Cellular Plasticity of Defa4Cre-Expressing Paneth Cells in Response to Notch Activation and Intestinal Injury

Abstract

Background & aims: Loss of leucine-rich repeat-containing G-protein-coupled receptor 5-positive crypt base columnar cells provides permissive conditions for different facultative stem cell populations to dedifferentiate and repopulate the stem cell compartment. In this study, we used a defensin α4-Cre recombinase (Defa4Cre) line to define the potential of Paneth cells to dedifferentiate and contribute to intestinal stem cell (ISC) maintenance during normal homeostasis and after intestinal injury.

Methods: Small intestine and enteroids from Defa4^Cre;Rosa26 tandem dimer Tomato (tdTomato), a red fluoresent protein, (or Rosa26 Enhanced Yellow Fluorescent Protein (EYFP)) reporter, Notch gain-of-function (Defa4^Cre;Rosa26 Notch Intracellular Domain (NICD)-ires-nuclear Green Fluorescent Protein (nGFP) and Defa4^Cre;Rosa26^{reverse tetracycline transactivator-ires} Enhanced Green Fluorescent Protein (EGFP);TetO^NICD), A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) loss-of-function (Defa4^Cre;ADAM10^flox/flox), and Adenomatous polyposis coli (APC) inactivation (Defa4^Cre;APC^flox/flox) mice were analyzed. Doxorubicin treatment was used as an acute intestinal injury model. Lineage tracing, proliferation, and differentiation were assessed in vitro and in vivo.

Results: Defa4^Cre-expressing cells are fated to become mature Paneth cells and do not contribute to ISC maintenance during normal homeostasis in vivo. However, spontaneous lineage tracing was observed in enteroids, and fluorescent-activated cell sorter-sorted Defa4^Cre-marked cells showed clonogenic enteroid growth. Notch activation in Defa4^Cre-expressing cells caused dedifferentiation to multipotent ISCs in vivo and was required for adenoma formation. ADAM10 deletion had no significant effect on crypt homeostasis. However, after acute doxorubicin-induced injury, Defa4^Cre-expressing cells contributed to regeneration in an ADAM10-Notch-dependent manner.

Conclusions: Our studies have shown that Defa4^Cre-expressing Paneth cells possess cellular plasticity, can dedifferentiate into multipotent stem cells upon Notch activation, and can contribute to intestinal regeneration in an acute injury model.

Keywords: Chemotherapy; Defensin; Enteroid; Intestinal Stem Cells; Notch; Paneth Cell; Regeneration.

Graphical abstract

Figure 1

Defa4^Cre-expressing cells are postmitotic Paneth cells. (A–F) Whole-mount and immunohistochemical analysis of small intestine from Defa4^Cre;Rosa26^tdTomato (N = 8) mice. (A) Representative whole-mount images of Tomato+ cells in the small intestine. (B) Immunofluorescent staining of Tomato+ cells with lysozyme. *Lysozyme+ cell that is Tomato-. (C) Quantification of percentage of Tomato+ that are Lysozyme+ cells and the percentage of lysozyme+ that are Tomato+ cells (N = 3 mice). (D–F) Immunofluorescent staining of Tomato+ cells with matrix metalloproteinase 7 (MMP7), lectin UEA-1, and EdU, respectively. (F) White arrow indicates Tomato+/EdU+ cell. (G) Immunofluorescent staining of Tomato+ cells with GFP in Lgr5^{EGFP-ires-CreER};Defa4^Cre;Rosa26^tdTomato crypts (N = 4 mice). White arrows indicate Tomato^Low+/GFP^Low+ cells. Scale bars: 20 μm. *P ≤ .05 and **P ≤ .01.

