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LETTER doi:10.1038/nature11399 Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice Lisa S.

Andersson1*, Martin Larhammar2*, Fatima Memic2*, Hanna Wootz2*, Doreen Schwochow1, Carl-Johan Rubin3, ¨ran Hja ¨lm3, Freyja Imsland3, Jessica L.

Petersen5, Molly E.

McCue5, Kalicharan Patra2, Thorvaldur Arnason4, Lisbeth Wellbring1, Go James R.

Mickelson5, Gus Cothran6, Nadav Ahituv7,8, Lars Roepstorff9, Sofia Mikko1, Anna Vallstedt2, Gabriella Lindgren1, Leif Andersson1,3* & Klas Kullander2* 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.

Horses show considerable variation in the pattern of locomotion.

The three naturally occurring gaits in all equids are, in order of increasing speed, walk, trot and canter/gallop.

Some horses can use alternate gaits, typically at intermediate speed, and ‘gaitedness’ is a trait upon which many specialized breeds have been developed.

Based on variation in footfall pattern, timing and cadence, these alternate gaits can be generally divided into four categories: pace, regular rhythm ambling, lateral ambling and diagonal ambling (Supplementary Notes and Supplementary Table 1).

Pace is a two-beat gait in which the horse moves the two legs on the same side of the body in a synchronized, lateral movement (Fig. 1a) in contrast to the trot, where the diagonal front and hind legs move forward and backward together (Fig. 1b).

Ambling gaits are four-beat gaits in which footfall pattern, foot placement and timing are often unique to specific breeds (Supplementary Notes and Supplementary Table 1).

To ¨ lt is a regular ambling gait characteristic of the Icelandic horse.

Many Icelandic horses also have the ability to pace and test scores for pace show a bimodal distribution (Fig. 1c) and high heritability, in the range 0.60–0.73 (ref. 3).

A genome-wide association analysis using 30 Icelandic horses classified as four-gaited (walk, to ¨ lt, trot and gallop) and 40 classified as five-gaited (walk, to ¨ lt, trot, gallop and pace) revealed a highly significant association between the ability to pace and a single nucleotide polymorphism (SNP; BIEC2_620109) at nucleotide position 22967656 on chromosome 23 (Fig. 1d).

The two flanking markers showed only weak association to the pacing phenotype, indicating that the mutation(s) underlying the association is located within the 684-kilobases 1 3 interval chr23:22628976–23315071.

We resequenced selected regions of the 684-kb interval in a panel of four- and five-gaited Icelandic horses.

The five-gaited horses were all homozygous for a minimal 438-kb haplotype (chr23:22877015–23315071), that was inferred to be identical-by-descent (IBD; Supplementary Table 2).

This region contains only three genes encoding different isoforms of the doublesex and mab-3 related transcription factors, DMRT1-3 (Fig. 1e).

The DMRT family of transcription factors carry a DM (dsx and mab-3) DNA-binding domain, conferring sequence-specific DNA binding distinct from a classical zinc-finger4.

We performed whole-genome resequencing of one four-gaited and one five-gaited Icelandic horse, homozygous for opposite alleles at the SNP associated with the ability to pace.

Average sequence coverage of 303 was obtained and polymorphisms identified in the critical 438-kb interval were compiled (Supplementary Table 3).

Homozygosity mapping using the sequenced five-gaited horse confirmed an IBD region of about 438 kb.

In this interval, we identified 65 sequence differences (60 SNPs and five small insertions/deletions) unique to the five-gaited horse when comparing data for the two horses and the reference genome (Supplementary Table 4); no structural rearrangements were detected.

We found five intronic or intragenic SNPs at sites showing some degree of evolutionary conservation, and a single base change at nucleotide position chr23:22999655 causing a premature stop at codon 301 in DMRT3 (DMRT3_Ser301STOP; Fig. 1f).

The allele is expected to encode a truncated protein lacking 174 amino acid residues of the full-length protein (Fig. 1g), of which 161 (92.5%) are identical between human and horse Dmrt3.

