Experiment
External ear hole closure in LG/J mice represents a model of regenerative response. The ability to regenerate tissue is heritable.
In the current study, an advanced intercross line (AIL) and a linkage mapping strategy were used to narrow the support intervals obtained in an F2 intercross analysis. Ear hole closure was examined in an Wustl:LG,SM-G34 AIL mapping population. Nineteen QTL were identified (NCBI Build 37/mm9). Genes within the support intervals were analyzed for differential expression in parental strain healing tissue, and both coding and non-coding SNPs were evaluated using genome sequences for the LG/J and SM/J strains.
The F34 population consisted of 1,169 mice in 137 full-sib families. At 3 weeks of age half of each litter was weaned onto a high or low fat diet. At 5 weeks of age, each animal had a 2mm hole punched in both right and left ears. The diameter of the hole was recorded 30 days after the hole punch for each animal. The measure of the healing phenotype for each animal was the average of healing on the left and right sides.
Both F33 parents and F34 offspring were genotyped for 3,072 SNPs; of those, 2,842 autosomal SNPs were used in the analysis. QTL interval mapping followed the procedures described by Haley and Knott. In addition to testing for the direct effects of QTL , significant locations for interactions with sex, diet or both were examined. All genes from the identified QTL regions were tested for differential expression between LG and SM at day 0 or day 7 using a two-tailed t-test. A select list of 34 candidate genes were analyzed for enriched common functions using the Ingenuity Pathway Analysis software. The significance threshold was set at p<0.001. Positional candidate genes in enriched common pathways and/or showing significant differential expression were evaluated for molecular variation.
Results: Table 1 Ear-healing QTL in the Wust:LG,SM-G34 AIL:
Eight of the 19 QTL identified were significant at the genome wide level:
QTL Heal50 (wound healing/ regeneration 50) mapped to Chromosome 1 peaking at 72,252,316 bp with a LOD score of 5.98, spanning a confidence interval between 71,676,295 and 73,411,001 bp with additive effects. Possible candidate genes detected for this locus were Fn1, Smarcal1 and Xrcc5 [Tables 2 and 3].
QTL Heal51 (wound healing/ regeneration 51) mapped to Chromosome 1 peaking at 134,931,146 bp with a LOD score of 5.18, spanning a confidence interval between 133,749,487 and 137,468,445 bp with additive effects. An interaction between genotype and diet was also detected at this locus. Possible candidate genes detected for this locus were Lgr6 and Ptprv [Tables 2 and 3].
QTL Heal52 (wound healing/ regeneration 52) mapped to Chromosome 7 peaking at 82,773,421 bp with a LOD score of 4.87, spanning a confidence interval between 81,045,510 and 86,086,593 bp with additive effects. An interaction between genotype and diet was also detected at this locus.
QTL Heal53 (wound healing/ regeneration 53) mapped to Chromosome 7 peaking at 87,330,462 bp with a LOD score of 4.97, spanning a confidence interval between 86,950,051 and 88,125,934 bp with additive effects. Possible candidate genes detected for this locus were Blm and Mesp2 [Tables 2 and 3].
QTL Heal54 (wound healing/ regeneration 54) mapped to Chromosome 11 peaking at 58,818,355 bp with a LOD score of 6.74, spanning a confidence interval between 57,795,593 and 59,802,286 bp with additive effects. Possible candidate genes detected for this locus were Hist3h2a, Hist3h2ba and Wnt3 [Table 2].
QTL Heal55 (wound healing/ regeneration 55) mapped to Chromosome 11 peaking at 99,864,706 bp with a LOD score of 5.99, spanning a confidence interval between 99,642,739 and 100,202,942 bp with additive effects. A possible candidate gene detected for this locus was Krt15 [Table 2].
QTL Heal56 wound healing/ regeneration 56) mapped to Chromosome 11 peaking at 101,318,869 bp with a LOD score of 5.49, spanning a confidence interval between 101,126,956 and 101,510,781 bp with additive effects. Possible candidate genes detected for this locus were Becn1, Brca1, and Rdm1 [Table 2 and 3].
