RET and the enteric nervous system

Ret Biology:  When we first started studying RET there were only a few papers describing this protein.  Now there are thousands of manuscripts about RET biology. In part this work is driven by the discovery that inactivating RET mutations cause Hirschsprung disease while activating mutations cause inherited cancer syndromes (MEN2A, MEN2B and FMTC). Here we summarize our contributions to understanding RET biology.
We became interested in RET because of a few studies by other investigators:
1. Ret deficient mice have no enteric neurons in the bowel and no kidneys. This paper began our interest in Ret biology. (Schuchardt and Pachnis Ret KO paper)
2. RET  mutations predispose to Hirschsprung disease in humans. Most people with Hirschsprung disease have only a single mutant Ret allele or a mutation that reduces Ret expression. Some of the earliest papers demonstrating the role of RET mutations in Hirschsprung disease are at these links: (Puliti paperYin paperAttie paperRomeo paperEdery paper)
3. Homozygous RET mutations (i.e., inactivating mutations in both chromosomal Ret copies) in humans also cause total intestinal aganglionosis, the most severe form of Hirschsprung disease (Shimotake paper). Fortunately this form of Hirschsprung disease is rare.
 
Work by Dr. Heuckeroth and colleagues demonstrated that Ret is essential for many aspects of normal development and for mature nervous system function.  This work began when Dr. Heuckeroth was a post-doctoral research fellow in Dr. Jeffrey Mibrandt's laboratory and continued in his own independent research laboratory since 1998.  Other close collaborators over many years include Dr. Eugene Johnson, Dr. Sanjay Jain, Dr. Hideki Enomoto, and Dr. John R. Grider.  Studies from these investigators led to:
 
1. Identification of the novel Ret ligands neurturin (led by Dr. Paul Kotzbauer; Neurturin discovery paper), persephin (a large collaborative effort; Persephin discovery paper), and artemin (led by Dr. Honma; Artemin discovery paper) and analysis of the mutant mice under Dr. Milbrandt's direction.
2. Identification of the Ret co-receptor GFRalpha2 (led by Dr. Robert Baloh in Dr. Milbrandt's lab; GFRalpha2 discovery paper), and demonstration of kidney and enteric nervous system defects in GFRalpha1 mutant mice (led by Dr. Hideki Enomoto in Dr. Milbrandt's lab; GFRalpha1 KO mouse paper).
3. Demonstration that Ret, Ret ligands and Ret co-receptors are important for normal development of the sympathetic and parasympathetic nervous system (led by Dr. Heuckeroth (Neurturin KO paper) and Dr. Enomoto (GDNF/NRTN parasympathetic paper)Ret sympathetic paper with Dr. Milbrandt).
  
Work by Dr. Heuckeroth and colleagues directly related to the enteric nervous system (ENS):  As Dr. Heuckeroth started his own research laboratory, he decided to focus efforts to understand the mechanisms that control enteric nervous system development. These studies are directly relevant to Hirschsprung disease, intestinal pseudo-obstruction syndrome and irritable bowel syndrome. We demonstrated:
 
1.  Neurturin and GDNF support ENS precursor proliferation, survival and neurite growth in culture (Heuckeroth GDNF/NRTN primary culture paper).  Persephin and endothelin-3 were also tested in these cultures, but did not increase neuron numbers.
2. Reduced numbers of acetylcholinesterase stained nerve fibers in the myenteric plexus of the bowel of neurturin deficient mice (Heuckeroth Neurturin KO paper). These mice also have abnormal bowel contractility.
3. Reduced numbers of enteric neurons in GDNF mutant mice and abnormal bowel contractility in GDNF+/-, Ret+/-, and GFRalpha1+/- mice (Gianino GDNF paper).  
4. Interestingly, unlike the rest of the developing nervous system where approximately 50% of newly generated neurons die because of inadequate trophic factor support, cell death is rare in the developing enteric nervous system.  Bid and Bax mutations do not affect enteric neuron number in mice (Gianino GDNF paper).
5. Although Ret heterozygous mice have almost normal ENS anatomy, mutations that reduce RET signaling by about 75% cause distal bowel aganglionosis that mimics human Hirschsprung disease (Jain RetDN paper).
6. RET signaling mutations can lead to long chains of ganglia that extend down the bowel in only one or two regions (Skinner Men2B partial rescue paper), a phenomena that may underlie some of the clinical challenges in defining the transition zone in human Hirschsprung disease.
7. Cultured enteric neuron precursors depend on RET induced activation of phosphatidylinositol 3-kinase (Pik3)/Akt/Forkhead signaling for GDNF stimulated survival (Srinivasan Pik3 paper).  Pik3 is also required for neurite growth supported by GDNF.
8. RET activates an intracellular signaling pathway that encourages ENS precursor migration and neurite growth via induction of cell polarity.  This same process determine axon number in ENS precursors. Using in vitro primary culture methods, lentiviral siRNA expression, fetal gut whole organ culture and protein immunoblot analysis, we outlined a signal transduction pathway from RET at the cell surface all the way to the cytoskeleton.  This model provides testable hypotheses and guides ongoing work. These studies implicated PKCzeta, GSK3, and Smurf1 in ENS development. (Polarity paper; Dr. Bhupinder Vohra)
9. Increased GDNF levels lead to more enteric neurons, but the timing and intensity of GDNF increases determines the types of neurons affected.  GDNF also appears to guide neurites from NAPDH diaphorase expressing neurons (Wang increased GDNF paper) within the ENS. This paper shows that a wide variety of ENS structural and functional defects can occur when Ret signaling is altered.  It also suggests that the intensity of ENS precursor proliferation is a major determinant of enteric neuron numbers. The Figure at right shows how different ways of increasing GDNF lead to different patterns of innervation in the mouse esophagus.
10. RET activates many different intracellular signaling pathways via phosphorylation of four interacellular tyrosine residues that act as docking sites for adaptor proteins. Mutating these tyrosine residues to phenylalanine selectively blocks a subset of Ret signaling pathways.  This paper shows that different signaling pathways have selective roles in normal development of the ENS, kidney, sympathetic and parasympathetic nervous system (Jain Ret signaling paper).
11. GDNF acutely enhances peristalsis by increasing substance P release in the ascending arm of the peristaltic reflex independent of the effect of GDNF on ENS development (Grider GDNF peristaltic reflex paper).
12. Vitamin A deficiency causes distal bowel aganglionosis that resembles human Hirschsprung disease and this effect is more severe in Ret mutant mice (Fu RA Gene Environment interaction paper).
13. Reducing cellular GTP levels with a medicine called mycophenolate causes distal bowel aganglionosis that resembles human Hirschsprung disease.  At low level of mycophenolate, aganglionosis only occures if mutations in Ret or Sox10 are also present (Lake MPA Gene-Environment interaction paper)