InteractiveFly: GeneBrief

Ret oncogene: Biological Overview | References

BIOLOGICAL OVERVIEW

Neurons develop highly stereotyped receptive fields by coordinated growth of their dendrites. Although cell surface cues play a major role in this process, few dendrite specific signals have been identified to date. An in vivo RNAi screen in Drosophila class IV dendritic arborization (C4da) neurons identified the conserved Ret receptor, known to play a role in axon guidance, as an important regulator of dendrite development. The loss of Ret results in severe dendrite defects due to loss of extracellular matrix adhesion, thus impairing growth within a 2D plane. Evidence is provided that Ret interacts with integrins (see Myospheroid) to regulate dendrite adhesion via rac1. In addition, Ret is required for dendrite stability and normal F-actin distribution suggesting it has an essential role in dendrite maintenance. Novel functions are proposed for Ret as a regulator in dendrite patterning and adhesion distinct from its role in axon guidance (Soba, 2015).

Accurate functional connectivity and sensory perception require proper development of the neuronal dendritic field, which ultimately determines the (sensory) input a specific neuron can receive and detect. Thus, coordinated dendrite growth and patterning is important for establishing the often complex, but highly stereotyped organization of receptive fields. Two of the organizing principles in dendrite development are self-avoidance and tiling. While self-avoidance describes the phenomenon of recognition and repulsion of isoneuronal dendritic branches, tiling refers to the complete yet non-redundant coverage of a receptive field by neighboring neurons of the same type. Both phenomena have been described in different systems across species including the mouse, zebrafish, medicinal leech, Caenorhabditis elegans, and Drosophila melanogaster (Soba, 2015).

Dendritic patterning by self-avoidance, tiling, and other mechanisms is thought to be mediated by cell surface receptors and cell adhesion molecules (CAMs), which play a pivotal role in integrating environmental and cellular cues into appropriate growth and adhesion responses. Many such receptors, prominently Robo and Ephrin receptors, have well understood roles in axon guidance. Although some of these axonal cues including Robo/Slit play a role in dendrite development as well, dendritic surface receptors and their functions are not fully characterized to date. Recent efforts have yielded some progress in this area. Down's syndrome cell adhesion molecule (Dscam) has been shown to regulate dendrite self-avoidance in Drosophila. Studies on protocadherins have revealed that they play an important role in dendrite self-avoidance in mammals. In C. elegans, sax-7/L1-CAM and menorin (mnr-1) form a defined pattern in the surrounding hypodermal tissue to guide PVD sensory neuron dendrite growth via the neuronal receptor dma-1. However, given the complexity and stereotypy of dendritic arbors within individual neuronal subtypes, it is important to search for additional signals for directing dendrite growth (Soba, 2015).

The Drosophila peripheral nervous system (PNS) has served as an excellent model which has helped to elucidate several molecular mechanisms regulating dendrite development. The larval PNS contains segmentally repeated dendritic arborization (da) neurons which have been classified as class I-IV according to their increasing dendritic complexity. All da neuron classes feature highly stereotyped sensory dendrite projections. Moreover, all da neurons exhibit self-avoidance behavior allowing them to develop their individual receptive fields without overlap. It has been demonstrated that all da neuron classes require Dscam for dendrite self-avoidance. In addition, the atypical cadherin flamingo and immunoglobulin super family (IgSF) member turtle might play a more restricted role in C4da neuron self-avoidance. Netrin and its receptor frazzled have also been shown to act in parallel to Dscam in class III da neurons ensuring their proper dendritic field size and location by providing an attractive growth cue which is counterbalanced by self-avoidance. For tiling, no surface receptor has been identified to date. However, the conserved hippo and tricornered kinases, and more recently the torc2 complex, have been implicated in C4da neuron tiling, as the loss of function of these genes results in iso- and hetero-neuronal crossing of dendrites (Soba, 2015).

Recent work has further shown that dendrite substrate adhesion plays an essential role in patterning. Da neuron dendrites are normally confined to a 2D space through interaction with the epithelial cell layer and the extracellular matrix (ECM) on the basal side of the epidermis. 2D growth of da neuron dendrites requires integrins, as loss of the α-integrin mew (multiple edomatous wing) or ß-integrin mys (myospheroid) results in dendrites being freed from the 2D confinement due to detachment from the ECM. Thus, they can avoid dendrites by growing into the epidermis leading to 3D crossing of iso- and hetero-neuronal branches (Han, 2012; Kim, 2012). Integrins are therefore essential to ensure repulsion-mediated self-avoidance and tiling mechanisms, which restrict growth of dendrites competing for the same territory. How integrins are recruited to dendrite adhesion sites and whether they cooperate with other cell surface receptors is unknown (Soba, 2015).

