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

ORIGINAL RESEARCH Cellular Plasticity of Defa4Cre-Expressing Paneth Cells in Response to Notch Activation and Intestinal Injury Jennifer C. Jones,1 Constance D. Brindley,2 Nicholas H. Elder,2 Martin G. Myers Jr,3 Michael W. Rajala,4 Christopher M. Dekaney,5 Eoin N. McNamee,6 Mark R. Frey,7 Noah F. Shroyer,8 and Peter J. Dempsey1,2 1 Cell Biology, Stem Cells and Development Graduate Program, 2Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, 6Mucosal Immunology Program, University of Colorado Medical School, Aurora, Colorado; 3Division of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan; 4Division of Gastroenterology, Department of Digestive Disease and Transplantation, Einstein Health Network, Philadelphia, Pennsylvania; 5Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina; 7Saban Research Institute, Children’s Hospital Los Angeles, Department of Pediatrics, Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, Los Angeles, California; 8Section of Gastroenterology and Hepatology, Department of Medicine, Baylor College of Medicine, Houston, Texas Defa4Cre; Rosa26tdTomato Defa4Cre; ADAM10flox/flox Defa4Cre; APCflox/flox Doxorubicin (DXR) Injury Model No lineage tracing No phenotype Paneth cell Notch Activation Defa4Cre; Rosa26NICD-ires-nGFP Defa4Cre; APCflox/flox; Rosa26NICD-ires-nGFP +4 cell Lgr5+ Dedifferentiation Lineage tracing Notch & Wnt Activation DXR Defa4Cre; Rosa26tdTomato Dedifferentiation Lineage tracing DXR Defa4Cre; ADAM10flox/flox; Rosa26tdTomato ADAM10 Deletion Inhibition of lineage tracing Adenoma formation SUMMARY Upon leucine-rich repeat-containing G-protein–coupled receptor 5–positive crypt base columnar loss, reserve stem cell populations can repopulate the stem cell niche. This study shows that defensin a4-Cre–expressing cells are fated to become mature Paneth cells during normal homeostasis, but Notch activation or doxorubicin injury causes their dedifferentiation into multipotent stem cells. 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 a4-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 Defa4Cre; Rosa26 tandem dimer Tomato (tdTomato), a red fluoresent protein, (or Rosa26 Enhanced Yellow Fluorescent Protein (EYFP)) reporter, Notch gain-of-function (Defa4Cre;Rosa26 Notch Intracellular Domain (NICD)-ires-nuclear Green Fluorescent Protein (nGFP) and Defa4Cre;Rosa26reverse tetracycline transactivator–ires Enhanced Green Fluorescent Protein (EGFP);TetONICD), A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) loss-of-function (Defa4Cre;ADAM10flox/flox), and Adenomatous polyposis coli (APC) inactivation (Defa4Cre;APCflox/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: Defa4Cre-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 Defa4Cre-marked cells showed clonogenic enteroid growth. Notch activation in Defa4Cre-expressing cells caused dedifferentiation to multipotent ISCs in vivo and was 534 Jones et al required for adenoma formation. ADAM10 deletion had no significant effect on crypt homeostasis. However, after acute doxorubicin-induced injury, Defa4Cre-expressing cells contributed to regeneration in an ADAM10–Notch–dependent manner. CONCLUSIONS: Our studies have shown that Defa4Creexpressing 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. (Cell Mol Gastroenterol Hepatol 2019;7:533–554; https:// doi.org/10.1016/j.jcmgh.2018.11.004) Keywords: Defensin; Paneth Cell; Intestinal Stem Cells; Regeneration; Enteroid; Notch; Chemotherapy. See editorial on page 619. T he epithelial cell layer lining the small intestine comprises repeating crypt-villus units with cycling leucine-rich repeat-containing G-protein–coupled receptor 5–positive (Lgr5þ) crypt base columnar (CBC) stem cells located in the crypt base. These Lgr5þ CBCs undergo constant renewal to replenish differentiated cells lining the intestine, which is required to maintain intestinal homeostasis and tissue integrity. The direct progeny of Lgr5þ CBCs are bipotent transit-amplifying (TA) progenitors, which are short-lived, rapidly cycling progenitors that give rise to all differentiated secretory (eg, Paneth cells, goblet cells, and enteroendocrine cells) and absorptive enterocyte cell types. Paneth cells are positioned between Lgr5þ CBCs at the crypt base and secrete granules containing antimicrobial products including lysozyme and defensins and, as part of the niche, provide important nutrient and stem cell niche signals. Although day-to-day homeostasis largely is maintained by Lgr5þ CBCs, intestinal injury and loss of the Lgr5þ CBC population has been shown to activate other reserve stem cell and progenitor populations that can dedifferentiate and revert to the stem cell state and contribute to crypt regeneration.1,2 Intestinal regeneration experiments using DNA damaging agents such as irradiation and chemotherapeutics or genetic ablation of Lgr5þ CBCs have shown that many different facultative stem cell populations can dedifferentiate and acquire stem cell behavior.1,2 Besides slowly cycling cells located about 4 cells up from the crypt base (þ4 cells) identified using different Cre recombinase fused to an estrogen receptor (CreER) reporter mice (eg, B lymphoma Mo-MLV insertion region 1 homolog [Bmi1þ], Mouse telomerase reverse transcriptase [mTertþ], Homeodomainonly protein homeobox [Hopxþ]) that can undergo injurydependent stem cell reversion, there is growing evidence that other progenitor populations as well as more committed cell types from enterocyte, endocrine, and goblet cell lineages show cellular plasticity.3–12 In addition, distinct label-retaining cells (LRCs) that express Paneth cell and enteroendocrine cell markers also are capable of dedifferentiation in vivo.13 Single-cell profiling of LRCs showed that short-term LRCs are composed of distinct endocrine and Pa (...truncated)