Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice

Journal name:
Nature
Volume:
488,
Pages:
642–646
Date published:
DOI:
doi:10.1038/nature11399
Received
Accepted
Published online

Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement1. These networks produce left–right alternation of limbs as well as coordinated activation of flexor and extensor muscles2. Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favourable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes part in neuronal specification within this subdivision, and is critical for the normal development of a coordinated locomotor network controlling limb movements. Our discovery positions Dmrt3 in a pivotal role for configuring the spinal circuits controlling stride in vertebrates. The DMRT3 mutation has had a major effect on the diversification of the domestic horse, as the altered gait characteristics of a number of breeds apparently require this mutation.

At a glance

Figures

  1. Identification of a DMRT3 mutation in horses.
    Figure 1: Identification of a DMRT3 mutation in horses.

    a, A pacing Icelandic horse, fore- and hindlegs on the same side of the body are synchronized. b, A trotting Icelandic horse, the diagonal fore- and hindlegs are synchronized. c, Distribution of breeding evaluation test scores for pace and trot in Icelandic horses. Score 5.0 indicates ‘gait not shown’. d, Genome-wide association analysis revealed a highly significant association between the ability to pace and SNP BIEC2_620109 on chromosome 23 (Praw = 1.7×10−9, corrected empirical P-value (EMP2) = 2.0×10−4, genome-wide significance). e, The 684kb genomic interval associated with the Gait locus; the minimum Gait IBD region (438kb) is shaded. f, Partial amino acid alignment of the predicted Dmrt3 protein in wild-type (WT) and mutant (MUT) horses and in other vertebrates. Horse nonsense mutation (red asterisk), sequence identities (dashes), insertions/deletions (dots). g, Schematics of wild-type and mutant Dmrt3. DM, zinc-finger like DNA binding module; DMA, protein domain of unknown function present in DMRT proteins.

  2. Characterization of motor coordination in mice lacking Dmrt3.
    Figure 2: Characterization of motor coordination in mice lacking Dmrt3.

    a, Dmrt3−/− mice showed decreased swimming duration (P<0.0001), and an increase in twitching movements compared to control and Dmrt3+/− (P = 0.0002); n = 5 control and Dmrt3−/−, n = 7 Dmrt3+/−. Time spent immobile was similar between genotypes (P = 0.13). b, Mice were tested for their ability to run (>5 step cycles) at different treadmill speeds (9, 15, 20, 25, 30cms−1) (n = 15 trials per genotype, five animals). Dmrt3−/− mice had difficulties at 20cms−1 (P = 0.04) with markedly reduced number of successful trials at 25cms−1 (P = 0.006) and 30cms−1 (P<0.0001) compared to controls. c, Schematic drawing of the gait parameters analysed in treadmill locomotion. df, At 20cms−1 Dmrt3−/− showed increased stride (d, forelimb: P<0.0001, hindlimb: P = 0.006), hind limb stance (e, P = 0.02) and swing (f, forelimb: P<0.0001, hindlimb: P = 0.001) time duration compared to control (n = 11–12 trials per genotype, five animals). g, Images of a P4 mouse airstepping. h, Dmrt3−/− mice showed a decreased number of alternating hindlimb steps during airstepping (P = 0.02 compared to controls; n = 4 per genotype, except controls n = 3). Wild-type littermate controls (white), Dmrt3+/− (grey), Dmrt3−/− (black). Mean±s.e.m. im, Fictive locomotion was recorded from ventral left (l) and right (r) lumbar (L) root 2 and 5 from neonatal spinal cords. i, j, Representative traces from control (i) and Dmrt3−/− (j) spinal cords. Time scale 10s. k, Dmrt3−/− mice displayed increased burst and interburst durations compared to control animals, analysis made on L2s (n = 6 per genotype, P = 0.02, burst; P = 0.001, interburst). l, m, Coherence power spectra analysis of left/right (l/r) and flexor/extensor (f/e) recordings (colour-graded scale; n = 3–5 per genotype). Time scale 100s.

  3. Characterization of Dmrt3-expressing cells in the mouse spinal cord.
    Figure 3: Characterization of Dmrt3-expressing cells in the mouse spinal cord.