DMRT3_Ser301STOP was evaluated as the candidate causative mutation.

We genotyped 352 additional Icelandic horses and found that all but one of the five-gaited horses were homozygous A/A for the DMRT3 nonsense mutation (Table 1); further investigation of competition records revealed that this single discordant horse was most likely phenotypically misclassified.

In contrast, only 31% of the four-gaited horses were homozygous A/A (P 5 2.4 3 10214).

Thus, homozygosity for the DMRT3 nonsense mutation is required for the ability to pace in this breed.

The observation that a considerable number of homozygous mutant horses are considered four-gaited may reflect phenotype misclassifications, but more likely incomplete penetrance due to other genetic factors, maturity and environmental effects, in particular training.

The DMRT3 genotype distribution across breeds was markedly dichotomous, with the mutation occurring at high frequency in all gaited breeds, whereas all tested non-gaited horses were homozygous wild type (Table 1), with the exception of horses used for harness Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, SE-75124 Uppsala, Sweden. 2Department of Neuroscience, Uppsala University, SE-75124 Uppsala, Sweden.

Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden. 4Faculty of Land and Animal Resources, The Agricultural University of Iceland, IS-311 Borgarnes, Iceland. 5College of Veterinary Medicine, University of Minnesota, St Paul, Minnesota 55108, USA. 6Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77483, USA. 7Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California 94143, USA. 8 Institute for Human Genetics, University of California San Francisco, San Francisco, California 94143, USA. 9Unit of Equine Studies, Swedish University of Agricultural Sciences, Uppsala, SE-75007 Sweden. *These authors contributed equally to this work. 6 4 2 | N AT U R E | VO L 4 8 8 | 3 0 AU G U S T 2 0 1 2 ©2012 Macmillan Publishers Limited.

All rights reserved LETTER RESEARCH a b c Number of horses 5,000 4,000 3,000 2,000 1,000 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.510.0 Scores from breeding field test 280 300 320 Trot Pace d 8 6 4 2 — g DM WT 474 aa MUT 300 aa DMA 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 5 1.7 3 1029, corrected empirical P-value (EMP2) 5 2.0 3 1024, genome-wide significance). e, The 684 kb genomic interval associated with the Gait locus; the minimum Gait IBD region (438 kb) 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. racing (see below).

Nearly all individuals from other gaited breeds were homozygous mutant, regardless of whether their four-beat alternate gait is characterized by lateral or diagonal couplets (Supplementary Table 1).

Thus, the DMRT3 mutation is permissive for the ability to perform alternate gaits, which can be either pace or four-beat ambling gaits.

Although this mutation must be advantageous for gaited horses, Table 1 | Allele frequency of the DMRT3 nonsense mutation among horse populations Breed n p(A) Icelandic horses* Four-gaited{ Five-gaited Random sample Other gaited horses Kentucky mountain saddle horse Missouri fox trotter Paso fino Peruvian paso Rocky mountain horse Tennessee walking horse Non-gaited horses Arabian horse Gotland pony North-Swedish draft horse Przewalski’s horse Shetland pony Swedish ardennes Swedish warmblood Thoroughbred Horses bred for harness racing Standardbred, trotter (Sweden) Standardbred, trotter (USA) Standardbred, pacer (USA) French trotter (France) 124 66 162 22 40 45 19 17 33 18 28 31 6 20 22 64 29 270 57 40 47 0.65 0.99 0.89 0.95 1.00 1.00 1.00 1.00 0.98 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.97 1.00 1.00 0.77 * These do not include the horses used in the initial genome-wide association and therefore provide a replication of the highly significant association. { Thirty-eight of the 124 four-gaited horses were homozygous A/A.

N, number of horses; p(A)5allele frequency of the DMRT3 nonsense mutation. it may be disadvantageous for others.

In fact, Icelandic horse homozygous mutants had inferior scores for gallop and trot (Supplementary Table 5).

Thus, there may be selection against the mutation in nongaited horses bred for dressage, show jumping or high-speed gallop.

