Genetic Basis Of Semi-lethal Creeper Trait In Chicken

The Creeper trait, a classical monogenic phenotype of chicken, is controlled by an autosomal dominant semi-lethal gene, Cp, which is mainly characterized by a pronounced shortness of the extremities. This trait has been widely cited in the genetics and molecular biology textbooks for illustrating autosomal dominant semi-lethal inheritance for decades. Researchers around the world have been studying the Cp gene for more than ninety years. However, the genomic mapping, structure, and function of the Cp gene are unknown.In the present study, Chinese Xingyi bantam chicken will be used as experimental populations and segregated families will be constructed to generate full-sib progeny. In the incubation experiment, we incubated fertile eggs from Creeper chickens inter se (Group I) and the control cross between Creeper and wild-type birds (Group Ⅱ). Early mortality and segregation ratio from two different matings were further analyzed. The results showed that 29.27% and 4.61% early embryonic mortality for the Creeper chickens and the control cross between Creeper and wild-type birds. Embryonic mortality of Creepers inter se expressed was around 25% higher than that of the control cross between Creepers and wild-type chickens. Ratio of Creeper and wild-type chicken follows 2:1 for Creeper inter se (χ2=0.0229, P=0.8796) while 1:1 for the crosses between Creeper and wild-type chickens (χ2=0.0292,P=0.8643). The observed phenotypes of Xingyi bantam and segregation analysis results are consistent with previous experiments. These findings further confirmed that Xingyi bantam breed carries the Cp gene.In order to perform genomic mapping of the Cp gene and reduce the noise of genetic background, segregation families were further constructed to produce their full-sib or half-sib progenies. In total,18 samples from 3 sire families, including 6 lethal embryos,6 Creeper and 6 wild-type chickens, were chosen for whole-genome resequencing. After quality control of reads, bioinformatics analyses will be performed genomic mapping of the Cp gene and identification of relevant causative mutation. Considering the linkage case for Creeper and Rose-comb (Cp-R-U), the genetic effects of the Cp gene will be determined. Among all the variations, only a 11,896 bp large deletion region (chr7: 21,798,705-21,810,600) covering the entire Indian hedgehog (IHH) gene was top-ranked in terms of the genetic effects, chromosome position, and mutation type in our analysis pipeline. Read depth was significantly lower in the deletion region than in both sides of the deletion region in the Creepers, and the read depth in the deletion region of the Creeper chickens was almost half of that in the wild-type chickens. All 6 Creeper chickens were shown to carry the same IHH deletion,IHH dominates cartilage differentiation, regulates chondrocytes proliferation and is essential for bone formation. Our results indicate that the deletion of IHH is the causative mutation for the semi-lethal Creeper trait. The breakpoint of the deletion region was confirmed by a diagnostic PCR test using a forward primer in the upstream and a reverse primer in the downstream of the deletion region. As expected, only 224 bp PCR product was obtained from the lethal embryos. An amplicon positioned within the deletion region yielded the expected 438 bp product in the wild-type chicken.PCR products from the heterozygotes had both 224 bp and 438 bp bands. Large scale segregation analysis demonstrated that the deletion of IHH was fully linked with early embryonic death and the Creeper trait.IHH plays crucial roles in regulating chondrocytes proliferation, cartilage differentiation, and bone formation. Expression profile of the IHH gene and its protein from different phenotypic individuals were analyzed by qRT-PCR and Western blot. The results showed that there is no expression in the lethal embryo at embryonic day 4 (E4). As compared to wild-type chickens, dramatic expression differences were detected between Creeper and wild-type chickens, and much lower expression was found in the Creeper fowls during different developmental stages. Along with the deletion of IHH in the Creeper fowls, we assume that IHH haploinsufficiency is insufficient to provide enough gene product to sustain normal function, thus resulted in the Creeper trait in the heterozygotes while dominant homogenous embryos were lethal during the early stage of embryonic development in chicken. Further experimental studies on heterozygotes are necessary to determine that the Creeper trait is the haploinsufficiency disorder in large populations.IHH signaling pathway has been shown to be of great significance in various developmental process, especially for cartilage development. To better understand the molecular mechanism underlying the semi-lethal Creeper trait in chicken, we investigated the expression pattern of IHH signaling pathway genes in the early embryos and tibial cartilage in three genotypic individuals. E4 embryos were clearly divided into three genotypes by the diagnostic PCR test, and then randomly chose to perform expression analysis. Relative expression of IHH, PTCH1 (patched), Smo (smoothened), HIP (hedgehog interacting protein), regulatory genes including Glil(glioma-associated oncogene homolog 1) and GH2 (glioma-associated oncogene homolog 2), OPN (osteopontin), and Col II (type II collagen) in the Creeper chickens were significantly lower than those in the wild-type chickens, suggesting that decreased expression of IHH signaling pathway genes were incapable of providing sufficient protein product to maintain normal function thus led to the Creeper trait in chickens.These pieces of evidence strongly suggest that the IHH gene is a causative gene for the Creeper trait. IHH signaling pathway is responsible for the Creeper trait formation by decreased expression in chickens.Taken together, this is the first report answering the longstanding riddle about the Creeper trait in animal genetics. Our data strongly suggest that the deletion of IHH in chicken chromosome 7 is the causative mutation for the Creeper trait and early embryo death in chicken.This work will add our Creeper phenotype to their chicken genetic resources and further develop an accurate molecular method of identifying the Creeper mutation, which will contribute to conservation and selection of Chinese Xingyi bantam and other Chinese indigenous breeds. Our findings will not only unravel the genetic basis of the longstanding Creeper phenotype mystery in chicken as the same gene underlying bone dysplasia in human and mouse, but also highlight its significance in animal development and human haploinsufficiency disorders.