    a, Dmrt3 mRNA expression pattern in adult spinal cord (P60). b, Dmrt3 mRNA expression in a restricted population of neurons migrating ventrally in the developing spinal cord at E11.5. c, A schematic spinal cord cross-section showing progenitor and transcription factor domains. d, Double immunolabelling of Dmrt3 and Pax7 shows that Dmrt3+ cells originate from the ventral-most part (bracket) of the dorsal domain (border indicated by line). Dmrt3+ cells overlap with the dI4/dI6/V0d marker Pax2, but not with the V0V/V0C/V0G marker Evx1 or the dI5 marker Lmx1b (compare brackets). Dmrt3+ cells overlap with the dI4/dI5/dI6 marker Lbx1. e, Double immunolabelling with Dmrt3 (arrowhead) and Wt1 (double arrow) show a partial overlap (arrow). f, Dmrt3+ interneurons (arrows) co-labelled with Viaat mRNA (green) but not with Vglut2 mRNA (green). g, Schematic of trans-synaptic muscle tracing of Dmrt3 neurons in the spinal cord (n = 6, ipsilateral; n = 5, contralateral). h, Double immunolabelling of Dmrt3 and green fluorescent protein (PRV152) show that both ipsi- and contralateral premotor interneurons overlap with Dmrt3+ cells (arrows). i, Quantification of Brn3a/Lbx1-positive neurons (control nsection = 16, Dmrt3−/− nsection = 21) and of Lbx1/Pax2-positive neurons (control nsection = 11, Dmrt3−/− nsection = 19). j, Immunolabelling of Wt1+ cells (red) in spinal cord sections from control and Dmrt3−/− E15.5 embryos. k, Quantification demonstrated that loss of Dmrt3 leads to an expanded Wt1+ cell subpopulation (control n = 43, Dmrt3+/− n = 36, Dmrt3−/− n = 46, ***P<0.0001). l, Schematic illustration of fate change in the Dmrt3 dI6 population of neurons. Mean±s.e.m. Scale bars: 400μm (a), 70μm (b, d, j), 50μm (e, f, h).

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Sequence Read Archive

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Author information

  1. These authors contributed equally to this work.

    • Lisa S. Andersson,
    • Martin Larhammar,
    • Fatima Memic,
    • Hanna Wootz,
    • Leif Andersson &
    • Klas Kullander

Affiliations

  1. Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, SE-75124 Uppsala, Sweden

    • Lisa S. Andersson,
    • Doreen Schwochow,
    • Lisbeth Wellbring,
    • Sofia Mikko,
    • Gabriella Lindgren &
    • Leif Andersson
  2. Department of Neuroscience, Uppsala University, SE-75124 Uppsala, Sweden

    • Martin Larhammar,
    • Fatima Memic,
    • Hanna Wootz,
    • Kalicharan Patra,
    • Anna Vallstedt &
    • Klas Kullander
  3. Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden

    • Carl-Johan Rubin,
    • Göran Hjälm,
    • Freyja Imsland &
    • Leif Andersson
  4. Faculty of Land and Animal Resources, The Agricultural University of Iceland, IS-311 Borgarnes, Iceland

    • Thorvaldur Arnason
  5. College of Veterinary Medicine, University of Minnesota, St Paul, Minnesota 55108, USA

    • Jessica L. Petersen,
    • Molly E. McCue &
    • James R. Mickelson
  6. Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77483, USA

    • Gus Cothran
  7. Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California 94143, USA

    • Nadav Ahituv
  8. Institute for Human Genetics, University of California San Francisco, San Francisco, California 94143, USA

    • Nadav Ahituv
  9. Unit of Equine Studies, Swedish University of Agricultural Sciences, Uppsala, SE-75007 Sweden

    • Lars Roepstorff

Contributions

L.S.A., S.M., G.L. and L.W. collected the horse material and/or performed the genome-wide association analysis. L.S.A., D.S., M.L., G.H. and L.A. planned, designed, performed and/or analysed horse experiments. M.L., F.M., H.W., K.P., A.V. and K.K. planned, designed performed and/or analysed mouse experiments. C.-J.R. performed bioinformatic analysis. T.A. analysed horse performance data. N.A., F.I., J.L.P., M.E.M., J.R.M. and G.C. contributed with materials. L.R. recorded horse gaits. L.A. led positional cloning and characterisation of horse DMRT3. K.K. led the mouse studies. K.K. and L.A. wrote the paper with contributions from all authors.

Competing financial interests

A patent application has been filed concerning the commercial use of some of the results presented in this paper.

Corresponding author

Correspondence to:

The Illumina reads have been submitted to the short reads archive (http://www.ncbi.nlm.nih.gov/sra); the accession number for the study is SRP012260 and accession numbers for individual data are: four-gaited horse, SRS309533; five-gaited horse, SRS309532. Sanger sequencing data have been submitted to GenBank (accession numbers JQ922365–JQ922395).

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Supplementary information

PDF files

  1. Supplementary Information (13.8M)

    This file contains Supplementary Figures 1-7, Supplementary Methods, Supplementary Notes, Supplementary References and legends for Supplementary Movies 1-4.

  2. Supplementary Tables (693K)

    This file contains Supplementary Tables 1-6.

Movies

  1. Supplementary Movie 1 (10.1M)

    This movie shows a side view of a heterozygous (CA) Swedish Standardbred trotter that performs a fairly symmetric and normal trot stride as well as a very asymmetric stride (see Supplementary Information file for full legend).

  2. Supplementary Movie 2 (11.5M)

    This movie shows a side-by-side view of wild-type and Dmrt3-/- mice (KO) mice during swimming - see Supplementary Information file for full legend.

  3. Supplementary Movie 3 (35.3M)

    This movie shows wild-type and Dmrt3-/- mice during treadmill locomotion - see Supplementary Information file for full legend.

  4. Supplementary Movie 4 (23.4M)

    This movie shows wild-type and Dmrt3-/- mice during airstepping - see Supplementary Information file for full legend.

Additional data