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First published online 4 August 2004
doi: 10.1242/dev.01232

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Rb family proteins differentially regulate distinct cell lineages during epithelial development

Department of Pathology and Immunology, and Division of Molecular Oncology in the Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA

View larger version (61K): [in a new window]   Fig. 1. Inducible lung epithelial-specific ablation of Rb. (A) Male mice heterozygous for both CC10-rtTA and tetCre transgenes, and homozygous for the floxed Rb gene allele (RbLoxP) were bred to female mice bearing a floxed Rb allele and a mutated null Rb allele (Rb-). Dams were treated with doxycycline (ovals), which activates the rtTA (arches) expressed specifically in lung epithelium under control of the CC10 promoter. Activated rtTA induces Cre expression leading to excision and functional loss of Rb. (B) PCR analysis on lung or tail DNA obtained from day 1 pups taken from doxycycline-treated dams, or control dams not treated with doxycycline. Representative results are shown. Recombination at the floxed Rb allele (RbRec) is dependent upon the presence of both CC10-rtTA and tetCre transgenes, and is detected in lung but not tail DNA. No recombination is detected in lung DNA obtained from control pups not treated with doxycycline. (C-F) Enzymatic staining for ß-gal (blue) in lungs from ROSA26 reporter mice harboring the CC10-rtTA and tetCre transgenes, and treated with doxycycline in utero. (C) Whole-mount staining of lungs from double transgenic day 1 pups (+) and control littermates (C) lacking one or both transgenes. (D,F) Staining of adult lung sections. Note that majority of ciliated and non-ciliated epithelial cells show staining. Inset in D highlights the scattered punctuate staining observed in the alveolar region, consistent with the localization of Type II cells. (E) Enzymatic staining for ß-gal (blue) followed by immunohistochemistry for CGRP (brown/black) in day 1 lung. Arrow marks a cell positive for both ß-gal activity and CGRP. br, bronchi/bronchioles; bv, blood vessel; a, alveoli.

View larger version (92K): [in a new window]   Fig. 2. Rb ablation results in epithelial abnormalities. (A-L) Morphological examination, TUNEL analysis and immunohistochemisty for Ki67 and PCNA in lungs from day 1 pups after Rb ablation (CC10-rtTA), from genetically identical pups without doxycycline treatment (No Dox), and from pups lacking one or both transgenes (Control). Arrows mark apoptotic cells (A), TUNEL-positive cells (D,E) and scattered PCNA-positive cells (K,L). Abbreviations are as in Fig. 1. (M) Quantification of epithelial apoptosis and proliferation as assessed by the percentage of TUNEL-positive and Ki67-positive cells, respectively. Rb ablation results in statistically significant increases in epithelial apoptosis and proliferation (*P<0.0002). Data represents the analysis of seven CC10-rtTA, eight control and four No Dox mice.

View larger version (109K): [in a new window]   Fig. 3. Rb ablation leads to cell lineage-specific effects in differentiation. (A-F) Immunohistochemical analysis for CCSP and CGRP in lungs from day 1 pups after Rb ablation (CC10-rtTA), from genetically identical pups without doxycycline treatment (No dox), and from pups lacking one or both transgenes (Control). Arrows mark neuroendocrine cells. Abbreviations are as in Fig. 1. Data represent analysis of seven CC10-rtTA, eight Control and four No Dox lungs.

View larger version (42K): [in a new window]   Fig. 4. Rb ablation leads to hypercellular neuroendocrine lesions. Mice were mated as detailed in Fig. 1A, and pregnant dams were treated with doxycycline throughout gestation. Doxycyline was discontinued on the day of birth. (A-D) Morphological examination and immunohistochemical analysis for CGRP in adult lungs after Rb ablation (CC10-rtTA), and in control littermates lacking one or both transgenes (Control). B represents a higher magnification of the boxed area in A. Arrows mark neuroendocrine cells. Note the remainder of the airway epithelium is essentially normal. Data is representative of three CC10-rtTA mice. (E,F) Adult lung sections before (E) and after (F) microdissection of the epithelium. (G) Microdissected epithelium on thermoplastic cap used for PCR analysis. Abbreviations are as in Fig. 1. (H) PCR analysis of microdissected epithelium from double transgenic adult mice (+) with RbLoxP/- (top) or RbLoxP/LoxP (bottom) alleles, and control littermates lacking one or both transgenes (C). Representative results are shown. Note loss of the RbLoxP allele and the emergence of the recombined allele (RbRec). L, ladder.

View larger version (88K): [in a new window]   Fig. 5. Rb family deficiency results in lung epithelial abnormalities. (A-D) Morphological examination of transgenic (CC10-T121; A,C) and control wild-type (WT; B,D) lungs. A pyknotic nucleus in transgenic lungs is marked with an arrow (C). Note lack of morphologic indicators of differentiation in transgenic lungs, including flattening of Type I cells (D, closed arrow), ciliated cells (D, open arrow) and apical cytoplasmic protrusions indicative of Clara cells (D, white arrowhead). Images are representative of seven CC10-T121 founder mice. (E-H) Immunohistochemistry for Ki67 (E,F) and TUNEL (G,H) analysis. Rare focal epithelial cells positive for Ki67 are marked with an arrow in wild-type lungs (F). Note the majority of Ki67 immunoreactivity is localized to mesenchymal cells in wild-type lungs (F, white arrowhead). Arrow indicates TUNEL-positive epithelial cells in transgenic lungs (G). No specific TUNEL staining is present in wild-type lungs (H). (I,J) Immunohistochemistry for PCNA. Scattered focal PCNA immunoreactive epithelial (arrows) and mesenchymal (white arrowhead) cells are indicated in wild-type lungs (J). Ki67, TUNEL and PCNA data is representative of four CC10-T121 founder mice. Abbreviations are as in Fig. 1.

View larger version (116K): [in a new window]   Fig. 6. Transgene expression correlates with epithelial abnormalities. (A) Western blot analysis of whole lung lysates, showing specific expression of truncated large T antigen (T121) in CC10-T121 transgenic (+) but not wild-type (-) lungs. Full-length large T antigen (Lg T) or small t antigen (Sm T) is not detected in transgenic lungs but is seen in control cell lines (C). NS, nonspecific bands. (B-E) Immunohistochemical analysis for T121 (B,D) or TK1 (C,E) expression in transgenic lungs. Arrows indicate transgene expression in alveolar Type II cells. (F-H) Morphological examination, Ki67 expression and TUNEL analysis in transgenic SPC-T121 lungs. Data is representative of seven founder CC10-T121 mice and two SPC-T121 mice. Transgene expression was detected in two of six CC10-TK1, and seven of eight SPC-TK1 transgenic lines; adult lungs are shown. Abbreviations are as in Fig. 1.

View larger version (98K): [in a new window]   Fig. 7. Rb family deficiency results in cell lineage-specific effects in differentiation. (A-F) Immunohistochemical analysis for CCSP and CGRP in transgenic and wild-type (WT) control lungs. Arrows mark neuroendocrine cells. Data is representative of three to six CC10-T121 founder mice and two SPC-T121 pups. (G) Immunohistochemistry for the transgene (T121) and CCSP in a CC10-T121 founder with a less severe phenotype. Note that transgene expression (green) and CCSP expression (red) do not overlap. Abbreviations are as in Fig. 1. (H) Quantification of CGRP-immunoreactive foci. CC10-T121 mice show increased CGRP-reactive foci when compared with wild-type (WT) (*P=0.0002) and SPC-T121 (*P=0.0008) mice. Data represent six CC10-T121 founder mice, four wild-type mice and two SPC-T121 mice.

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