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 4

Rb1 cKO mice have an increased number of BrdU+ and Ki67+ myenteric neurons in the distal small bowel, but BrdU+ or Ki67+ myenteric neurons were not detected in controls.

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Rb1 cKO mice have an increased number of BrdU+ and Ki67+ myenteric neur...
Mice were injected with BrdU daily from P9 to P16 and then sacrificed at P20. (A–D) Immunohistochemistry for PGP9.5 (red), SOX10 (purple), BrdU (green), and Ki67 (green) was used to detect neurons, glia, DNA synthesis, and cycling cells, respectively. BrdU+ and Ki67+ neurons and glia were readily detectable in Rb1 cKO mice. (B) The arrows highlight a large neuron with BrdU incorporation into the nucleus. (D) The arrows highlight a Ki67+ neuron. Arrowheads highlight (B) BrdU+ and (D) Ki67+ glia. BrdU+ and Ki67+ neurons were not detected in WT mice. (E and F) Quantitative analysis (n = 200 cells per bar). Scale bar: 50 μm. *P < 0.05 versus control.