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First published online 11 March 2008
doi: 10.1242/jcs.022913

Journal of Cell Science 121, 969-978 (2008)
Published by The Company of Biologists 2008
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Targeted transgenic expression of the mutation causing Hutchinson-Gilford progeria syndrome leads to proliferative and degenerative epidermal disease

Hanna Sagelius1, Ylva Rosengardten1, Mubashir Hanif1, Michael R. Erdos2, Björn Rozell3, Francis S. Collins2 and Maria Eriksson1,*

1 Department of Biosciences and Nutrition, Karolinska Institutet, Karolinska University Hospital, Huddinge, Novum, SE-14186 Stockholm, Sweden
2 Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, 50 South Drive, Bethesda, MD 20892, USA
3 Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, SE-14186 Stockholm, Sweden


Figure 1
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  		Fig. 1. Minigenes of human lamin A. (A,B) Constructs used for tetop-LAwt (A) and tetop-LAG608G (B) transgenic mice. Both constructs contain a minigene, including the coding region of lamin A (exons 1-10, intron 11 and exon 12), and differ only in nucleotide 1824, where tetop-LAwt+ carries the wild-type lamin A sequence, and the tetop-LAG608G+ carries the most common HGPS mutation, 1824C>T. tetop, Tet-operon; IRES, internal ribosomal entry site; eGFP, coding region for enhanced green fluorescent protein. (C) The tet-off system used in this study. Bi-transgenic animals, tetop-LAG608G+; K5tTA+, express human LA, LAdel50 (progerin) and green fluorescent protein (GFP). In the presence of doxycycline (DOX) the transcription is turned off.

 

Figure 2
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  		Fig. 2. Transgene expression in bi-transgenic mice. (A) RT-PCR of dorsal/ventral skin from bi-transgenic mice and controls using a human LA and LAdel150-specific assay. Presence and absence of transgene is indicated with + and –, respectively. Lanes 1 and 2 are RT-PCR from cell line of HGPS parent AG03504 and HGPS AG03506, respectively. Lane 14 contains cDNA from ventral skin of FVB/N wild-type mice. Lanes 3, 12 and 13 are no-RT control samples for AG03506, tetop-LAG608G+; K5tTA+ and tetop-LAwt+; K5tTA+, respectively. Lane 15 is a no-template control. M, 100 bp ladder (Invitrogen). RT-PCR for TATA-binding protein (TBP) served as a control for the RT (bottom panel). (B,C) Western blot on protein extracts from dorsal/ventral skin of bi-transgenic animals and controls. (B) Filters were simultaneously incubated with antibodies for detection of human lamin A/C (mab3211) and β-actin. Separate western blots, including the same amount of protein extract, were run for detection of green fluorescent protein (GFP, ab290). (C) Enhanced protein separation western blots. An antibody to the N-terminal region of lamin A/C detects lamin A/C of both human and mouse origin, sc-6215 (left). sc-6214 (right) detects prelamin A. (B,C) Same filters were incubated with an antibody to β-actin. F1 line and PCR genotype are indicated above each lane. + and – indicate presence of LA minigene or K5tTA, and absence of K5tTA, respectively. Lane 1 contains protein extracts from HGPS cell line AG03506. Lane 8 is protein extract from a FVB/N wild-type mouse.

 

Figure 3
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  		Fig. 3. Transgenic minigene expression is present in keratinocytes of the interfollicular epidermis and the hair follicle. (A-I) F1 line VF1-07 tetop-LAG608G+; K5tTA+. (J-L) FvB/N wild-type mice. (M-O) F1 line SF1-04 tetop-LAwt+; K5tTA+. Immunofluorescence with the cytokeratin 5 (CK5) antibody in green (A,G,J), human lamin A/C antibody in red (B,E,H,K,N) and DAPI (D,M). (C,F,I,L,O) Merged panels, including DAPI. Dorsal skin tissue sections from different ages of mice: 18 weeks (A-C), 9 weeks (D-F), 9 weeks end-stage (G-I), 4 weeks (J-L) and 22 weeks (M-O). (K) No staining with the human lamin A/C antibody to FVB/N wild-type dorsal skin shows that the antibody is specific to human lamin A/C. Transgenic mice were supplied with doxycycline until time of weaning – day 21. Scale bars: 50 µm.