We found a high frequency of the DMRT3 mutation in horses bred for harness racing (Table 1).

These horses have the ability to trot or pace at high speed without breaking into a gallop, the natural gait at high speed for horses.

The American Standardbred was established in the 19th century and bred for harness racing.competitions are held separately in trot or pace and assortative mating based on preferred gait has subdivided the breed into two populations, pacers and trotters.

In contrast to the pattern in Icelandic horses, where homozygosity for DMRT3_Ser301STOP was associated with the ability to pace, both Standardbred pacers and trotters are homozygous for the mutation.

Thus, the mutation may promote the ability to trot or pace at high speed and genetic modifiers determine the gait to which the horse is best suited.

The Swedish Standardbred is largely developed from the American Standardbred but is not completely fixed for the DMRT3 mutation, probably owing to the import of French trotters, a breed with a fairly high frequency of the wild-type allele (Table 1).

The segregation of the two alleles in the Swedish Standardbred provided an opportunity to examine the effect of the mutation on racing performance.

The DMRT3 mutation was associated with superior breeding values (BV) for racing performance (BVCA 5 95.7 6 1.7, n 5 17; BVAA 5 109.0 6 0.8, n 5 206; P , 0.0001) and increased earned prize money (XCA 5 48,000 6 US$35,000, n 5 17; XAA 5 161,000 6 US$24,000, n 5 206; Pone-sided 5 0.007).

We also genotyped 61 horses from one racing camp in a blind test; two of these had major difficulties in sustaining trot at high speed (Supplementary Fig. 1a, b and Supplementary Movie 1) and were heterozygous C/A, whereas all others were homozygous A/A (P 5 0.0005).

Whereas the horse discovery demonstrates that DMRT3 has an effect on gait coordination, studies of its possible role in locomotor 3 0 AU G U S T 2 0 1 2 | VO L 4 8 8 | N AT U R E | 6 4 3 ©2012 Macmillan Publishers Limited.

All rights reserved — Ctrl Dmrt3–/– 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 Dmrt31 cells originate from the ventral-most part (bracket) of the dorsal domain (border indicated by line).

Dmrt31 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).

Dmrt31 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, Dmrt31 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 5 6, ipsilateral; n 5 5, contralateral).

H, Double immunolabelling of Dmrt3 and green fluorescent protein (PRV152) show that both ipsi- and contralateral premotor interneurons overlap with Dmrt31 cells (arrows).

I, Quantification of Brn3a/ Lbx1-positive neurons (control nsection 5 16, Dmrt32/2 nsection 5 21) and of Lbx1/Pax2-positive neurons (control nsection 5 11, Dmrt32/2 nsection 5 19).

J, Immunolabelling of Wt11 cells (red) in spinal cord sections from control and Dmrt32/2 E15.5 embryos.

K, Quantification demonstrated that loss of Dmrt3 leads to an expanded Wt11 cell subpopulation (control n 5 43, Dmrt31/2 n 5 36, Dmrt32/2 n 5 46, ***P , 0.0001).

L, Schematic illustration of fate change in the Dmrt3 dI6 population of neurons.

Mean 6 s.e.m.

Scale bars: 400 mm (a), 70 mm (b, d, j), 50 mm (e, f, h). Expression levels between mutant and wild-type homozygotes were similar and DMRT3 mRNA was found in a small population of neurons located in the ventral horn and around the central canal in both wildtype and mutant horses (Supplementary Fig. 1c–e).

Furthermore, 3 0 AU G U S T 2 0 1 2 | VO L 4 8 8 | N AT U R E | 6 4 5 ©2012 Macmillan Publishers Limited.

All rights reserved RESEARCH LETTER transfection experiments and an electrophoretic mobility shift assay indicated that the mutant Dmrt3 protein maintain its cellular localization and DNA-binding profile (Supplementary Fig. 1f, g).

It may therefore be a dominant negative form with normal DNA binding but defective protein interactions.

