The KIT gene is associated with the english spotting coat color locus and congenital megacolon in Checkered Giant rabbits (Oryctolagus cuniculus)

Abstract

The English spotting coat color locus in rabbits, also known as Dominant white spotting locus, is determined by an incompletely dominant allele (En). Rabbits homozygous for the recessive wild-type allele (en/en) are self-colored, heterozygous En/en rabbits are normally spotted, and homozygous En/En animals are almost completely white. Compared to vital en/en and En/en rabbits, En/En animals are subvital because of a dilated ("mega") cecum and ascending colon. In this study, we investigated the role of the KIT gene as a candidate for the English spotting locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of En/En rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (En/en) with nine Checkered Giant (En/en) and two en/en does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the KIT gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the English spotting coat color phenotype (θ=0.00 LOD =75.56). KIT gene expression in cecum and colon specimens of En/En (pathological) rabbits was 5-10% of that of en/en (control) rabbits. En/En rabbits showed reduced and altered c-kit immunolabelled ICC compared to en/en controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (P<0.05) and substance P (P<0.01) immunoreactive neurons in En/En vs. en/en. Electron microscopy analysis showed neuronal and ICC abnormalities in En/En tissues. The En/En rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon.

Figure 1. F1 and backcrossed families obtained from Checkered Giant parental animals.

Black squares and circles  =  rabbits with en/en genotype at the English spotting locus; spotted squares and circles  =  rabbits with En/en genotype; white squares and circles  =  rabbits with En/En genotype. ID of the animals is reported in blue or red (for those used as parental animals). The genotype of the g.93948587T>C SNP is reported for all animals. Rabbits that are circled in green have been used to collect specimens for anatomo-histochemical, gene expression analyses and resequencing (rabbits with n. 1–9). Rabbits that are circled in orange have been inspected after slaughtering. Animals with underlined ID showed hard feces in the intestine.

Figure 2. Typical appearance of the intestine in En/En and en/en weaned rabbits.

Macroscopic features of the gut in the rabbit model investigated in this study. Note the markedly dilated ascending colon in En/En (pathological) compared to en/en rabbits (control).

Figure 3. Rabbits with En/En, En/en and en/en genotypes at the English spotting locus.

The genotypes are identified by the three coat color phenotypes, respectively.

Figure 4. Relative KIT gene expression levels in the ascending colon of En/En and en/en rabbits.

In En/En rabbits KIT expression was significantly lower than in en/en rabbits (5±2%; P<0.00001, t-test).

Figure 5. Representative images of rabbit colonic mucosa in En/En vs en/en.

A) En/En; B) en/en. The arrow indicate c-kit immunoreactivity in the tunica muscularis of the ascending colon in an en/en rabbit.

Figure 6. Representative images of colon whole mounts in En/En and en/en rabbit myenteric plexuses.

A) En/En myenteric ganglia (arrows) with human neuronal protein immunoreactive (HuC/D-IR) neurons. B) En/En myenteric ganglia (arrows) with nitric oxide synthase immunoreactive (nNOS-IR) neurons, (arrowheads) nNOS-IR nerve bundles. C) en/en myenteric ganglia (arrows) with HuC/D-IR neurons. D) en/en myenteric ganglia (arrows) with nNOS-IR neurons, (arrowheads) nNOS-IR nerve bundles arranged within primary, secondary and tertiary nerve strands. In En/En (A and B) the ganglia and nerve bundles are less dense, have a lower number of HuC/D-IR and nNOS-IR neurons compared to en/en rabbits (C and D). E) and F): differences between En/En vs en/en SP-IR neurons (arrow), ganglia and nerve bundles (arrowhead) in the morphology of myenteric plexus in the ascending colon. In En/En (E) ganglia (arrow) are small and have a lower number of substance P immunoreactive (SP-IR) neurons than en/en rabbits (F). A–D scale bars 200 µm; E–F scale bars 100 µm.

Figure 7. Mean density of immunoreactive myenteric neurons in En/En vs en/en rabbits.

Different immunoreactive neurons are reported: human neuronal protein (HuC/D) (A: En/En vs en/en, P<0.05), nitric oxide synthase (nNOS) (B) and substance P (SP) (C: En/En vs en/en, P<0.01) myenteric labeled neurons. Results are expressed as mean ± standard error of the mean.

Figure 8. Electron microscopy of Interstitial cells of Cajal (ICC).

en/en (A and B) control and En/En (C and D) pathological animals. A) ascending colon: an intramuscular ICC close to a nerve bundle (NB) and smooth muscle cells (SMC). Arrows indicate cell-to-cell contacts between ICC and SMC. x12,500. B) ascending colon: ICC at the submucosal border of the circular muscle layer showing in the cytoplasm several cisternae of the smooth endoplasmic reticulum and filaments, and caveolae along the plasma membrane; on the upper side, a nerve bundle and, on the lower side, the smooth muscle cells (SMC). x12,500. C) ascending colon: an intramuscular ICC with swollen mitochondria and extremely dilated cisternae of rough endoplasmic reticulum; on the upper side, a blood capillary (BC); on the right side, a nerve bundle (NB). The arrowhead indicates a nerve ending near the ICC. x15.000. D) descending colon: several intramuscular ICC with large intracytoplasmatic vacuoles; the arrows indicate cell-to-cell contacts between ICC and SMC. x7,500.

Figure 9. Electron microscopy of myenteric neurons.

en/en control (A) and En/En (B and C) pathological animals. A) descending colon: the neuron has an extended Golgi apparatus (G), thin RER cisternae, free ribosomes, and small oval shaped mitochondria. x10,000. B) descending colon: detail of the cytoplasm of a neuron with dilated RER cisternae and swollen mitochondria; G: Golgi apparatus. x15,000. C) ascending colon: a myenteric ganglion with three cells identifiable as neuroblasts (Nbl). These cells are small and have a scarce cytoplasm mainly containing free ribosomes. Glia: glial cells. x10,000. Intragangliar nerve endings (NE): pathological animals. D) descending and E) ascending colon. Most of the NE has an anomalous content: few or abnormally featured synaptic vesicles, lamellar bodies, lysosomes. D) x15,000; E) x20,000.