Experiment
The adrenal gland of mice contains a transient zone between the adrenal cortex and the adrenal medulla: the X-zone. There are clear strain differences in terms of X-zone morphology. Nulliparous females of the inbred mouse DDD/SgnRbrc (DDD) strain develop adrenal X-zones containing exclusively vacuolated cells, whereas females of the inbred mouse C57BL/6J (B6) strain develop X-zones containing only non-vacuolated cells. The X-zone vacuolation is a physiologic process associated with the X-zone degeneration and is tightly regulated by genetic factors.
In the current study, a quantitative trait locus (QTL) analysis for the extent of X-zone vacuolation was performed for two types of F2 female mice: F2 Ay mice and F2 non-Ay mice. Identifying the genes responsible for the differences in the extent of X-zone vacuolation could provide clues for a better understanding of the critical role of this zone. The murine X-zone and the human fetal zone are considered analogous structures.
The DDD.Cg-Ay congenic is a mouse strain carrying the Ay allele at the agouti locus and was used for this study because a close association between the X-zone morphology and the agouti locus genotype has been suggested. The Ay allele is dominant and homozygous lethal; therefore, living Ay mice are invariably heterozygotes. Female B6 mice were crossed with DDD.Cg-Ay males to produce the F1 generation. F1 mice carrying the Ay locus were intercrossed with F1 non-Ay mice to produce the F2 generation.
F2 females were weaned at 4 weeks of age. At 16 weeks of age, mice were euthanized and their adrenal glands were removed. The degree of X-zone vacuolation was evaluated according to a four grade system:
Grade '0' - indicated that the X-zone contained only non-vacuolated cells; corresponding to the X-zone morphology of the parental B6 non-Ay strain.
Grade '3' - the layer of vacuolated cells in an X-zone of Grade 3 was the thickest among the grades; corresponding to X-zone morphology in the parental DDD non-Ay strain.
Grade '2' - the layer of vacuolated cells in an X-zone of Grade 2 was thicker than that in an X-zone of Grade 1.
Grade '1' - the layer of vacuolated cells in an X-zone of Grade 1 was apparently the thinner that that in Grade 2.
Grade 0 as indicated above was recognized as containing non-vacuolated cells only and X-zones of Grades 1-3 were recognized as containing vacuolated cells. The grade of X-zone vacuolation was treated in three different ways: as a parametric trait, as a non-parametric trait, and as a binary trait. QTLs that were identified as significant in at least one of these three analyses were considered to be significant.
Genomic DNA was isolated from the tails of mice. Microsatellite sequence length polymorphisms were identified after PCR amplification of genomic DNA. QTL analysis was done using R/qtl. Threshold LOD scores for suggestive (P < 0.63) and significant (P < 0.05) linkages were determined by performing 1,000 permutations for each trait. For significant QTLs, a 95% confidence interval (CI) was defined by a decline of 1.5-LOD.
Of a total of 298 F2 females, 148 were F2 non-Ay and 150 were F2 Ay mice were analyzed.
When the grade was analyzed as a parametric trait in F2 non-Ay mice, three significant QTLs were identified on chromosomes 1, 2, and X:
QTL Xzvdq1 (X-zone vacuolation in DDD mice QTL 1) mapped to Chromosome 1 with a LOD score 3.40 at 40 cM nearest marker D1Mit10. The 95% CI spanned from 17 to 69 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq1.
QTL Xzvdq2 (X-zone vacuolation in DDD mice QTL 2) mapped to Chromosome 2 with a LOD score of 5.26 at 73 cM nearest marker D2Mit285. The 95% CI spanned from 65 to 90 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq2.
QTL Xzvdq3 (X-zone vacuolation in DDD mice QTL 3) mapped to Chromosome X with a LOD score of 3.72 at 51 cM nearest marker DXMit64. The 95% CI spanned from 32 to 57 cM. The DDD allele was associated with the lower grade of vacuolation at Xzvdq3.
Curator Note: Because different mapping populations were used, because vacuolation was treated in different ways and because differing locations and peak LOD scores were identified we have assigned each significant QTL identified in the current study a unique identifier.
When the grade was analyzed as a non-parametric trait in F2 non-Ay mice, three significant QTLs were identified on chromosomes 1, 2, and 6:
QTL Xzvdq7 (X-zone vacuolation in DDD mice QTL 7) mapped to Chromosome 1 with a LOD score of 3.20 at 39 cM nearest marker D1Mit303. The 95% CI spanned from 14 to 68 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq7.
QTL Xzvdq8 (X-zone vacuolation in DDD mice QTL 8) mapped to Chromosome 2 with a LOD score of 6.24 at 76 cM nearest marker D2Mit285. The 95% CI spanned from 68 to 87 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq8.
