The RET gene encodes RET protein, which triggers intracellular signaling pathways for enteric neurogenesis, and RET mutation results in Hirschsprung's disease

Enteric neurons and ganglia are derived from vagal and sacral neural crest cells, which undergo migration from the neural tube to the gut wall. In the gut wall, they first undergo rostrocaudal migration followed by migration from the superficial to deep layers. After migration, they proliferate and differentiate into the enteric plexus. Expression of the Rearranged During Transfection (RET) gene and its protein RET plays a crucial role in the formation of enteric neurons. This review describes the molecular mechanism by which the RET gene and the RET protein influence the development of enteric neurons. Vagal neural crest cells give rise to enteric neurons and glia of the foregut and midgut while sacral neural crest cells give rise to neurons of the hindgut. Interaction of RET protein with its ligands (glial cell derived neurotrophic factor (GDNF), neurturin (NRTN), and artemin (ARTN)) and its co-receptors (GDNF receptor alpha proteins (GFRα1-4)) activates the Phosphoinositide-3-kinase-protein kinase B (PI3K-PKB/AKT), RAS mitogen-activated protein kinase (RAS/MAPK) and phospholipase Cγ (PLCγ) signaling pathways, which control the survival, migration, proliferation, differentiation, and maturation of the vagal and sacral neural crest cells into enteric neurons. Abnormalities of the RET gene result in Hirschsprung's disease.


Introduction
The enteric nervous system is part of the autonomic nervous system and is comprised of a complex array of interconnected neurons in ganglia located throughout the gut wall. The majority of neurons of the gut wall are derived from the vagal neural crest cells, with a minor contribution from the sacral neural crest cells [1][2][3]. These neural crest cells undergo massive migration, proliferation, and differentiation, an event that starts at about embryonic day 8.5 in animals and after the third week of intrauterine life in humans [4]. Several genes play an important role during the development of enteric neurons including the Rearranged During Transfection (RET) gene [5].

Origin and development of the enteric nervous system
The enteric nervous system is derived from the vagal and sacral neural crest of somite levels 1-7 and 28 [6]. These crest cells give rise to enteric neurons and ganglia of the pre-umbilical and post-umbilical parts of the gut wall [7]. They initially undergo a single wave of rostrocaudal migration along the gut wall [8,9]. Only a small number of neural crest cells are required for rostrocaudal colonization in the gut wall [10,11]. Next, they migrate to the unoccupied site of the developing gut and proliferate [12]. The migration of immature enteric neuroblasts in the gut wall takes place, on average, at a speed of 15 µm/h [13], which is slow compared to the undifferentiated vagal enteric neural crest cells [13,14]. A second wave of migration occurs from the periphery to the deep layers of the gut wall through connective tissue pathways, forming the myenteric plexus first, followed by the submucosal plexus [15]. In birds, the enteric neural crest cells first migrate toward the submucosa, forming the submucosal plexus, which then migrates outwards between muscle layers to form the myenteric plexus [16]. During the morphogenesis and differentiation of enteric neural crest cells into enteric neurons, several proteins [17,18] play essential roles. For the differentiation of vagal and sacral neural crest cells into enteric neurons, the RET gene and the encoded RET protein play a pivotal role [19,20].
The RET protein is a receptor tyrosine kinase. The RET gene was originally described as a human oncogene, but it was later established that RET plays a crucial role in the development of enteric neurons and defects in the human RET gene result in the syndrome known as Hirschsprung's disease [21].

Location of the RET gene
The RET gene lies in the long arm of chromosome 10 (10q11.2) and contains 21 exons [22] and 18 or 5 introns [23,24]. The DNA sequence of this gene was originally found to be rearranged within 3T3 fibroblast cell line following transfection with DNA from lymphoma cells [25].

Structure of the RET protein
The RET gene encodes a receptor tyrosine kinase transmembrane protein [26]. The RET protein has three different isoforms (RET51, RET43, and RET9), which differ in the C-terminal amino acids [27]. Two isoforms, RET9 and RET51, also differ in their intracellular domains [28,29]. RET is comprised of 1114 amino acids [30] and has three domains. The N-terminal domain is extracellular and consists of 29-635 amino acids [30]. It has four cadherin-like domains (CLDs) and cysteine-rich regions [31][32][33]. The CLDs each consist of 110 amino acids [34] and CLD2 and CLD3 each have a Ca 2+ binding site, which is required for maintaining the integrity of the RET protein [35]. The cysteine-rich regions contain 120 residues and are connected to the transmembrane domain [34]. The hydrophobic transmembrane domain of RET spans the cell membrane [31,33] and consists of 636-657 amino acids [30]. It mediates extracellular calcium-binding for maturation of the immature 150-kDa RET protein in the endoplasmic reticulum to the mature 170-kDa protein and its migration to the cell membrane [36,37]. Finally, the cytoplasmic domain is a tyrosine (Tyr) kinase domain and consists of 657-1114 amino acids [30]. It contains 16 tyrosine residues (six in RET9, 18 in RET51, whereas Tyr 1090 and Tyr 1096 are present only in RET51) [31,32]. This domain also contains catalytic protein kinases, a distinct regulatory sequence of 14-18 tyrosine residues, and serine and threonine phosphorylation sites [38,39]. There are 18 tyrosine residues, two in the juxtamembrane domain, 11 in the kinase domain, and five in the carboxyl terminal tail [40]. In addition, this domain also has phosphopeptide motifs that provide a binding and docking site for cytoplasmic downstream signaling proteins, such as Src homolog 2 (SH2) and phosphotyrosine-binding domain (PTB) [41].

Jun-mediated signaling pathway
The JNK pathway is required for the normal migration of enteric neural crest cells. Several MAPKKKs together with MEKK1-4, MLK3, and Tak1 phosphorylate and activate MKK4 and MKK7 [70]. MKK4 and MKK7 then catalyze the phosphorylation of C-Jun N-terminal kinase (CJN Kinase) [71]. This further activates MAPKKK via the small G-protein, RAC. RAC further activates MLK3, MEKK1, and MEKK4 [72][73][74], and finally activates the JNK pathway. This JNK pathway then causes the phosphorylation and activation of several transcription factors, including C-Jun, Jun A, Jun B, ATF2, and EIK, and these enable enteric neural crest cell survival [70] and migration [75,76].

Abnormalities of the RET gene
Deletion of the RET gene or mutations in the exons and introns that result in changes in the intracellular and extracellular domains of RET lead to Hirschsprung's disease (Tables 1-11).   Table 4. Deletions in the RET gene.

Mutations Effects Outcomes
RET isoforms mutations and defects RET9 [124] Lack of enteric ganglion in colon [124] Mutation of tyrosine 1062 of RET9 to phenylalanine [125] Deficient in enteric nervous system [125]

Conclusions
Vagal and sacral neural crest cells migrate in a rostrocaudal direction where they colonize in an orderly manner in the foregut, midgut, and hindgut following signaling by the receptor tyrosine kinase RET protein. This protein promotes the survival of enteric neurons, as well as proliferation and differentiation of multipotent enteric progenitor cells present in the gut wall. Developmental studies in model organisms and genetic studies of Hirschsprung's disease have provided a detailed understanding of enteric nervous system development via expression of the RET gene. In summary, the RET gene encodes a tyrosine kinase receptor, RET, which is required for the normal formation of enteric neurons. Mutation of the RET gene leads to dysfunctional RET binding to the GDNF, ARTN, and NRTN ligands resulting in Hirschsprung's disease.