To identify novel receptors required for generating complex, stereotypical dendritic fields, an in vivo RNAi screen was performed for cell surface molecules in C4da neurons. The Drosophila homolog of Ret (rearranged during transfection) was identified as a patterning receptor of C4da dendrites. Loss of Ret function in C4da neurons severely affects dendrite coverage, dynamics, growth, and adhesion. In particular, dendrite stability and 2D growth are impaired resulting in reduced dendritic field coverage and abnormal 3D dendrite crossing, respectively. These defects can be completely rescued by Ret expression in C4da neurons. It was further shown that Ret interaction with integrins is needed to mediate C4da dendrite-ECM adhesion, but not dendrite growth. These data suggest that Ret together with integrins acts through the small GTPase rac1, which is required for dendrite adhesion and 2D growth of C4da neuron dendrites as well. This study thus describes a novel role for the Ret receptor in dendrite development and adhesion by direct receptor crosstalk with integrins and its downstream signals (Soba, 2015).

This study provides evidence that Ret is a regulator of dendrite growth and patterning of C4da neurons. Ret is a conserved receptor tyrosine kinase (RTK) expressed in the nervous system of vertebrates (Pachnis, 1993; Schuchardt, 1994) and D. melanogaster (Sugaya, 1994; Hahn, 2001), and has been shown to have a number of important functions in nervous system development and maintenance: it regulates motor neuron axon guidance (Kramer, 2006), dopaminergic neuron maintenance and regeneration (Kowsky, 2007; Kramer, 2007), and mechanoreceptor differentiation and projection to the spinal cord and medulla (Bourane, 2009; Luo, 2009). Ret signaling is activated by binding to glial cell line derived neurotrophic factor (GDNF) family ligands and their high affinity co-receptors, the GDNF family receptors (GFRα) (reviewed in Runeberg-Roos, 2007). Ret also plays an important role in human development and disease as loss of function mutations of Ret lead to Hirschprung's disease displaying colonic aganglionosis due to defective enteric nervous system development (Amiel, 2008). Conversely, Ret gain of function mutations are causal for autosomal dominant MEN2 (multiple endocrine neoplasia type 2) type medullary thyroid carcinoma (Lairmore, 1993; Almeida, 2012; Soba, 2015 and references).

Prior to this study, Ret has not been implicated in dendrite development. This study shows that Ret is required specifically for 2D growth of C4da neurons by regulating integrin dependent dendrite-ECM adhesion. Normally, C4da neuron dendrites are virtually always in contact with the ECM and the basal surface of the epithelium lining the larval cuticle, and thus tightly sandwiched between the two compartments. In both integrin and Ret mutants, dendrite-ECM adhesion is impaired. Ret and integrins can co-localize in dendrites and thus likely form a functional complex that could induce and maintain adhesion of dendrites to the ECM. Since Ret loss of function primarily leads to detached terminal dendrite branches, it is tempting to speculate that Ret might be required to recruit integrins to sites of growing dendrites to promote ECM interaction. This is supported by the colocalization of Ret and integrins on the dendrite surface. Their cooperative interaction could thus ensure proper adhesion of growing branches and, conversely, the fidelity of self-avoidance and tiling (Soba, 2015).

These results also highlight the importance of integrating different guidance and adhesion cues to achieve precise neuronal patterning. This has so far only been studied in axon guidance in vivo. Interestingly, vertebrate Ret has been shown to cooperate with Ephrins to ensure high fidelity axon guidance in motor neurons by mediating attractive EphrinA reverse signaling (Kramer, 2006; Bonanomi, 2012). Similar mechanisms may conceivably be employed for growing dendrites, which also encounter a multitude of attractive, repulsive, and adhesive cues that have to be properly integrated. Besides pathways acting independently or in a parallel fashion, an emerging view is that receptors exhibit direct crosstalk to integrate incoming signals. So far, only parallel receptor pathways like Dscam and Netrin-Frazzled signaling in class III da neurons or Dscam/integrins have been identified co-regulating dendrite morphogenesis. The current data show that the Ret receptor and integrins integrate dendrite adhesion and growth by collaborative interaction of the two cell surface receptors. The molecular and genetic link between Ret and integrins suggests that in this case direct receptor crosstalk plays a major role in their function. How exactly these cell surface receptors cooperate and interact remains to be elucidated. Integrins have been shown to display extensive crosstalk with other signaling receptors, including RTKs. Although integrins are involved in adhesion of virtually all cell types, the underlying signaling and recruitment of integrins to sites of adhesion in vivo is complex and not completely understood. It has been suggested that integrin and growth factor receptor crosstalk can occur by concomitant signaling, collaborative activation, or direct activation of associated signaling pathways. For example, matrix-bound VEGF can induce complex formation between VEGFR2 and β1-integrin with concomitant targeting of β1-integrin to focal adhesions in endothelial cells. The current findings of biochemical interaction and colocalization of Ret with the α/β-integrins mys and mew in C4da neuron dendrites argue in favor of direct receptor interaction and subsequent activation of a common signaling pathway (Soba, 2015).

