Scale bars: 50 μm (A–G) and 150 μm (D–H). Significant effect of genotype ***p ≤ 0.001.
- Figure 4.
Aberrant commissural fibers and interneuron positioning in the ventral lumbar spinal cord of early postnatal (P0–P1) hop mice. A, Schematics of different strategies for tracer application to detect CINs. Midline tracing using FDA to show local crossing CINs (B, C), local tracing using FDA to label short projecting CINs (D–E') or intersegmental FDA and RDA tracing to show ascending (aCINs), descending (dCINs), and bifurcating CINs (adCINs) in (F–G'). B, C, Transverse sections within the L2 level of spinal cords and density maps (H, I) show interneuron clustering on the midline in hop mice (C, I) as opposed to two ventromedial clusters in controls (B, H). N, Quantification of traced cells show similar number of cells found (p ≥ 0.05; n = 9–12 sections from 3 mice per genotype). D–E', Transverse sections within the L2 level traced on L2 (D, E) shows that the fibers freely cross the midline in hop (E') compared with control mice (D'). J, K, Density maps illustrate diffuse and aberrant positioning of CINs on the midline in hop mice compared with controls. O, Quantification of traced cells show similar CIN numbers (p ≥ 0.05; n = 6–9 sections from 3 mice per genotype). F, G', Aberrant fibers cross over the midline in spinal cords of hop mice (G, G') ventral from the central canal and the amount of long-range projecting CINs are reduced compared with controls. L, M, Density plots of aCINs (red) and dCINs (green) in control (L) and hop (M) spinal cords. P, Quantification of traced cells show a significant reduction of intersegmental projections over the midline (RDA: p ≤ 0.001, d = 2.92, FDA: p ≤ 0.001, d = 2.96; aCIN, dCIN, and adCIN: p ≤ 0.001, d = 4.46; n = 8–11 sections from 4 mice per genotype). For intersegmental tracing on cervical spinal refer to Extended Data Figure 4-1. White box in D, F indicates the area of close-up (D', G'). The outline of the spinal cord is indicated by a dashed line (B, C), central canal and midline are indicated by a dotted line (F', G'). Scale bars: 100 μm (B–G) and 200 μm (D'–G'). Significant effect of genotype ***p ≤ 0.001.
- Figure 5.
The distinct spinal cord phenotype in hop mice is present at embryonic stages. A, B, Photomicrographs of immunohistochemistry staining on E15.5 hop and control spinal cords with NeuN (green) and Map2 (red) showing aberrant fibers and misplaced neurons disrespecting the ventral midline. C, D', White box in A, B indicates ROI for statistical analysis of aberrant positioned NeuN+ cells and fiber crossing (C, NeuN+ cells: p < 0.001, d = −5.46; D', Map2 intensity as ratio to control: p < 0.001, d = −2.81; n = 5 per genotype). White line in A', B' indicates the thickness of the ventral commissure, quantified length as ratio to controls in D (p < 0.001, d = −5.71; n = 5 per genotype). E–H, RDA retrograde commisural interneuron (CIN) tracing on E15.5 (E, F) and E12.5 (G, H) spinal cord. Dotted line indicates midline and outline of the spinal cord. E'–H', Density plots visualizing medial displacement of traced CIN in hop (n = 12 sections from 3 embryos per genotype). I–P, In situ hybridization on E15.5 (I, J, M, N) and E12.5 (K, L, O, P) lumbar spinal cords using probes detecting Netrin1 and EphrinB3. Arrow heads point to regions of ventral Netrin1 and EphrinB3 mRNA expression. Q, R, Staining intensity (background corrected ratio) is significantly reduced in hop compared with control spinal cords for both axon guidance molecules and time points (Netrin; E12.5 p < 0.001, d = 5.86, n = 7 per group, E15.5: p < 0.001, d = 3.47, n = 5 per group; EphrinB3; E12.5 p < 0.001, d = 6.46, n = 7 per group, E15.5: p < 0.001, d = 5.19, n = 10 per group). Scale bars: 50 μm (A–H) 100 μm (I–P). Significant effect of genotype ***p ≤ 0.001.
- Figure 6.