Figure 2

Enteroids generated from small intestinal crypts of Defa4^Cre-expressing cells are capable of sporadic Tomato+ lineage tracing. (A) Phase-contrast and immunofluorescent images of jejunal enteroid expressing Tomato+ cells within bud structures. (B) Immunofluorescence staining showing co-localization of Tomato expression with lysozyme and UEA-1 in enteroids. (C) Quantification of crypts generating enteroids showing only Paneth cell expression (ie, no lineage tracing) vs Tomato+ lineage tracing (N = 3 mice). Right: top panels, 5-day-old crypt culture undergoing complete Tomato+ lineage tracing. Bottom panels, Tomato+ lineage tracing within individual buds of a 12-day-old enteroid. (D) Flow cytometric analysis of different Tomato and EdU-expressing cell populations isolated from enteroids. Right: Quantification of individual gated cell populations (N = 4). Scale bars: 100 μm. Tom, Tomato.

Figure 3

FACS-sorted Tomato+ cells are capable of clonogenic enteroid growth. (A) Representative flow cytometric gating of EpCAM+ and Tomato expression used for FACS sorting of single-cell suspensions of isolated jejunal Defa4^Cre;Rosa26^tdTomato crypts. Left: Control, rat IgG2a–fluorescein isothiocyanate antibody. Right: Rat anti-EpCAM–fluorescein isothiocyanate antibody. (B) Phase-contrast and immunofluorescent images of FACS-sorted Tomato^Low+ (left) and Tomato^Hi+ (right) cells. (C) Quantification of clonogenic growth of FACS-sorted Tomato- and Tomato+ cells grown in Basement Membrane Extract (BME) (N = 5). (D) Phase-contrast and immunofluorescent images show clonogenic organoid growth and Tomato expression in individual FACS-sorted Tomato- and Tomato+ cells. Scale bars: (B) 10 μm; (D) 100 μm. Tom, Tomato.

Figure 4

Notch activation in Defa4^Cre-expressing cells induces robust lineage tracing in vivo and in vitro. Immunohistochemical analysis of the ileum from (A, C, and E) Defa4^Cre;Rosa26^EYFP (N = 3) and (B, D, and F) Defa4^Cre;Rosa26^{NICD-ires-nGFP} (n = 5 and n = 2 ≥ 71 wk) mice. (A) Immunofluorescent staining of GFP expression. (B) Immunofluorescent staining of NICD (nGFP+) expression. (C and D) H&E staining. (E and F) Immunofluorescent staining of EdU+ cells. (G) Quantification of EdU+ cells by crypt cell position (N = 3 per genotype). (H) Quantification of crypt depth and villus height (Defa4^Cre;Rosa26^EYFP, N = 3; Defa4^Cre;Rosa26^{NICD-ires-nGFP}, N = 5). (I) Ileal enteroids derived from Defa4^Cre;Rosa26^{NICD-ires-nGFP}. Left and middle: Whole-mount fluorescent and phase-contrast images, respectively. Right: Immunofluorescent staining of NICD (nGFP+) in frozen section of enteroid. Scale bars: 20 μm. *P ≤ .05 and **P ≤ .01.

Figure 5

Notch activation in Defa4^Cre-expressing cells leads to loss of secretory cell differentiation and increased expression of the stem cell marker Olfm4. Histologic and immunofluorescent staining of the small intestine from Defa4^Cre;Rosa26^EYFP and Defa4^Cre;Rosa26^{NICD-ires-nGFP} mice. (A) PAS/Alcian blue staining. Dashed line denotes wild-type crypt in jejunum of Defa4^Cre;Rosa26^{NICD-ires-nGFP} intestine. (B) Immunofluorescent staining of GFP or NICD (nGFP+) with cell type–specific markers, lysozyme, MUC2, chromogranin A, and villin. (C) Immunofluorescent staining of GFP or NICD (nGFP+) with the stem cell marker Olfm4. Scale bars: 50 μm.

Figure 6

Inducible Notch activation in adult Defa4^Cre-expressing cells allows for dedifferentiation and lineage tracing. (A) Schematic of inducible Notch activation in adult Defa4^Cre-expressing cells. In the presence of doxycycline, reverse tetracycline transactivator will bind the tetracycline response element (TRE) in TetO^NICD allele and activate NICD expression in Defa4^Cre-expressing cells. Defa4^Cre;Rosa26^{rtTA-ires-EGFP};TetO^NICD mice (N = 5) received 2 mg/mL doxycycline in water for 2 weeks. (B) Immunofluorescent staining with GFP antibody. Top: No doxycycline (N = 4). Bottom: Two weeks on doxycycline (N = 5). Scale bars: 20 μm.