Integrins and RTKs like Ret do share some of the same intracellular signaling components. These comprise, among others, the MAPK (mitogen-activated protein kinase) pathway, Pi3-Kinase (Pi3K), and Rho family GTPases including Rac1. Previous studies provide evidence for Ret-integrin-Rac1 interplay in vitro showing that Ret can enhance integrin mediated adhesion (Cockburn, 2010) and induce Rac1 dependent lamellipodia formation (Fukuda, 2002) in cell culture models. In primary chick motor neurons, Rac1 is involved in neurite outgrowth on the integrin substrates laminin and fibronectin. Interestingly, Rac1 has previously been shown to regulate dendrite branching in C4da neurons, however a role in dendrite adhesion in vivo has not been described before. This study shows that Rac1 is required for dendrite-ECM adhesion similarly to what has been described for integrins, and Ret and integrin dependent adhesion was genetically linked with Rac1 function. In Drosophila, MAPK, Src and PI3K can be activated by constitutively active Ret overexpression in the compound eye (Read, 2005; Dar, 2012). Moreover, novel inhibitors of Ret signaling targeting Raf, Src, and S6-Kinase (S6K) prevent lethality induced by Ret over-activation in a Drosophila multiple endocrine neoplasia (MEN2) model (Dar, 2012). Interestingly, S6K has been shown to be involved in dendrite growth but not tiling in C4da neurons (Koike-Kumagai, 2009). It remains to be shown if these pathways play a direct role in Ret function in dendrite adhesion and growth (Soba, 2015).

Notwithstanding important commonalities, Ret function in C4da neurons cannot be fully explained by crosstalk with integrins and rac1. Reduced dendritic field coverage, likely due to the observed increase in dendrite turnover, is only evident in Ret but not in integrin or rac1 mutant C4da neurons. Moreover, increasing integrin expression in a Ret mutant background did not rescue dendrite coverage defects, albeit it prevented dendrite crossing. These findings indicate that Ret has additional functions in dendritic branch growth and stability that require as yet unknown extracellular and intracellular mediators. This is also supported by the aberrant F-actin localization in neurons lacking Ret. In this study, Ret dependent intracellular effectors are likely important for F-actin assembly to support directed dendrite growth and stabilization, and their localization and activity might be deregulated in the absence of Ret (Soba, 2015).

Drosophila Ret is a highly conserved molecule, its cognate vertebrate ligand GDNF, however is not (Airaksinen, 2006). In addition, Drosophila Ret can neither bind GDNF nor transduce GDNF signaling, although it has been shown to contain a functional tyrosine kinase domain (Abrescia, 2005). In mammals, the GFRα co-receptors are essential components of GDNF/Ret signaling (Runeberg-Roos, 2007). A Drosophila GFR-like homolog (dGFRL) has recently been characterized and was found to function and interact with the NCAM homolog FasII (Kallijärvi, 2012). Therefore, it appears that Ret's functional interaction partners in dendrite development differ significantly from the previously described co-factors in other systems. It is interesting to speculate that a yet undiscovered Ret ligand is involved in Ret mediated dendrite growth and branch stabilization, which might have implications for mammalian Ret function as well: due to its role in the maintenance of dopaminergic neurons and motor axon growth in mouse, adhesion related signaling via integrins could well be important during these processes. Moreover, the formation of a dorsal root ganglia derived mechanosensory neurons and their afferent and efferent fiber growth and innervation depends on Ret expression. It will be interesting to investigate the functional interplay of Ret and integrins in central and peripheral target innervation and neurite maintenance in these systems, given the interdependent function of Ret and integrins in sensory dendrite growth as shown in this study (Soba, 2015).

In summary, this study describes a novel role for the Ret receptor in dendrite branch growth and stability in Drosophila C4da neurons. This role involves cell-autonomous effects of Ret on ECM adhesion, and F-actin localization in these neurons. Moreover, dendritic adhesion defects attributable to Ret have been linked to integrin and rac1 function featuring a novel and possibly conserved mode of action for Ret in dendrite development (Soba, 2015).

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. This study reports a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. These findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homoeostatic contexts. They also suggest an epithelial contribution to Ret loss-of-function disorders such as Hirschsprung disease (Perea, 2017).

