Figure 1.
Hpt/+ and +/+ female mice at 19 weeks of age.
The Hpt/+ mouse shows patchy alopecia on the trunk and head.
Figure 2.
Renal histopathology ofHpt/+ and +/+ female mice at 1, 4, and 52 weeks of age.
Representative glomeruli from Hpt/+ and +/+mice, at 1 week, 4 weeks, and 52 weeks of age. There are no marked histopathological changes between Hpt/+ and +/+ glomeruli at 1 week of age. At 4 weeks of age, Hpt/+ glomeruli show enlargement, diffuse mesangial matrix expansion (D) and a few dilated glomerular capillaries (arrowheads). At 12 months of age, Hpt/+ glomeruli show diffuse (D) and nodular (N) mesangial matrix expansion with mesangiolysis (L), capillary aneurysms (arrowheads), and thickening of glomerular (long arrows) and capsular (short arrows) basement membranes. Hematoxylin and Eosin (H&E), Periodic Acid Schiff (PAS), Jones Methenamine Silver (JMS) stains, and type IV collagen immunohistochemistry are shown; x600.
Figure 3.
A (male), B (female). Blood urea nitrogen (BUN) levels in individual Hpt/+ and +/+ mice. C (male), D (female). Urine albumin:creatinine ratio (ACR) in individual Hpt/+ and +/+ mice. Renal function progressively declines in the Hpt/+ animals. BUN values are significantly elevated (p<0.05) in Hpt/+ male (A) and female (B) mice compared with sex-matched +/+ controls at all time points (unpaired two-tailed T-test with Welsh’s correction). Urinary ACR is significantly elevated (P<0.05) as early as one month of age in males (C) and by three months of age in females (D) (unpaired two-tailed T-test with Welsh’s correction).
Figure 4.
High resolution mapping of the Hpt mutation.
Fine mapping of the Hpt mutation by segregation analysis of the HPT/LeJ-Hpt/+ x CAST/EiJ) F1 hybrid x C3HeB/FeJ cross. All of the 182 N2 offspring typed for the Chromosome 4 markers had one allele derived from C3HeB/FeJ (A) and the other allele derived from either C57BL/6J (B) or CAST/EiJ (C). Shown are the AB (gray) and AC (white) genotypes of 25 markers in the six most informative mutant N2 mice (Hpt/+, genotype designation AB) recombinant between D4Mit176 and D4Mit202. The markers D4Mit 352, hptssr22, hptssr23, and hptssr25 are non-recombinant with the Hpt mutation in all six mice, and the new flanking markers D4Mit 199 and hptssr50 refine the candidate gene interval to a 6.7 Mb region.
Figure 5.
A) Sybr Green qPCR expression analysis of genes in the 6.7 Mb candidate region of Chromosome 4.
Relative gene expression levels of 62 genes from the adult kidneys of Hpt/+ mice (1.5–15 months of age) within the candidate interval are shown (n = 3). Only Tal1 showed a 18-fold increase, whereas no greater than 3-fold change was observed among other genes in the interval. The SybrGreen-TAL1 Probe is located in the non-coding cDNA region of exon 5 (see Fig.7A). B) Tissue-specific expression of Tal1. Relative gene expression levels of Tal1 in Hpt/+ versus +/+ animals using Sybr Green qPCR (n = 3). Tal1 is overexpressed in the kidney, skin, thymus and brain of Hpt/+ mice, while there is no significant expression difference in liver or spleen. As there were no statistically significant differences in the expression values of 4 day, 14 day, 6 month, or 10–15 month Hpt/+ mice tested, expression values for each genotype were pooled from all age groups.
Figure 6.
Tal1 Southern blot and IAP insertion.
There are 4 EcoRI sites in the murine Tal1 gene (labeled E1–E4). Southern blot was performed on genomic DNA from 15 dpc embryos from Hpt/Hpt, Hpt/+ and +/+ mice using a cDNA probe spanning exons 2–3 (A). Blotting revealed two bands in wildtype embryos: 10.1 kb (fragment E3–E4), and 5.2 kb (fragment E1–E2). Hpt/+ embryos show a third band, at ∼4.6 kb, representing a fragment created by the insertion of a new EcoRI site in intron 4. Hpt/Hpt embryos show only the 5.2 kb and 4.6 kb fragments (B). 1 µg of total RNA was reverse transcribed and PCR was performed to evaluate IAP insertion. Specific primers (Tables S3&4) were used to amplify exons 3 and 4, and IAP and exon 5 (C and D). Whereas the higher molecular weight band in Hpt/+ indicates IAP insertion, the lower molecular weight indicates wildtype transcript (C). Four primer sets flanking IAP and exon 5 were used on 17-day-old Hpt/+ and +/+ kidney cDNA to ascertain if there was a fusion product of IAP RNA with subsequent exons. ntc, no template control (D). When the product of primer set 1 (Table S4) was sequenced, the 3′ end of the IAP was spliced with exon 4 and part of exon 5 (E). Shown is the protein product that might result from using the alternative translation initiation site in exon 4, which is downstream of the IAP insertion (F).
Figure 7.
Real-time qPCR analysis of Tal1 expression using Taqman probes.
The Taqman-65 and -33 probes span exons 3 and 4, and exons 4 and 5, respectively (A). While the increased expression of Tal1 in Hpt/+ mice versus +/+ controls is significant with both probe sets in all organs tested, the more 3′ Taqman-33 shows greater up-regulation than the Taqman-65 probe located more 5′(B).
Figure 8.
Tal1 protein levels in kidney tissues.
Western blot analysis of kidney tissues from 2.5- and 5-week-old +/+ and Hpt/+ mice. 40 µg of protein was loaded onto each lane, and probed with c-terminal Tal1-specific antibody. A subtle but significant increase in Tal1 protein levels was observed in 2.5-week-old Hpt/+ mice compared with +/+ control mice, however we did not detect any shortened protein products using this antibody. Actin was used as a loading control. ns, non-specific bands (A). Densitometry quantification of Tal1 protein expression in the kidneys of 2.5-week-old Hpt/+ and +/+ mice (B). *p<0.05.