 

Figure 4
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  		Fig. 4. Progressive pathological changes from epidermal overexpression of lamin A and lamin Adel50 in sections from dorsal skin. (A-G,J-L) Haematoxylin and Eosin staining. (A) Normal structure of the skin in late anagen phase is shown in a section from dorsal skin of FVB/N wild-type mice. (B-G) F1 line VF1-07, tetop-LAG608G+; K5tTA+, 9-week-old mice. Intermediate severe and mild stage evident in the same section (B; left and right, respectively). Intermediate severe stage with severe epidermal hyperplasia, extensive immature hyperparakeratosis, and enlargement and displacement of sebaceous glands (B, enlarged in C,E,F), and mild stage (B,D,G). (H,I) Immunohistochemistry using and antibody to adipophilin show staining of lipid-containing cells of the sebaceous unit in FVB/N wild-type (H) and F1 line VF1-07, tetop-LAG608G+; K5tTA+ (I) 17-week-old mice. (J) End-stage disease characterized by epidermal hypoplasia, resting hair follicle, associated hypoplastic sebaceous gland, absence of hypodermis and a well-developed fibrosis of the dermis. (K) F1 line VF1-07 tetop-LAG608G+; K5tTA– at 11 months (for comparison with J). (L) Bi-transgenic animals from the SF1-04 line show a well-defined structure of the skin, as shown in a dorsal skin section from a 24.5-week-old mouse. Transgenic mice were supplied with doxycycline until time of weaning – day 21. Scale bars: A,C-G,J-L, 100 µm; B, 500 µm; H,I, 50 µm.

 

Figure 5
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  		Fig. 5. Epidermal overexpression of lamin A and lamin Adel50 leads to aberrant expression of keratin 5 and keratin 6, and increased proliferation, as shown by immunohistochemistry with antibodies to keratin 5 (CK5), keratin 6 (CK6) and phospho-histone H3 (PHH3). (A,B,F) FVB/N wild type in late anagen stage. (C-E,G,H) Bi-transgenic animals from F1 line VF1-07 of 9 and 18 weeks. (A) CK5 normal expression pattern in the basal cells of the interfollicular epidermis, the outer root sheet and the peripheral cells of the sebaceous glands. (B) CK6 normal expression pattern in the inner root sheet. (C) In progerin-expressing mice, CK5 expression is also detected in the suprabasal population of keratinocytes and is expressed equally across the sebaceous glands. (D,E) CK6 expression is also detected in the interfollicular epidermis and sebaceous glands. (F) Normal PHH3 expression in the bulb of a late anagen hair follicle. (G,H) Increased proliferation in cells of the interfollicular epidermis and the hair follicle. Transgenic mice were supplied with doxycycline until time of weaning – day 21. Scale bars: C-E, 100 µm; A,B,F-H, 50 µm.

 

Figure 6
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  		Fig. 6. Expression of epidermal differentiation markers. Dorsal skin section from FVB/N wild type in late anagen phase (A,D,G,J), and bi-transgenic animals of the F1 line VF1-07 in intermediate stage (B,C,E,F,H,I,K,L). Immunofluorescence with the cytokeratin 1 (CK1), cytokeratin 10 (CK10), filaggrin and loricrin antibodies in green and merged with DAPI (blue). High-magnification view of interfollicular epidermis show expression of CK1 and CK10 in a few cells of the basal layer (C and F, respectively). Transgenic mice were supplied with doxycyline until time of weaning – day 21. BL, basal layer; D, dermis. Scale bars: 50 µm.

 

Figure 7
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  		Fig. 7. Reduced growth rates and premature death of tetop-LAG608G bi-transgenic animals. Body weights (grams, A-C) or percent survival to age (weeks, D-F) were plotted for F1 line VF1-07. Each panel includes data from three different genotypes: (A) tetop-LAG608G+; K5tTA+ (+/+); (B) tetop-LAG608G+; K5tTA– (+/–); and (C) tetop-LAG608G–; K5tTA– (–/–). The difference in survival for bi-transgenic animals is noted: (D) +/+ (n=12, median survival=14 weeks) and +/– (n=10), {chi}2=12.79, P=0.0003. (E) +/+ (n=5, median survival=7 weeks) and +/– (n=9), {chi}2=13.4, P=0.0003. (F) +/+ (n=6, median survival=29 weeks) and +/– (n=9), {chi}2=7.943, P=0.0048. No statistically significant difference in survival was noted between +/– and –/– animals (D-F). Intercross breeding pairs were supplied with doxycycline that was removed at day of weaning – day 21 (A,D) or day of birth – day 0 (B,C,E,F). (C,F) Results when animals were fed on the cage floor with water-dissolved pellets from postnatal day 21 and onwards. (G) Photograph of 7-week-old bi-transgenic and single transgenic animals.

 

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© The Company of Biologists Ltd 2008