These findings in Drosophila indicate that Ret is expressed not only by enteric neurons, but also by the adult somatic stem cells of the intestinal epithelium. In contrast to known Ret functions in other progenitor cell types -- for example, in spermatogonia or the hematopoietic system -- Ret is not required for the survival of adult somatic stem cells in the intestine, but sustains both their homeostatic and regenerative proliferative capacity. Gain- and loss-of-function experiments point to the existence of positive feedback between Ret and Wg signalling. Despite abundant genetic evidence that Wg signalling promotes stem cell proliferation in flies, the source of Wg has remained unclear. Using new, improved tools to visualise Wg expression, the current findings lend further support to recent data (Tian, 2016) indicating that the source of Wg ligand is not the stem cells themselves, despite the striking Ret-driven upregulation of Wg on their surface. How might Ret signalling in adult intestinal progenitors lead to Wg protein upregulation in these cells without affecting its transcript? Two possible ways in which it might do so is by upregulating the expression of Wg receptor(s) on their surface, and/or by promoting signalling from stem cells to the Wg-producing cells at the intestinal boundaries and/or the visceral muscles (Buchon, 2013; Tian, 2016) to increase Wg release/trafficking (Perea, 2017).

Epithelial Ret is not a peculiarity of the fly intestine; Ret is also expressed in the developing intestinal epithelium of mice, prior to the maturation of enteroendocrine or Lgr5-positive stem cells. Although immunohistochemical analyses have not revealed a specific Ret-positive cell population at this stage, ex vivo experiments using epithelial cultures devoid of enteric neuron or mesenchyme point to an intrinsic role for Ret at this stage in promoting epithelial maturation. The mechanism underlying the maturation-promoting effects of Ret may involve positive feedback between Ret and Wnt signalling similar to those found in flies. Indeed, the Wnt pathway target Axin2 is reduced in epithelial cultures derived from Ret51 mice and upregulated when wild-type FEnS are treated with the Ret ligand GDNF: a treatment that also promotes their branching. These data are consistent with the previous finding that elevated Wnt signalling promotes FEnS to organoid maturation and is reminiscent of the Wnt11/Ret autoregulatory loop promoting ureteric branching during kidney development. The relevant source of Wnt driving tissue maturation is currently unknown and is most likely not epithelial. The Drosophila finding that Wg ligand upregulation is not transcriptional underscores the importance of considering tissue crosstalk and non-autonomous signalling in any future studies addressing Wnt contributions to epithelial maturation in mice (Perea, 2017).

At first sight, the Ret effects on developmental maturation in mice appear to be different from its homeostatic role in flies. However, this study found that Ret continues to be expressed in the adult small intestine, where Ret expression is prominent in a subset of enteroendocrine cells positive for the secretory marker chromogranin-A. Based on their position, these cells may correspond to enterochromaffin cells: intriguing cells that contribute 90% of the serotonin in circulation, control gastrointestinal motility and secretions and have recently been shown to be chemosensory. A very recent study has blurred the distinction between enteroendocrine cells and their precursors by revealing expression overlaps between markers of enteroendocrine precursor identity and differentiated fate (including chromogranin-A), and by suggesting that differentiated enteroendocrine cells can have stem cell-like properties (Yan, 2017). This is exciting because it suggests that, whilst the cellular classification of Ret-positive cells based on known markers may differ between flies (ISCs) and mice (enteroendocrine), Ret-enabled stem cell functionality may contribute to regeneration in both epithelia. Intriguingly, endocrine tumours derived from the small intestine, ileal carcinoids, secrete serotonin and have been reported to express high levels of Ret. Conditional deletion of Ret in adult intestinal epithelium will, in future, determine its contribution to enteroendocrine fate and will help establish possible enteroendocrine contributions to intestinal homeostasis and tumour formation (Perea, 2017).

Consistent with ex vivo transfection studies in mammalian cells, pointing to physical association between Ret and c-Src, this study found that Src kinase Src42A is required downstream of Ret to activate Wg signalling. These findings therefore strengthen the link between two pathways previously known to control ISC proliferation in flies-Src and Wg, and may provide a physiological context for the previously reported, Src-dependent mitogenic effects of mutated, oncogenic Ret in the Drosophila developing retina. The finding that, in the Drosophila intestine, Src kinases control expression of a mitogenic module consisting of String/Cdc25 and cyclin E provides a simple link between Ret activation and its pro-proliferative effects. Overactive Src kinases do, however, lead to intestinal tumours so mechanisms must be in place to limit the positive Ret/Wg feedback loop so that it sustains homeostatic proliferation, but does not result in tumour formation. Availability of Wg ligand may be an extrinsic mechanism, but the focal adhesion kinase Fak may provide a cell-intrinsic break downstream of Ret/Src activation. Consistent with this idea, Ret expression leads to both Src42A and Fak phosphorylation, but this study found that the two kinases have opposing effects on proliferation: Src42A promotes proliferation downstream of Ret, whereas Fak blocks it. Hence, despite the fact that blocking Fak function may represent a therapeutic opportunity in some cancers, the current findings are more aligned with a previous study (Macagno, 2014) that suggested that, at least in the context of Ret-driven tumorigenesis, Fak can act as a tumour suppressor. In future, it will also be of interest to explore how the Ras/Raf/Erk pathway, activated by Ret in other contexts and previously shown to affect ISC proliferation in flies, intersects with Src/Fak/Wg signalling in response to Ret activation (Perea, 2017).