Abnormal Shh and lumbar spinal cord patterning in hop mice. A–D, Photomicrographs of Shh immunohistochemistry staining on transverse spinal cord sections at E12.5 (A, B) and E10.5 (C, D). Notochord and floorplate phenotype at E12.5 in low power (A, B) and close up from control (A', A'') and hop (B', B'') spinal cords. Dashed lines outline the spinal cord and notochord. The distance between the notochord and spinal cord is indicated by stars (A, B) and is reduced in hop spinal cords (G; p < 0.001, d = 7.23, n = 15 per group). A', B', A malformed notochord together with reduced amounts of Shh (H; p = 0.002, d = 2.50, n = 5 per group). Lack of floorplate cells and severely reduced Shh expression in the spinal cord (A'', B'') of hop mice compared with controls (H; p = 0.016, d = 1.93, n = 5 per group). C, D, Similarly, we found reduced amounts of Shh at E10.5 in hop spinal cords compared with controls (H; p < 0.001, d = 1.36, n = 15 per group). E, F, Ttc26 immunostaining on E12.5 spinal cord transverse sections. For clarity, blue nuclear counterstain with DAPI has been omitted. I, The expression of Ttc26 is found in the notochord at similar intensity levels both in control and hop spinal cords. J–V, Immunostainings on E12.5 spinal cord sections reveal a missing Nkx2.2 progenitor domain and a migration defect of neurons in the lumbar spinal cord of hop mice. J, K, The expression of the ventral most V3 progenitor marker Nkx2.2 is absent in lumbar sections from hop mice (K). White box indicates area of higher magnification (J', K'). V, Nkx2.2 phenotype at different rostro-caudal levels exemplified with photomicrographs and visualized using density plots. The midline is indicated by lines (n = 4 sections per level). L–V, Density plots show misplaced cells and lack of a ventral midline for additional progenitor markers as specified below each panel (n = 3). For representative photomicrographs, refer to Extended Data Figure 6-1. W, Schematic summary of defects found in the hop mice in the lumbar part of the spinal cord including weak Shh expression, misplaced and malformed notochord, a dorsally shifted central canal, absence of floorplate and p3 domains as well as absence of a ventral midline. pdINs, progenitor domains for dorsal interneurons; FP, floor plate. The outline of the spinal cord and midline are indicated by dashed lines. FP, floorplate; NC, notochord. Scale bars: 100 μm (A, B, J, K, M–V), 25 μm (A', B') 30 μm (A'', B'', J', K'), and 50 μm (C, D). Significant effect of genotype *p < 0.05, **p < 0.01, ***p ≤ 0.001.
- Figure 7.
Introduction of a point mutation in Ttc26 and analysis Ttc26Y430X mutants. For initial validation of the hop mouse mutation, refer to Extended Data Figure 7-1. A, We then applied the CRISPR/Cas9 system to generate mice with a point mutation in exon 15 of the Ttc26 gene, generating a substitution of tyrosine to a premature stop codon. By simultaneous microinjection of guideRNA, hCas9 mRNA and a single-stranded donor oligonucleotide (ssDON) into mouse oocytes, mouse chromosome six harboring the Ttc26 gene was modified (left). The exon (black) and intron (red) organization is indicated together with Sanger sequencing traces of wild-type (bottom) and the mutated allele (top) highlighting the premature stop mutation. The introduction of a PvuII site for screening purposes (blue) and the location of the guideRNA are indicated. B, C, Images of E12.5 (B) and E16.5 (C) Ttc26Y430X mice showing preaxial polydactyly (arrowheads, star). D–F, Photomicrographs of Hematoxylin and Eosin staining on E16.5 transverse spinal cord sections. D, Lumbar spinal cord of control mice showing a well delineated central canal (upper black arrow), a present ventral funiculus with a border between white and gray matter (dashed line) and a present ventral spinal artery (lower arrow). E, Lumbar spinal cord of Ttc26Y430X mice showing a poorly delineated dorsally shifted central canal (upper arrow), an absent ventral funiculus (upper star) and an absent ventral spinal artery (lower arrow). F, Cervical spinal cord of Ttc26Y430X mice showing a well delineated central canal (upper arrow) and a present ventral funiculus with a clear midline border (lower arrow). G–L, Photomicrographs of immunohistochemistry experiments on E16.5 transverse lumbar spinal cord sections of control and Ttc26Y430X mice. G, NeuN staining showing a poorly defined ventral midline in Ttc26Y430X mice, with misplaced neurons within the white matter of the ventral funiculus. Enlargements on the right show cells positioned at the location of the missing midline in mutant but not control spinal cord sections (G, H). I, J, Cholinergic neurons as identified by their VAChT expression (red) were found in the medial and lateral motor neuron cluster, as well as in neurons around the central canal. Dmrt3-expressing interneurons were found medioventrally from the central canal. In mutant Ttc26Y430 spinal cords, medial and lateral motor neuron nuclei were located at their expected positions. In contrast, central canal cholinergic neurons and Dmrt3-expressing interneurons were found ventral of the central canal and positioned in close proximity or even on top of the presumed position of the midline. I, J, VAChT immunostaining showing no apparent differences regarding position and size of motor neurons between Ttc26Y430 and control mice. I', J', Central canal VAChT-expressing cells, as well as (K, L) Dmrt3-expressing cells, were ventrally and medially displaced in Ttc26Y430 spinal cords compared with controls. J', L', Enlargement of the ventromedial area of the spinal cord demonstrating misplaced cells in Ttc26Y430 spinal cords (J', arrows). Scale bars: 100 μm (D–H), 50 μm (I–L), and 100 μm (I'–L'). For comparative analysis in the rabbit, refer to Extended Data Figure 7-2.
Extended Data
Extended Data Figure 1-1
Mid-sagittal hemisection does not disrupt the ipsilateral alternating burst pattern. A, Neurograms illustrating a typical pattern recorded from a hop mouse. B, Pattern recorded from the preparation following a midsagittal hemisection. Note the preservation of the L3–L5 pattern suggesting that flexor-extensor coordination is maintained. C, Schematic illustrating stimulation and recording arrangement for cauda equina stimulation. Cauda equina evoked rhythmic pattern (4 Hz, 10-s train) showed segmental synchronous pattern and ipsilateral alternating pattern. Download Figure 1-1, TIF file.
