A single gene mutation is enough to prevent rabbits from getting about using their characteristic jumping gait, according to the results of research headed by Miguel Carneiro PhD, of the Universidade do Porto, and Leif Andersson, PhD, at Uppsala University.

Their studies in an unusual strain of domesticated rabbit that can’t jump—but instead gets about by lifting its back legs off the ground and walking on its front paws—pinpointed the animals’ inability to move about like normal rabbits to a single mutation in the transcription factor gene RORB. The studies showed that the RORB gene mutation is causal for the sauteur rabbit’s inability to hop, and results in a drastic reduction in RORB-positive neurons in the spinal cord, as well as defects in differentiation of populations of spinal cord interneurons, which ultimately results in limb discoordination.

Reporting on their work in PLOS Genetics, the team wrote, “This discoordination dramatically impairs efficient locomotion, and as a consequence, individuals from this strain adapt their locomotion behavior for longer and/or faster movements by lifting the hindlimbs off the ground and move supported solely by their forelimbs, similarly to a human acrobat when walking on hands.”

The results also provide new insights into the different ways that animals with backbones move. Carneiro, Andersson and colleagues describe their findings in a paper titled, “A loss-of-function mutation in RORB disrupts saltatorial locomotion in rabbits.”

Most mammals can switch between different gaits (e.g. walking, running, trotting, galloping), to change speed, or to aid travel across different terrains, the authors explained. Different species have different gait patterns, partly dependent on whether they move around on two legs, or four legs.

Rabbits and hares have a characteristic jumping gait, by which they alternate rhythmical movement of the forelimbs, and then synchronous bilateral movement of the hindlimbs. Its known as saltatorial locomotion, and kangaroos and some rodent species typically hop or jump about using a similar type of movement.

But while we can describe these different gaits in animals, What isn’t well understood is how they are controlled, and fine-tuned at the molecular and genetic level, the team continued. “Despite intense interest in the biomechanical, morphological, and physiological adaptations that characterize distinct types of locomotion in vertebrates, the genetic, molecular, and developmental bases underlying differences between individuals and species have seldom been reported.”

The researchers looked more closely at jumping-related genes in a strain of domesticated rabbit known as sauteur d’Alfort. Rather than adopt the jumping gait that is typical of rabbits, the sauteur has a strange gait at both slower and faster speeds. “At slow speed, during the swing phase, they lift excessively their hindlimbs. At higher speed, the movements of the hindlimbs, instead of being synchronized, show a slight shift and the sauteur rabbits never perform the jumping,” the team noted. And when they want to move faster, the sauteur rabbit lifts its back legs off the ground completely, and walks on just  its front paws. “… individuals from this strain adapt their locomotion behavior for longer and/or faster movements by lifting the hindlimbs off the ground and move supported solely by their forelimbs, similarly to a human acrobat when walking on hands,” the team continued. Sauteur rabbits are also born blind because of retinal dysplasia, and start developing cataracts after their first year of life.

To carry out their studies the team crossed a male sauteur rabbit with a female wild type rabbit. “… we crossed a single male of the sauteur D’Alfort strain, expected to be homozygous (sam/sam), with a single female of the New Zealand white breed homozygous for the wild-type allele (+/+).” And by comparing the offspring genomes and jumping abilities, the researchers identified the cause of the underlying developmental defect as a specific mutation in the RAR related orphan receptor B (RORB) gene.

The sauteur d’alfort strain and associated phenotypes. (A) Typical posture of a sauteur rabbit (sam/sam) adopted when jumping (i.e., moving faster or across longer distances). Hindlegs are lifted from the ground, the body is held vertically, and locomotion is achieved through the alternate use of the forelegs. (B) Ocular malformations observed both in sam/sam and +/sam individuals include bilateral papillary colobomas, reduction in pupillary reflexes, bilateral cataracts with lesions in various components of the eye, glaucoma, and/or entropion and ectropion. [Taken by (A) R. Cavignaux; (B) S. Boucher: Carneiro M et al., 2021, PLOS Genetics]

Typically, the RORB protein is found in many regions of the rabbit nervous system, but immunohistochemical (IHC) analyses showed that the RORB mutation leads to a sharp decrease in the number of neurons in the spinal cord that produce RORB. “In the spinal cord from rabbits heterozygous for the sauteur allele (+/sam), the number of neurons expressing RORB was approximately 25% lower than in the wild-type animals. In contrast, in rabbits homozygous for the sauteur allele (sam/ sam), the expression of RORB was undetectable by IHC,” they wrote. “This suggests that the high proportion of abnormal transcripts in the spinal cord of sauteur rabbits results in a drastic reduction of RORB-positive neurons when compared to wild-type and heterozygous rabbits. This defect may cause the anomalous motor phenotype observed in sauteur rabbits.”

The researchers’ investigations confirmed that the RORB mutation was directly responsible for the sauteur d’Alfort animals’ weird walk, and demonstrate that a functional RORB gene is necessary for rabbits—and likely other hopping animals—to perform their characteristic jumping gait. “This study provides a rare example of an abnormal gait behavior mapped to a single base change and the first description of a gene required for saltatorial locomotion … It further demonstrates the importance of the RORB protein for the normal wiring of the spinal cord …”

Interestingly, the findings build on previous studies showing that mice lacking the RORB gene had a duck-like walk. “The causality of the RORB splice site mutation in sauteur rabbits is further supported by the phenotypic overlap with Rorb knock-out mice, which show retinal degeneration and a duck-like gait,” the team noted. “In conclusion, this study demonstrates that a mutation in the RORB gene is the cause of the locomotion phenotype observed in sauteur rabbits, likely through aberrant differentiation of spinal interneurons.”