Both Wnt and Src pathways can have strong effects on proliferation, differentiation and/or tumorigenesis in the murine intestine. Src is required for mouse intestinal tumourigenesis following upregulation of Wnt signalling. Based on functional findings in Drosophila and expression data in mice, a possible contribution of the Ret- and CgA-positive cells to this process deserves further investigation. It will also be of interest to investigate how Src/Fak kinase signalling contributes to the maturation of foetal intestinal epithelial cells and whether this is important in the development of intestinal disorders (Perea, 2017).

Ret expression is one of the defining features of enteric neurons. This study has found another evolutionarily conserved and physiologically significant site of Ret expression: the intestinal epithelium. The presence of Ret in these two gastrointestinal cell types of very different developmental origin raises the possibility that the development and/or physiology of enteric neurons, intestinal epithelial progenitors and, in mammals, Ret-expressing intestinal lymphoid cells is coordinated. Such coordination may, for example, help ensure a match between the size of the intestinal epithelium, the number of innervating neurons during development and the transition from an immature foetal epithelium into a functional epithelium involved in nutrient uptake and interorgan signalling. In mammals, Ret ligands of the glial cell line derived neurotrophic factor (Gdnf) family may orchestrate Ret signalling in these three tissues. In flies (which lack these Ret ligands), integrins have been shown to interact with Ret in sensory neurons. Interfering with integrin expression in the Drosophila intestine can have different effects on intestinal progenitor proliferation, survival and/or orientation depending on whether the integrins are removed from the progenitors or their niche-the visceral muscles. Intriguingly, integrin downregulation in adult intestinal progenitors reduces their normal proliferation and can suppress their overproliferation in response to overactive Wingless signalling: phenotypes strikingly similar to those resulting from Ret downregulation. In the light of the known links between integrins and Fak/Src signalling in both normal and cancer cells and the effects that this study has found for Src and Fak downstream of Ret activation, Ret could provide a new route for the integrin activation of the Src/Fak complex. Alternatively, the recent finding that GDF15, a divergent member of the TGF-β superfamily, signals through a GDNF family receptor α-like in a Ret-dependent way also raises the intriguing possibility that TGF-β-like ligands modulate Ret signalling in the intestinal epithelium, potentially linking intestinal regeneration with the known GDF15 roles in food intake/body weight (Perea, 2017).

The crucial requirement for Ret in enteric nervous system development is underscored by disorders such as HSCR, in which Ret loss of function leads to almost complete absence of enteric innervation in varying lengths of the distal gut. Whilst the contribution of enteric aganglionosis to HSCR is unquestionable, the current findings raise the possibility that, if the epithelial expression of Ret is conserved in humans, dysregulation of epithelial signalling may contribute to disorders that, like HSCR, result from Ret mutation. Epithelial Ret signalling might also contribute to other aspects of gastrointestinal physiology previously shown to be affected by reduced Ret function, such as intestinal motility, gut-microbiota interactions and the compensatory response to massive small bowel resection. Interestingly, HSCR is typically diagnosed around birth due to defects in gastrointestinal functions. This coincides with the first demands on intestinal function, which could reflect not only neuronal defects related to peristalsis, but also defects associated with the transition from a foetal into a functional adult epithelium. Given that many of the pathways that drive tissue expansion and the maintenance of non-differentiated progenitor populations during foetal development are deregulated in cancer, a possible contribution of Ret signalling to colorectal tumours also deserves further investigation (Perea, 2017).

