Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction
Ming Fu, … , J. William Harbour, Robert O. Heuckeroth
Ming Fu, … , J. William Harbour, Robert O. Heuckeroth
Published November 1, 2013
Citation Information: J Clin Invest. 2013;123(12):5152-5164. https://doi.org/10.1172/JCI67653.
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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 6

Most giant neurons in distal small bowel myenteric plexus express nNOS.

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Most giant neurons in distal small bowel myenteric plexus express nNOS. ...
(A–F) Whole-mount myenteric plexus stained with antibodies to nNOS (green, NO-producing neurons), calretinin (red, excitatory motor neurons), PGP9.5 (red, all neurons), and lamin B2 (green, nuclear lamina) or by NADPH-d histochemistry (NO-producing neurons). (G–I) Nuclear area for DSI and colon neurons based on lamin B2 staining. Graphs show cumulative frequency for nuclear area. Each point represents the percentage of neurons with nuclear area smaller than the x axis value in μm2. (A–D) Typical images of calretinin- and nNOS-expressing neurons in the distal small bowel (A and B) or colon (C and D) myenteric plexus of control (A and C) orRb1 cKO (B and D) mice. (E and F) Triple labeling for PGP9.5, lamin B2, and NADPH-d in DSIs of control and Rb1 cKO mice. The arrows highlight a large neuron. (G) Quantitative analysis shows that many NADPH-d–expressing neurons have very large nuclei in Rb1 cKO mice. Since NADPH-d–expressing neurons are common, it is not surprising that enough PGP9.5+ neurons have large nuclei to shift the curve. (H) Colon myenteric plexus neurons are very close to normal size in Rb1 cKO mice. (I) In contrast to NO-producing neurons, calretinin-expressing myenteric neurons are near normal size in the small intestines of Rb1 cKO mice. Scale bar: 50 μm. (G–I) WT, n = 200 cells; Rb1 cKO, n = 300 cells per line.