Extended Data Figure 2-1
Brain commissure morphology appears normal in hop mice. Photomicrographs of horizontal sections from adult control (A, C, E, G, I, K) and hop (B, D, F, H, J, L) tissue. A, B, The corpus callosum (CC) and hippocampal commissure (HC) showed no gross defects. C, D, G, H, The anterior commissure (AC) also appeared normal. E, F, Sections showing the habenular commissure (HaC), the posterior commissure (PC), and the subcommissural organ (sc), all three present and with normal appearance in both control and hop mice. I, J, The pontine nuclei (pn) is also present in both genotypes. K, L, No aberrant commissure was found between the roof of the fourth ventricle (FV), and cerebellum (ce) at the junction of midbrain and hindbrain, in control and hop mice. Scale bars: 1000 μm (A–D, G, H) and 500 μm (E, F, I–L) Download Figure 2-1, TIF file.
Extended Data Figure 4-1
Normal CINs and axon midline crossing in the cervical spinal cord of early postnatal (P0–P1) hop mice. A–F, Intersegmental tracing using FDA/RDA to locate ascending (aCINs), descending (dCINs), and bifurcating CINs (adCINs). No apparent defects in hop mice (D–F') compared to controls (A–C') on cervical sections were found. Download Figure 4-1, TIF file.
Extended Data Figure 6-1
Embryonic patterning defects in hop spinal cords. Photomicrographs of immunohistochemistry experiments on E12.5 spinal cord transverse sections using antibodies against transcription factors as indicated at the top. White boxes in A–E indicate areas of higher magnification in the respective panel below (A'–E'). The stainings show misplaced cells and lack of ventral midline for all tested progenitor markers. The outline of the spinal cord, central canal, and midline are indicated by dashed lines. Scale bars: 100 μm (A–J), 50 μm (A', F'), and 30 μm (B', G'–J'). Download Figure 6-1, TIF file.
Supplementary Material
Extended Data Figure 7-1. Validation of the hop mouse mutation. The exon/intron organization of Ttc26 is indicated together with Sanger sequencing traces of wild type and hop highlighting the premature stop mutation in exon 15 (out of 18) of Ttc26. Genomic DNA from a hop homozygote (stock 002718) and BALB/cByJ control (stock 001026) was obtained from The Jackson Laboratory’s DNA Resource (n = 3 per genotype). Exomes were captured at the Yale Center for Genome Analysis, using the NimbleGen SeqCap EZ Mouse Exome and following NimbleGen protocols. Captured pools were sequenced (75 bp, paired-end) on an Illumina HiSeq 2000 using previously described methods (Choi et al., 2009). We obtained ∼91 million (BALB/cByJ) to ∼113 million (hop) high-quality reads. Illumina reads were first trimmed based on their quality scores to remove low-quality regions using the program Btrim (Kong, 2011). A cutoff of 20 for average quality scores within a moving window of size 5-bp was used. Minimum acceptable read length was 25 bp. Other parameters of Btrim were set to defaults. The preprocessed reads were then aligned to the mouse genome reference sequence (mm9) using the BWA mapping program (Li and Durbin, 2009). The mapping results were converted into SAMtools pileup format using SAMtools programs (Li et al., 2009). PCR duplicates were removed using the rmdup command from SAMtools, resulting in ∼84x (BALB/cByJ) or ∼100x (hop) coverage across the exome. More than 97% of all bases included in the exome showed at least 8x coverage and >90% of the bases showed at least 20x coverage. Single nucleotide variations (SNVs) were called using SAMtools’ pileup command. Further filtering was performed using in-house scripts to exclude those SNV calls that had less than 3 reads or a SNP score less than 20. Annotation was added based on the UCSC RefSeq gene model (http://genome.ucsc.edu/; Pruitt et al., 2009). Based on SNV homozygosity mapping, the interval was narrowed to ∼700 kb on chromosome 6 (37.9–38.6 Mb), which fell within the previously mapped hop genomic region. After filtering for known SNPs and repeat regions, we identified 17 possible point mutations, 16 of which were located in introns or 3′ untranslated regions. Download Figure 7-1, TIF file.
Extended Data Figure 7-2
Relatively high number of missense mutations in the rabbit Ttc26 gene. A, Amino acid alignment of TTC26 sequences from eight mammals made using MUSCLE with missense mutations in rabbit marked (pink boxes). B, Photomicrograph of HE stained lumbar spinal cord section from wild-type adult rabbit (box shown magnified region). The morphology is similar to the wild-type mouse spinal cord, with a well delineated central canal (upper arrow), a ventral funiculus with a clear border between white and grey matter and a ventral spinal artery (lower arrow). C–G, Immunohistochemistry performed on postnatal rabbits towards NeuN (C), Chat (D), Lbx1 (E), Parvalbumin (F), and Pax2 (G) show a clear bilateral separation of the spinal neuronal populations. Scale bars: 200 μm (C–G). Download Figure 7-2, TIF file.
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