The Drosophila Ret gene functions in the stomatogastric nervous system with the Maverick TGFβ ligand and the Gfrl co-receptor

The RET receptor tyrosine kinase is crucial for the development of the enteric nervous system (ENS), acting as a receptor for Glial cell line-derived neurotrophic factor (GDNF) via GFR co-receptors. Drosophila has a well-conserved RET homolog (Ret) that has been proposed to function independently of the Gfr-like co-receptor (Gfrl). This study found that Ret is required for development of the stomatogastric (enteric) nervous system in both embryos and larvae, and its loss results in feeding defects. Live imaging analysis suggests that peristaltic waves are initiated but not propagated in mutant midguts. Examination of axons innervating the midgut reveals increased branching but the area covered by the branches is decreased. This phenotype can be rescued by Ret expression. Additionally, Gfrl shares the same ENS and feeding defects, suggesting that Ret and Gfrl might function together via a common ligand. This study identified the TGFβ family member Maverick (Mav) as a ligand for Gfrl and a Mav chromosomal deficiency displayed similar embryonic ENS defects. These results suggest that the Ret and Gfrl families co-evolved before the separation of invertebrate and vertebrate lineages (Myers, 2018)

The RET (rearranged during transfection) receptor tyrosine kinase is the leading susceptibility locus for Hirschsprung's disease (HSCR), a congenital lack of neurons in the distal regions of the digestive tract. HSCR arises due to the abnormal migration and survival of enteric neuron precursors derived from the neural crest, which has been classified as a neurocristopathy. RET is also found to have a role in kidney development and in a subset of neuroendocrine cancers. The ligands for RET are members of the Glial cell line-derived neurotrophic factor (GDNF) family, which act by binding to a GDNF family receptor (GFR) to activate intracellular RET signaling, or the Neural cell adhesion molecule (NCAM). GDNF is an important component of vertebrate brain development and maintenance, with clinical relevance to Parkinson's disease (Myers, 2018)

GDNF ligands appeared with the emergence of jawed fish and GFRs underwent a gene expansion at the same time. This expansion coincides with the appearance of the neural crest, a distinguishing structure for vertebrates. Homologs of the RET and GFR receptors are present in invertebrates but are thought to function independently of each other, with GFRs operating in conjunction with Fas2/NCAM rather than with a soluble ligan. In Drosophila, the RET gene (Ret) is expressed by enteric neurons and epithelial progenitor cells of the adult midgut and is required for homeostasis of these populations (Perea, 2017). In the Drosophila embryo, Ret is expressed in the developing stomatogastric nervous system (SNS), a population of cells that delaminate and migrate along the developing gut to form the enteric nervous system (ENS), and Ret is also expressed in the Malpighian tubules, the fly equivalent of the kidney. A previous study observed expression of Gfrl promoter fragments in the developing SNS, suggesting that Ret and Gfrl might function together in this tissue (Hernandez, 2015). Using CRISPR this study generated Drosophila Ret alleles and found defects in embryonic SNS formation and larval SNS function. These phenotypes led identification of the novel TGFβ family member Maverick (Mav) as an invertebrate GFR/Ret ligand and a candidate for the ancestor of GDNF. The results reveal remarkable similarities in the signaling mechanisms used to generate the insect SNS and the vertebrate ENS (Myers, 2018)

This study describes the effects of mutating the Ret gene in Drosophila and uncovered an evolutionarily conserved role in the development of the ENS. The incorrect positioning of SNS cells in the Drosophila embryo resembles hypoganglionic ENS phenotypes seen when RET is mutated in vertebrates. In HSCR, the most distal nerves of the digestive tract are affected. Likewise, in Ret mutant larvae the most distal nerves of the SNS, located on the midgut, have an altered anatomy and the larvae show defects in food ingestion. The phenotype resembles the neurotrophic effects of decreased serotonin or CNS dopamine signaling during midgut nerve formation, which also leads to increased axon branching and decreased feeding (Myers, 2018)

Although defects are visible in the embryonic SNS, there appear to be two separate lethal phases. Some first instar larvae display feeding defects and die. This is particularly evident in the original alleles that carry the background recessive lethal mutation, and the possibility is being investigated that the background lethal mutation specifically enhances the Ret mutations. Subsequent larval feeding defects often do not emerge until 2-4 days after hatching. Larvae with food in their guts can be observed foraging, suggesting that the larvae have problems with food ingestion. This is supported by observations of mutant larvae with food throughout their midguts, but with peristaltic defects in the anterior midgut. Initially a neurodegenerative defect similar to Wallerian degeneration was expected, but the axon defect was not suppressed by reducing dSarm activity. A model is currently favored in which initial SNS defects are amplified as the larva dramatically increases its mass several hundred fold. To keep pace with the expanding midgut, Ret may be required to promote axon growth, guidance, or be fulfilling a pro-synaptic role. These functions have been observed for RET and GDNF (Myers, 2018)

The midgut axon phenotype resembles defasciculation of the nerves and Gfrl genetically interacts with the fasciculation molecule Fas2, so Ret/Gfrl could potentially be modulating fasciculation as has been observed for other signaling systems. Alternatively, defasciculation may be a consequence of growth cones searching for sources of ligand, as proposed for Netrin and Bolwig's nerve. Decreased midgut innervation and function may provide negative feedback to upstream gut signaling, decreasing the ability to pass food through the pharynx and esophagus. The midgut axons may also be required to maintain communication with downstream enteroendocrine cells. An alternative hypothesis raised by the similarity of the Ret and Pink1 phenotypes is that the midgut neurons are running out of energy due to mitochondrial dysfunction (Myers, 2018).