QTL Xzvdq4 (X-zone vacuolation in DDD mice QTL 4) mapped to Chromosome 6 with a LOD score of 3.26 at 67 cM nearest marker D6Mit256. The 95% CI spanned from 50 to 75 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq4.
When the grade was analyzed as a binary trait in F2 non-Ay mice, one significant QTL was identified on chromosomes X:
QTL Xzvdq9 (X-zone vacuolation in DDD mice QTL 9) mapped to Chromosome X with a LOD score of 3.80 at 50 cM nearest marker DXMit64. The 95% CI spanned from 39 to 57 cM. The B6 allele was associated with the higher grades of vacuolation at Xzvdq9.
When the grade was analyzed as a parametric trait in F2 Ay mice, two significant QTLs were identified on chromosomes 6 and 12:
QTL Xzvdq10 (X-zone vacuolation in DDD mice QTL 10) mapped to Chromosome 6 with a LOD score of 8.85 at 60 cM nearest marker D6Mit256. The 95% CI spanned from 55 to 70 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq10.
QTL Xzvdq5 (X-zone vacuolation in DDD mice QTL 5) mapped to Chromosome 12 with a LOD score of 3.42 at 52 cM nearest marker D12Mit259. The 95% CI spanned from 22 to 62 cM. The B6 allele was associated with the higher grades of vacuolation at Xzvdq5.
When the grade was analyzed as a non-parametric trait in F2 Ay mice, two significant QTLs were identified on chromosomes 2 and 6:
QTL Xzvdq11 (X-zone vacuolation in DDD mice QTL 11) mapped to Chromosome 2 with a LOD score of 3.40 at 87 cM nearest marker D2Mit285. The 95% CI spanned from 72 to 101 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq11.
QTL Xzvdq12 (X-zone vacuolation in DDD mice QTL 12) mapped to Chromosome 6 with a LOD score of 7.71 at 65 cM nearest marker D6Mit256. The 95% CI spanned from 53 to 73 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq12.
When the grade was analyzed as a binary trait in F2 Ay mice, one significant QTL was identified on chromosomes 6:
QTL Xzvdq13 (X-zone vacuolation in DDD mice QTL 13) mapped to Chromosome 6 with a LOD score of 4.36 at 59 cM nearest marker D6Mit256. The 95% CI spanned from 37 to 71 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq13.
When the Ay and the non-Ay progeny from the same F2 cross were combined single QTL scans revealed significant QTL when the grade was analyzed as either parametric or non-parametric on chromosomes 1, 2, and 6.
In the parametric analysis of the combined F2 progeny:
QTL Xzvdq14 (X-zone vacuolation in DDD mice QTL 14) mapped to Chromosome 1 with a LOD score of 3.95 at 34 cM nearest marker D1Mit303. The 95% CI spanned from 20 to 58 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq14.
QTL Xzvdq15 (X-zone vacuolation in DDD mice QTL 15) mapped to Chromosome 2 with a LOD score of 5.03 at 85 cM nearest marker D2Mit285. The 95% CI spanned from 64 to 98 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq15.
QTL Xzvdq16 (X-zone vacuolation in DDD mice QTL 16) mapped to Chromosome 6 with a LOD score of 10.90 at 85 cM nearest marker D6Mit256. The 95% CI spanned from 56 to 72 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq16.
In the non-parametric analysis of the combined F2 progeny:
QTL Xzvdq17 (X-zone vacuolation in DDD mice QTL 17) mapped to Chromosome 1 with a LOD score of 3.55 at 35 cM nearest marker D1Mit303. The 95% CI spanned from 17 to 59 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq17.
QTL Xzvdq18 (X-zone vacuolation in DDD mice QTL 18) mapped to Chromosome 2 with a LOD score of 6.54 at 86 cM nearest marker D2Mit285. The 95% CI spanned from 67 to 96 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq18.
QTL Xzvdq19 (X-zone vacuolation in DDD mice QTL 19) mapped to Chromosome 6 with a LOD score of 10.58 at 66 cM nearest marker D6Mit256. The 95% CI spanned from 17 to 59 cM. The DDD allele was associated with the higher grades of vacuolation at Xzvdq19.
When the agouti locus genotype (at D2Mit285) was included as an interactive covariate an additional significant QTL was identified on Chr 8 mapping to D8Mit191 with a combined LOD score of 4.85. The locus was named Xzvdq6, X-zone vacuolation in DDD mice QTL 6.
Next the relative contributions of each QTL when considered together were evaluated using multiple QTL models [Table 5]. Most of the QTL identified previously were confirmed. The proportions of the total phenotypic variance accounted for in the F2 non-Ay, F2 Ay, and combined F2 mice were 42.9%, 32.6%, and 34.8%, respectively.
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