p63-expressing cells are the stem cells of developing prostate, bladder, and colorectal epithelia

Abstract

The tumor protein p63 (p63), and more specifically the NH2-terminal truncated (ΔN) p63 isoform, is a marker of basal epithelial cells and is required for normal development of several epithelial tissues, including the bladder and prostate glands. Although p63-expressing cells are proposed to be the stem cells of the developing prostate epithelium and bladder urothelium, cell lineages in these endoderm-derived epithelia remain highly controversial, and rigorous lineage tracing studies are warranted. Here, we generated knock-in mice expressing Cre recombinase (Cre) under the control of the endogenous ΔNp63 promoter. Heterozygote ΔNp63(+/Cre) mice were phenotypically normal and fertile. Cre-mediated recombination in ΔNp63(+/Cre);ROSA26(EYFP) reporter mice faithfully recapitulated the pattern of ΔNp63 expression and were useful for genetic lineage tracing of ΔNp63-expressing cells of the caudal endoderm in vivo. We found that ΔNp63-positive cells of the urogenital sinus generated all epithelial lineages of the prostate and bladder, indicating that these cells represent the stem/progenitor cells of those epithelia during development. We also observed ΔNp63 expression in caudal gut endoderm and the contribution of ΔNp63-positive cells to the stem/progenitor compartment of adult colorectal epithelium. Because p63 is a master regulator of stratified epithelial development, this finding provides a unique developmental insight into the cell of origin of squamous cell metaplasia and squamous cell carcinoma of the colon.

Keywords: genitourinary tract; hindgut; large intestine.

Fig. 1.

Generation of ∆Np63^+/Cre knock-in (KI) mice. (A) Schematic representation of the strategy used to generate mice that express Cre under the ∆Np63 promoter. Cre recombinase followed the PGK-Neo selection cassette was inserted in p63 intron 3 located on chromosome 16 so that the ATG of Cre replaces the ATG of ∆Np63. (B) Southern blot analysis of ES cells electroporated with ∆Np63-Cre targeting vectors. DNA was digested with SphI and, after blotting, hybridized with probe a for 5′ arm homologous recombination (HR) screening. The membrane was then stripped and rehybridized with probe b for 3′ arm HR screening. The 9-kb band (a and b) represents the wt allele, the 5.7-kb band (a) represents the 5′ arm targeting event, and the 5-kb band (b) represents the 3′ targeting event. Lanes 2 and 3 in a and b show the expected bands, indicating successful HR. (C) PCR-based assay for mouse genotyping. (D) Macroscopic features of ∆Np63^+/Cre and ∆Np63^Cre/Cre P0-1 mice.

Fig. 2.

Cre-mediated recombination mirrors the expression pattern of ∆Np63 in ∆Np63^+/Cre;ROSA26^EYFP 13.5 dpc embryos. IHC analyses of ΔNp63 and EYFP expression in 13.5 dpc ∆Np63^+/Cre;ROSA26^EYFP embryos show that EYFP is expressed selectively in ΔNp63-positive tissues. (A) Representative images of serial sagittal sections of whole embryos. Higher magnifications of the selected areas are also shown. (B) Representative images of serial sections from various organs. (Scale bar: 50 μm.)

Fig. 3.

∆Np63-negative umbrella cells of ∆Np63^+/Cre;ROSA26^EYFP mice express EYFP, demonstrating that they form from ∆Np63-positive stem cells of the primitive bladder. (A–C) Representative images of sagittal sections of the bladder from ∆Np63^+/Cre;ROSA26^EYFP mice at 13.5 dpc (A), 15.5 dpc (B), and 7 wk of age (C) triple-stained for p63/EYFP/uroplakin III (UroIII). White stars indicate p63⁺ EYFP⁺ uroplakin III⁻ cells. Yellow arrowheads point to p63⁺ uroplakin III⁺ EYFP⁺ superficial cells and white arrowheads show p63⁻ uroplakin III⁺ EYFP⁺ umbrella cells. Dotted lines represent basal membrane. (Scale bar: 20 μm.)

Fig. 4.

∆Np63-negative prostate luminal and neuroendocrine cells of ∆Np63^+/Cre;ROSA26^EYFP mice express EYFP, demonstrating that they derive from ∆Np63-positive stem cells. Representative images of sagittal UGS sections from 13.5 dpc (A) and 15.5 dpc (B) ∆Np63^+/Cre;ROSA26^EYFP embryos double-stained for EYFP/p63. Stars indicate p63⁺ EYFP⁺ cells. White arrowheads point to p63⁻ EYFP⁺ cells. (C) Representative images of transversal sections of prostatic buds from P0-1 ∆Np63^+/Cre;ROSA26^EYFP mice double-stained for EYFP/p63. Higher magnifications of the selected areas are also shown. The blue box highlights the proximal region; the red box highlights the distal region of the buds. The white arrowhead points to a p63⁻ EYFP⁺ cell. (D) Representative images of the prostate from 7-wk-old ∆Np63^+/Cre;ROSA26^EYFP mice stained for p63/CK8/EYFP. Higher magnifications of the area selected by the red box are shown. White stars indicate p63⁺ EYFP⁺ cells. The white arrowhead points to a p63⁻ CK8⁺ EYFP⁺ luminal cell. (E) Representative images of the prostate from 7-wk-old ∆Np63^+/Cre;ROSA26^EYFP mice stained for synaptophysin (Syn)/EYFP. The yellow arrowhead shows a Syn⁺ EYFP⁺ neuroendocrine cell. L indicates the lumen; dotted lines represent basal membrane. (Scale bar: 20 μm.)

Fig. 5.

∆Np63-negative cells of the large intestine of ∆Np63^+/Cre;ROSA26^EYFP mice express EYFP, demonstrating that they derive from ∆Np63-positive stem cells of the caudal endoderm. (A) Representative images of sagittal sections of the urorectal region of ∆Np63^+/Cre;ROSA26^EYFP mice at 11.5 dpc (Left), 13.5 dpc (Center), and 15.5 dpc (Right) immunostained for ∆Np63. Higher magnifications of the selected areas are shown. The red box highlights the proximal region; the blue box highlights the distal region of the developing gut. (Scale bar: Upper, 100 μm; Lower, 20 μm.) (B) Representative images of sagittal sections of the urorectal region at 13.5 dpc (Left) and distal hindgut at 15.5 dpc (Right) double-stained for EYFP/p63. White arrowheads indicate p63⁺ EYFP⁺ cells; yellow arrowheads point to p63⁻ EYFP⁺ cells. (Scale bar: Left, 100 μm; Right, 50 μm.) In both A and B, c indicates the cloaca, dg indicates the developing gut, u indicates the UGS, and b indicates the bladder. (C) Representative images of tissue sections of the large (Left) and small (Right) intestine from 7-wk-old ∆Np63^+/Cre;ROSA26^EYFP mice immunostained for EYFP. (Scale bar: 100 μm.) (D) Representative images of the adult colonic epithelium double-stained for EYFP/Alcian Blue. The black arrow indicates an EYFP⁺ Alcian Blue⁺ goblet cell; the black arrowhead points to an EYFP⁺ Alcian Blue⁻ enterocyte. (Scale bar: 10 μm.) (E) Representative images of the adult colonic epithelium double stained for EYFP/lysozyme. The yellow arrowhead points to an EYFP⁺ lysozyme⁺ Paneth cell. Scale bar, 20 μm. (F) Representative images of the adult colonic epithelium double stained for EYFP/Syn. The white arrowhead indicates a synaptophysin⁺ EYFP⁺ enteroendocrine cell. (Scale bar: 20 μm.) The dotted line represents the basal membrane.