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doi: 10.1242/10.1242/dev.00333

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Structural-proliferative units and organ growth: effects of insulin-like growth factor 2 on the growth of colon and skin

William R. Bennett^*, Tracey E. Crew, Jonathan M. W. Slack and Andrew Ward

Center for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK
^* Present address: UK Centre for Tissue Engineering, Room 3.446, Stopford Building, Biological Sciences, University of Manchester, Manchester M13 9PT, UK

View larger version (112K): [in a new window]   Fig. 1. Visualisation of SPUs in H253 female hemizygotes. Specimens are stained with X-Gal to reveal ß-galactosidase-positive (blue) cells. (A) The colon at 12 weeks of age, showing monoclonality of crypts. (B) The skin, epidermal wholemount viewed from exterior surface showing columns of blue cells extending through the epidermis.

View larger version (21K): [in a new window]   Fig. 2. Growth of the colon in Igf2 knockout, wild-type and K:Igf2 mice. (A) Lumenal area measured at 2-4 weeks of age. (B) Semi-log plots of crypt numbers measured at 2-4 weeks of age. (C) Comparison of crypt heights measured at 2 weeks and 12 weeks of age (K:Igf2 samples indicated by K2). Each animal was sampled in triplicate and results averaged (medians indicated by horizontal bars, interquartile range by boxes and full range by vertical capped bars); n6 for each genotype tested. Kruskal-Wallis test for significance: 2-week (columns 1-3) P<0.0001; 12-week (columns 4-6) P=0.0284.

View larger version (113K): [in a new window]   Fig. 3. Crypt fission in the colon (the sample shown is wild type, 2-week postnatal). Bifurcating crypts are indicated by arrowheads.

View larger version (14K): [in a new window]   Fig. 4. Colon crypt expansion in Igf2 knockout, wild-type and K:Igf2 mice. (A) Crypt fission rates at 2-4 weeks of age. (B) Proliferative index measured at 2 weeks and 12 weeks of age (K:Igf2 samples indicated by K2). At least 6 crypts were sampled in each animal and the results averaged (medians indicated by horizontal bars, interquartile range by boxes and full range by whiskers); n6 for each genotype tested. Kruskal-Wallis test for significance: 2-week (columns 1-3) P=0.0069; 12-week (columns 4-6) P=0.0293.

View larger version (66K): [in a new window]   Fig. 5. PCNA and TUNEL labelling of colonic crypts. (A-D) PCNA staining of Igf2 knockout (A), wild type (B), K:Igf2 samples (C) and. negative control (D; wild-type sample with primary antibody omitted). The proportion of the crypt that is PCNA positive is greater in the K:Igf2. The knockout crypt shown is quite short but within the normal range of sizes. Scale bars: 25 µm. Arrowheads indicate the upper limit of PCNA-positive cells. (E,F) Crypt from a wild-type animal viewed for TUNEL (E; showing a single apoptotic cell) and DAPI (F, showing all nuclei) staining. Scale bars: 50 µm.

View larger version (66K): [in a new window]   Fig. 6. PCNA labelling of skin in transverse section from Igf2 knockout (A; some of the PCNA positive cells are indicated by black triangles and some of the PCNA negative cells by white triangles), wild-type (B) and K:Igf2 (C) mice.

View larger version (17K): [in a new window]   Fig. 7. Epidermal expansion in Igf2 knockout, wild-type and K:Igf2 mice. Mice were sampled on the day of birth (neo), at 2 weeks (2-wk) and at 12 weeks (12-wk) of age. (A) Epidermal cell number (nuclear counts). Each animal was sampled in triplicate and results were averaged (medians indicated by horizontal bars, interquartile range by boxes and full range by vertical capped bars); n6 for each genotype tested. Kruskal-Wallis test for significance: neonatal (columns 1-3) P=0.0039; 2-week (columns 4-6) P=0.0002; 12-week (columns 7-9) P=0.0015. (B) Epidermal proliferative index. Each animal was sampled in triplicate and results were averaged (medians indicated by horizontal bars, interquartile range by boxes and full range by vertical capped bars); n5 for each genotype tested. Kruskal-Wallis test for significance: neonatal (columns 1-3), P=0.2837 (not significant); 2-week (columns 4-6) P=0.0007; 12-week (columns 7-9) P=0.0693 (not significant).