Genetic mosaic analysis based on Cre recombinase and navigated laser capture microdissection

Abstract

Defining molecular interactions that occur at the interface between "normal" and "abnormal" cell populations represents an important but often underexplored aspect of the pathogenesis of diseases with focal origins. Here, we illustrate an approach for conducting such analyses based on mosaic patterns of Cre recombinase expression in the adult mouse intestinal epithelium. Transgenic mice were generated that express Cre in the stem cell niche of crypts located in specified regions of their intestine. Some of these mice were engineered to allow for doxycycline-inducible Cre expression. Recombination in all pedigrees was mosaic: Cre-expressing crypts that supported recombination in all of their active multipotent stem cells were located adjacent to "control" crypts that did not express Cre at detectable levels. Cre-mediated recombination of a floxed LacZ reporter provided direct evidence that adult small-intestinal crypts contain more than one active multipotent stem cell, and that these cells can be retained in both small-intestinal and colonic crypts for at least 80 d. A method was developed to recover epithelial cells from crypts with or without recombination for subsequent gene expression profiling. Stained sections of intestine were used to create electronic image templates to guide laser capture microdissection (LCM) of adjacent frozen sections. This navigated form of LCM overcomes problems with mRNA degradation encountered when cells are marked directly by immunohistochemical methods. Combining Cre-engineered genetic mosaic mice with navigated-LCM will allow biology and pathobiology to be explored at the junction between normal and perturbed cellular cohorts.

Figure 1

Regional features of FabplCre-mediated recombination in the intestinal epithelium. Whole mounts, prepared from 6-week-old bi-transgenic mice, were stained with X-Gal to visualize LacZ as blue. (Bars = 1.08 mm.)

Figure 2

Mosaic patterns of Cre recombination. (A–C) Six-week-old Fabpl^{−596 to +21}Cre/R26R mice. (A) X-Gal- and NFR-stained section from the proximal third of the small intestine. Crypts (e.g., arrows) are monophenotypic: they contain a wholly LacZ+ or LacZ− population of epithelial cells. (B) X-Gal-stained whole mount of proximal small intestine showing striped villi (e.g., arrow). (C) Rare crypts contain segregated cohorts of LacZ+ and LacZ− cells that extend to adjacent villi (arrows and arrowheads point to members of the goblet cell and enterocytic lineages, respectively). We refer to crypts with this distinct sidedness as having a Cheron phenotype, after the protagonists Lokai and Bele in the StarTrek episode “Let that be your last battlefield.” (D and E) X-Gal-stained sections from the proximal colon of 6-week-old Fabpl^{4X at −132}Cre/R26R mice. (D) Monophenotypic crypts. (E) A small fraction of crypts exhibit a distinctive pattern of segregation of LacZ+ cells to their base. (Bar in B = 1.08 mm; elsewhere, 25 μm.)

Figure 3

Doxycycline-inducible Cre recombination. (A and B) Proximal small intestines from Fabpl^{4X at −132}rtTA/tetO-Cre/R26R mice analyzed 20 d and 80 d after cessation of doxycycline treatment. Insets show that monophenotypic LacZ+ crypts are present in each case. (C) Ceca from mice killed 20 d and 80 d after treatment. (Bars in A and B = 1.08 mm; C = 0.55 mm; Insets = 25 μm.)

Figure 4

Navigated-LCM. (A) Montage of captured electronic images of an X-Gal- and NFR-stained section of terminal ileum from a Fabpl^{4X at −132}Cre/R26R mouse. (B–E) LCM of a cluster of neighboring monophenotypic LacZ+ and LacZ− crypts: (B) electronic image template; (C) adjacent NFR-stained section targeted for LCM; (D) removal of crypts; (E) captured crypts.

Figure 5

PCR genotyping and RT-PCR profiling of gene expression in juxtaposed LacZ+ and LacZ− crypt epithelial cells recovered by navigated-LCM. (A) R26R genotyping. Primers 1,2 were used to detect the intact locus, primers 1,3, the recombined locus. DNA from monophenotypic LacZ+ crypts yield a 525-bp PCR product from the recombined R26R locus (Left) and no detectable 250-bp product from the intact locus (Right). (B) PCR of cDNA showing that the FabplCre mRNA transcript is detectable only in LacZ+ crypts, whereas E-cadherin mRNA is present in both LacZ+ and LacZ− crypts.

Figure 6

Comparison of the integrity of mRNA isolated after navigated-LCM and immuno-LCM. (A) Real-time RT-PCR. Each cDNA was prepared by using oligo(dT) and RNA from ≈20,000 LCM ileal villus epithelial cells. After 40 cycles of PCR with primers that recognize sequences from the 5′ end of E-cadherin, reaction products were resolved by agarose gel electrophoresis. (B) Effects of navigated (n)- and immuno (i)-LCM protocols on levels of various RNA species. Oligo(dT)-primed cDNA was generated from equivalent number of ileal cells that had no antibody exposure (zero time point; n-LCM conditions) or had been subjected to i-LCM conditions. Real-time RT-PCR used aliquots of cDNA representing ≈200 crypt-villus units and primers directed to regions derived from the 5′ ends of all RNAs surveyed. (C) Real-time RT-PCR assays of Apc mRNA integrity in RNA prepared from intact ileum and from cells scraped from ileal sections subjected to n-LCM or i-LCM conditions. See text for definition of the integrity index.