QTL Heal57 (wound healing/ regeneration 57) mapped to Chromosome 13 peaking at 106,712,772 bp with a LOD score of 7.96, spanning a confidence interval between 105,104,329 and 107,727,570 bp with additive effects. A possible candidate gene detected for this locus was Adamts6 [Table 2 and 3].
Eleven of the 19 QTL Identified were significant only at the chromosome-wise level:
QTL Heal58 (wound healing/ regeneration 58) mapped to Chromosome 1 peaking at 140,498,264 bp with a LOD score of 3.70, spanning a confidence interval between 139,536,983 and 142,773,121 bp with additive effects. Ptprc was identified as a candidate gene at this locus [Table 2 and 3].
QTL Heal59 (wound healing/ regeneration 59) mapped to Chromosome 7 peaking at 70,741,322 bp with a LOD score of 4.63, spanning a confidence interval between 66,490,265 and 72,631,840 bp with additive effects. An interaction between genotype and diet was also detected at this locus. Fan1 and Ndn were identified as candidate genes at this locus [Table 2
QTL Heal60 (wound healing/ regeneration 60) mapped to Chromosome 9 peaking at 62,013,984 bp with a LOD score of 4.66, spanning a confidence interval between 61,092,166 and 62,280,288 bp with additive effects. Kif23 and Coro2b were identified as candidate genes at this locus [Table 2].
QTL Heal61 (wound healing/ regeneration 61) mapped to Chromosome 9 peaking at 95,077,076 bp with a LOD score of 3.79, spanning a confidence interval between 93,516,401 and 96,892,086 bp with additive effects. Atr and Rnf7 were identified as candidate genes at this locus [Table 2 and 3].
QTL Heal62 (wound healing/ regeneration 62) mapped to Chromosome 10 peaking at 128,553,075 bp with a LOD score of 3.53, spanning a confidence interval between 127,751,828 and 128,553,075 bp. An interaction between genotype and diet was also detected at this locus. Cdk2, Cs, Itga7, Myl6, Obfc2b and Smarcc2 were identified as candidate genes at this locus [Table 2 and 3].
QTL Heal63 (wound healing/ regeneration 63) mapped to Chromosome 12 peaking at 73,203,378 bp with a LOD score of 3.96, spanning a confidence interval between 70,704,786 and 73,595,736 bp with additive effects.
QTL Heal64 (wound healing/ regeneration 64) mapped to Chromosome 13 peaking at 70,313,984 bp with a LOD score of 3.97, spanning a confidence interval between 69,468,028 and 71,022,095 bp with additive and dominant effects. Interactions between genotype and diet and genotype and sex were also detected at this locus. Adamts16 was identified as a candidate gene at this locus [Table 2 and 3].
QTL Heal65 (wound healing/ regeneration 65) mapped to Chromosome 14 peaking at 49,832,566 bp with a LOD score of 3.40, spanning a confidence interval between 47,748,393 and 50,848,141 bp with additive and dominant effects. Atg14 was identified as a candidate gene at this locus [Table 2].
QTL Heal66 (wound healing/ regeneration 66) mapped to Chromosome 18 peaking at 38,525,762 bp with a LOD score of 3.65, spanning a confidence interval between 37,267,434 and 39,270,239 bp with additive and dominant effects. Interactions between genotype and sex and genotype and sex and diet was also detected at this locus. Diap1, Fgf1 and Hdac3 were identified as candidate genes at this locus [Table 2 and 3].
QTL Heal67 (wound healing/ regeneration 67) mapped to Chromosome 18 peaking at 42,341,135 bp with a LOD score of 3.73, spanning a confidence interval between 40,489,461 and 42,861,162 bp with additive and dominant effects. Interactions between genotype and diet and genotype were also detected at this locus.
QTL Heal68 (wound healing/ regeneration 68) mapped to Chromosome 13 peaking at 44,011,475 bp with a LOD score of 3.60, spanning a confidence interval between 43,794,001 and 45,574,479 bp with additive effects. Mcc was identified as a candidate gene at this locus [Table 2 and 3].
Analysis Tools