This analysis enabled identification of the divergent TGFβ Mav as the elusive ligand for Drosophila Ret. The expression pattern of mav is consistent with a role in embryonic SNS development. Although the Mav ligand is concentrated in certain regions of the foregut and may create localized gradients, the broad expression pattern suggests that the Ret/Gfrl signaling pathway could be permissive rather than instructive during SNS precursor migration. Embryonic Ret signaling could primarily transduce a neurotrophic signal, and apoptosis has been observed in the migrating SNS precursors. In vertebrates, models in which GDNF/Ret signaling promotes proliferation rather than cell migration have been proposed to explain development of the nervous system. Experiments are underway to distinguish between these models in the fly. Although Gfrl expression has not yet been observed in the SNS, Gfrl could be acting in a soluble form or in trans. Gfrl promoter fragments continue to drive expression in the anterior midgut of the larvae in support of the trans model. Despite extensive sequence divergence in the extracellular domain of Ret, domain differences in GFRs and low homology of Mav to the GDNF family, the molecular logic of the protein complex appears preserved. In vertebrates, RET and GFR form a preassembled complex, and GDNF binds GFR to activate RET. Molecular data are strikingly similar, as this study found that Drosophila Ret and Gfrl can functionally interact in the absence of Mav, and that Mav interacts strongly with Gfrl, but only very weakly with Ret. In flies, Mav modulates synapse formation at the neuromuscular junction of body wall muscles. Ret is not expressed in body wall muscles , and Mav is likely to be signaling through activin/BMP type 1 receptors. A Mav homolog, Panda, has been found in the sea urchin Paracentrotus lividus, where it plays a role in dorsoventral axis formation and is also likely to be signaling through type 1 receptors. Mav and Panda both lack a key leucine residue, so their binding to type 1 receptors might be weaker than other ligands. Candidate Ret and Mav homologs have been found in Strongylocentrotus purpuratus, suggesting that Mav homologs might interact with both type 1 and Ret receptors in sea urchins (Myers, 2018).

Ret exhibits highly dynamic mRNA expression in the embryo. Ret is also expressed in adult midgut precursors at an earlier stage in development, as well as in discrete cells in the CNS, PNS and Malpighian tubules. mav mRNA is expressed weakly in the foregut primordium and at later stages in the pharynx, esophagus and proventriculus. Analysis of an epitope-tagged Mav expressed at endogenous levels indicates strong expression in the epithelial region from which the SNS precursor clusters delaminate and expansion to match the pattern of the mRNA, becoming concentrated near the sites at which the SNS neurons stop migrating (junction of the pharynx and esophagus, proventriculus). mav is also expressed in the epidermis and visceral mesoderm. Apart from promoter fragments driving reporters, Gfrl expression has not been observed in the SNS. Gfrl could therefore be expressed at low levels, or the protein might be acting in trans or in a soluble form. Gfrl promoter fragments continue to drive expression in the anterior midgut of the larvae (Myers, 2018).

Despite promiscuity in binding between TGFβ and their receptors in vertebrates, GDNF family members have not been reported to bind BMP/TGFβ receptors, suggesting that the ability to interact with more than one receptor was lost during evolution. The GDNF family of ligands, including GDNF, Neurturin, Artemin and Persephin, all appeared when fish gained jaws, as homologs cannot be identified in the published Agnatha sequences. GDNF ligands are distinguished by a highly conserved DLGLGY motif, part of one of two fingers that mediate binding to GFRα. This motif is not present in Mav or Panda. The change may have increased affinity or specificity for GFRs and additional changes might have prevented crosstalk with Activin/BMP type 1 receptors. Mav and Panda are similar to GDF-15, a TGFβ placed in the subfamily containing GDNF. GDF-15 is an inflammatory cytokine, and although it activates SMAD signaling, GDF-15 does not have an identified receptor. GDF-15 has GDNF-like neurotrophic activity for dopaminergic neurons, so it would be interesting to test GDF-15 for binding to GFRs (Myers, 2018).

The limited sequence data available suggest a model in which a divergent TGFβ acquired an ability to bind GFRs and activate Ret, which was followed by extensive co-evolution of the extracellular components. However, the downstream signaling pathways appear to be conserved, so the Ret SNS phenotypes open the door to invertebrate genetic analysis of this clinically important signaling pathway. Particularly exciting is the possibility of functional suppressor screens to identify mutations that could compensate for a lack of Ret signaling. Drosophila has already been used to identify genetic modifiers and a candidate drug to counteract oncogenic Ret signaling (Myers, 2018).

