Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction
Ming Fu, Solange Landreville, Olga A. Agapova, Luke A. Wiley, Michael Shoykhet, J. William Harbour, Robert O. Heuckeroth
Ming Fu, Solange Landreville, Olga A. Agapova, Luke A. Wiley, Michael Shoykhet, J. William Harbour, Robert O. Heuckeroth
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Research Article Gastroenterology

Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction

Abstract

The retinoblastoma 1 (RB1) tumor suppressor is a critical regulator of cell cycle progression and development. To investigate the role of RB1 in neural crest–derived melanocytes, we bred mice with a floxed Rb1 allele with mice expressing Cre from the tyrosinase (Tyr) promoter. TyrCre+;Rb1fl/fl mice exhibited no melanocyte defects but died unexpectedly early with intestinal obstruction, striking defects in the enteric nervous system (ENS), and abnormal intestinal motility. Cre-induced DNA recombination occurred in all enteric glia and most small bowel myenteric neurons, yet phenotypic effects of Rb1 loss were cell-type specific. Enteric glia were twice as abundant in mutant mice compared with those in control animals, while myenteric neuron number was normal. Most myenteric neurons also appeared normal in size, but NO-producing myenteric neurons developed very large nuclei as a result of DNA replication without cell division (i.e., endoreplication). Parallel studies in vitro found that exogenous NO and Rb1 shRNA increased ENS precursor DNA replication and nuclear size. The large, irregularly shaped nuclei in NO-producing neurons were remarkably similar to those in progeria, an early-onset aging disorder that has been linked to RB1 dysfunction. These findings reveal a role for RB1 in the ENS.

Authors

Ming Fu, Solange Landreville, Olga A. Agapova, Luke A. Wiley, Michael Shoykhet, J. William Harbour, Robert O. Heuckeroth

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Figure 8

NO donor and Rb1 silencing increased cell cycle entry in ENS precursors.

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NO donor and Rb1 silencing increased cell cycle entry in ENS precursors....
(A–H) E12.5 midgut slices were cultured (B, D, F, and H) with or (A, C, E, and G) without the NO donor DETA NONOate and (C, D, G, and H) with lentivirus expressing shRNA against Rb1 or (A, B, E, and F) a control lentivirus for 48 hours. (D and H) Arrowheads indicate Rb1 shRNA lentivirus–infected ENS precursors with enlarged nuclei in the presence of NO donor. Antibodies to RET (blue) and PH3 (red) were used to identify neural crest–derived cells and cells in the cell cycle, respectively. (I) Quantitative analysis of neural crest–derived (RET+), virus-infected (GFP+) cells in the cell cycle (PH3+). (J) Quantitative analysis of nuclear area for RET+GFP+ cells after virus infection and NO donor or control culture. In box-and-whisker plot, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate 10th and 90th percentiles. Scale bar: 20 μm. *P < 0.001 for Rb1 shRNA and for DETA NONOate versus controls, 2-way ANOVA.