The remarkable association between the DMRT3 nonsense mutation and gaitedness across horse breeds, combined with the demonstration that mouse Dmrt3 is required for normal development of a coordinated locomotor network in the spinal cord, allow us to conclude that DMRT3_Ser301STOP is a causative mutation affecting the pattern of locomotion in horses.

The horse phenotype indicates that Dmrt3 neurons not only have a critical role for left/right coordination but also for coordinating the movement of the fore- and hindlegs.

The mutation facilitates lateral gaits, ambling and pace, and inhibits the transition from trot or pace to gallop.

Homozygosity for the mutation is required, but not sufficient for pacing, as many Standardbred trotters and some Icelandic horses that are homozygous mutant do not pace.

In mice, a complete loss of Dmrt3 on one allele does not lead to any detectable phenotype, whereas in Icelandic horses, heterozygosity for the DMRT3_Ser301STOP mutation promotes to ¨ lt, supporting our hypothesis that the mutant protein in horses acts as a dominant negative form.

Dmrt3 neurons are present in the horse and mouse spinal cord, and in the mouse, they develop into premotor inhibitory interneurons projecting ipsi- and contralaterally.

Inhibitory commissural connections have been suggested as major constituents of left-right phasing during locomotion23–25, and in cat, such interneurons have been implicated in mediation of the crossed reflexes and in the selection of different motor patterns26,27.

Thus, Dmrt3 neurons have a character and position in spinal cord circuitry that concurs with gait coordination. 11.

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Supplementary Information is available in the online version of the paper.

Acknowledgements Thanks to S.

Mikulovic and E.

Restrepo for valuable input, C.

Birchmeier for Lbx1 antibody, L.

Enquist and J.

Martin for PRV152, S.

Ewart for horse ˚ gerup for access to race horses.

The work was supported by grants samples, and B.

A from the Swedish Foundation for Strategic Research, the Swedish Research Council Formas (221-2009-1631), Swedish Research Council Medicine and Health (2007-3630/4479, 2010-4394), Swedish Society for Medical Research (H.W.), National Institute of Child Health & Human Development R01HD059862 (N.A.), and the Swedish Brain Foundation.

Sequencing was performed by the SNP&SEQ Technology Platform, supported by Uppsala University and Hospital, SciLife Lab – Uppsala and the Swedish Research Council (80576801 and 70374401).computer resources were supplied by UPPMAX.


Is a Royal Swedish Academy of Sciences Research Fellow supported by a grant from the Knut and Alice Wallenberg Foundation.

Author 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.


Performed bioinformatic analysis.


Analysed horse performance data.

N.A., F.I., J.L.P., M.E.M., J.R.M.

And G.C.

Contributed with materials.


Recorded horse gaits.


Led positional cloning and characterisation of horse DMRT3.


Led the mouse studies.


And L.A.

Wrote the paper with contributions from all authors.

Author Information The Illumina reads have been submitted to the short reads archive (; 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).

Reprints and permissions information is available at

This paper is distributed under the terms of the Creative Commons Attribution-Non-Commercial-Share Alike licence, and the online version of the paper is freely available to all readers.

The authors declare competing financial interests: details are available in the online version of the paper.

Readers are welcome to comment on the online version of the paper.

Correspondence and requests for materials should be addressed to L.A. ( METHODS SUMMARY A summary of the methods can be found in the Supplementary Information and includes detailed information on study populations, genotyping methods and genome-wide association analysis, genome resequencing and calling of genetic variants, Dmrt3-null mice, immunohistochemistry, in situ hybridization of mouse and horse tissue, spinal cord and muscle tracing, extracellular physiology, behaviour recordings and statistical analyses, expression analysis using mouse and horse tissue, transfection experiments and electrophoretic mobility shift assays. Received 18 April; accepted 5 July 2012.

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Dev. 33, 27–37 (1990). 1. 6 4 6 | N AT U R E | VO L 4 8 8 | 3 0 AU G U S T 2 0 1 2 ©2012 Macmillan Publishers Limited.

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