Figure 7

Notch activation induces crypt hyperplasia and adenoma formation in Defa4^Cre; APC^flox/floxmice. (A) Survival curve for Defa4^Cre;APC^flox/flox (N = 7) and Defa4^Cre;APC^flox/flox;Rosa26^{NICD-ires-nGFP} (N = 10) mice. (B) H&E analysis and β-catenin staining. Left and middle: Ileum. Right: proximal jejunum. (C) Immunofluorescent staining of cell proliferation (EdU) and Olfm4 within the ileum. Upper right: PAS/Alcian blue staining. Scale bars: 50 μm.

Figure 8

ADAM10 deletion in Defa4^Cre-expressing does not alter Paneth cell localization or intestinal homeostasis. Analysis of small intestine and enteroids from Defa4^Cre;ADAM10^flox/flox;Rosa26^tdTomato mice (N = 3). (A) Immunofluorescent staining of Tomato+ cells with lysozyme. Right: Analysis of ADAM10 recombination in isolated crypts and FACS-sorted Tomato+ cells. Mouse genotypes: (1) wild-type Defa4^Cre;Rosa26^tdTomato and (2) Defa4^Cre;ADAM10^flox/flox;Rosa26^tdTomato. (B) Immunofluorescent staining of Tomato+ cells with MUC2 and chromogranin A (CHGA). Right: PAS/Alcian blue staining. (C) Immunofluorescent staining of Tomato+ cells with EdU and Olfm4. (D) Quantification of jejunal crypts generating enteroids showing only Paneth cell expression (ie, no lineage tracing) vs Tomato+ lineage tracing (N = 3). Right: Enteroids showing complete Tomato+ lineage tracing. (E) Immunofluorescent staining of Tomato+ lineage traced enteroids with ADAM10. Middle: Analysis of ADAM10 recombination in Tomato+ lineage traced enteroids. Right: RT-PCR analysis of ADAM10 exon 8–9 expression. Mouse genotypes: (1) wild-type Defa4^Cre;Rosa26^tdTomato and (2) Defa4^Cre;ADAM10^flox/flox;Rosa26^tdTomato. Scale bars: (A–C and E) 20 μm; (D) 100 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild-type.

Figure 9

Defa4^Cre-expressing cells can dedifferentiate and contribute to regeneration after DXR-induced injury. Analysis of DXR-treated (A and B) Defa4^Cre;Rosa26^tdTomato and (C) Defa4^Cre;ADAM10^flox/flox;Rosa26^tdTomato mice 7–10 days after treatment. (A) Tomato+ lineage tracing in the jejunum of untreated and DXR-treated Defa4^Cre;Rosa26^tdTomato mice. Right: Quantification of Tomato+ lineage tracing events within a 5-cm region of proximal jejunum (control, N = 8; DXR, N = 18). (B) Immunofluorescent staining of Tomato+ lineage tracing events with cell type–specific markers: lysozyme (Paneth cells), MUC2 (goblet cells), chromogranin A (CHGA) (enteroendocrine cells), and proliferation (EdU) and stem cell markers, Olfm4. (C) Tomato+ lineage tracing in jejunum of DXR-treated Defa4^Cre;ADAM10^flox/flox;Rosa26^tdTomato co-stained with ADAM10. Right: Immunofluorescent staining of ADAM10 shown as a single channel from the middle panel. (D) Quantification of Tomato+ lineage tracing events within a 5-cm region of proximal jejunum from Defa4^Cre;Rosa26^tdTomato (N = 18) mice and Defa4^Cre;ADAM10^flox/flox;Rosa26^tdTomato (N = 18) mice. Scale bars: 100 μm. *P ≤ .05 and **P ≤ .01. Ctl, control; NA, not applicable; ND, not detected.