It is concluded Ret has an evolutionarily conserved role in the formation and function of the ENS. The GDNF signaling pathway has its origins in TGFβ signaling (Myers, 2018).

Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants

Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. This study investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. It was shown that signaling active version of Ret (Ret^MEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of Ret^MEN2B significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD (Klein, 2014).

The receptor tyrosine kinase Ret is already known to be required for long-term survival of nigral dopamine neurons in mice, and stimulation with its ligand GDNF protects dopamine neurons from cell death in a variety of toxin-based rodent and primate models of PD. The present work found that a signaling-active version of the Drosophila homolog of Ret suppresses degeneration of muscle tissue and mitochondrial abnormalities in Pink1 mutants. Interestingly, park mutants were not rescued. In human SH-SY5Y cells, stimulation of endogenous Ret by GDNF rescued both morphological and bioenergetic defects of mitochondria in PINK1-depleted cells. Pink1 and Parkin were previously shown to interact genetically in Drosophila in what was proposed to be a linear pathway, and a significant body of work has described how Pink1 and Parkin function to initiate mitophagy of impaired mitochondria, and arrest of mitochondrial trafficking. However, in the cell culture model of this study, Ret signaling did not induce mitophagy or Parkin recruitment, arguing that Ret rescues PINK1 deficits independently of Parkin. A recent study demonstrated that Pink1 mutants in contrast to park mutants have decreased function of complex I of the electron transport chain, suggesting that Pink1 is required for maintaining efficient complex I enzymatic activity and that this function is upstream of mitochondrial remodeling. This study found that Ret rescued both the impairment of complex I activity, and partially the mitochondrial morphology in Pink1 mutants, suggesting that complex I is a target of Ret signaling. Previous studies of complex I inhibition or genetic depletion have shown mild morphological impairments in Drosophila muscle, contrary to the stronger phenotype of Pink1 mutants. Therefore, it was somewhat unexpected that restoring complex I activity would be sufficient to rescue also morphological defects. One interpretation is that the Pink1 mutant morphological phenotype is more severe due to a synergistic effect of deficits in remodeling/mitophagy and complex I activity, which in this study was partially rescued. Another possibility is that Ret signaling not only targets complex I, but also morphology in a Parkin-independent manner (Klein, 2014).

Extrapolated to mammalian models, the results suggest a novel mechanism by which the GDNF family of neurotrophic factors may promote survival of dopamine neurons in PD. Several of the mammalian models where the neuroprotective effects of GDNF treatment were initially discovered, were in fact models of mitochondrial dysfunction, either directly via complex I inhibition by MPTP treatment. In light of the current findings, it would be interesting to investigate whether or not GDNF improves complex I activity in these model systems. GDNF has been tested in models of α-synuclein overexpression, a pathology that is not known to cause complex I deficiency, but did not show any neuroprotective effects, fitting with the current hypothesis (Klein, 2014).

The current findings support recent evidence showing that Pink1 has an important function related to complex I activity, which is independent of its function in recruiting Parkin to the outer mitochondrial membrane upon loss of membrane potential. This model is consistent with a partial rescue of Pink1 deficiencies, e.g., by either overexpressing Parkin or the yeast complex I equivalent NADH dehydrogenase, or, in the current work, Ret^MEN2B. In addition, the current findings are consistent with a recent study showing that Pink1-deficient flies but not Parkin-deficient flies can be rescued by TRAP1, which also seems to have beneficial effects on complex I activity (Klein, 2014).

The pathways by which Ret signaling targets complex I and rescues Pink1 mutants requires further investigation. Also, the mechanism by which Pink1 regulates complex I remains elusive, it may regulate for example gene expression, phosphorylation status or assembly. Gene expression analysis showed that most subunits are unchanged by Ret^MEN2B, but interestingly one subunit was moderately downregulated in Pink1 mutants and upregulated by Ret^MEN2B, which may improve function. However, the possibility cannot be excluded that Ret signaling targets complex I, and perhaps other metabolic components, by different means (Klein, 2014).

Brain-derived neurotrophic factor (BDNF) protects mouse cortical neurons against drug-induced excitotoxicity, an effect that was blocked by the complex I inhibitor Rotenone and a MEK1/2 inhibitor, suggesting that BDNF signaling via the Ras/Erk pathway can regulate complex I function (Markham, 2012). The signaling properties and functions of Drosophila Ret are not characterized in great detail, but it is structurally homologous to mammalian Ret and can, to some extent, activate the same signaling pathways (Abrescia, 2005). Mammalian Ret on the other hand, has been extensively characterized and is known to activate a number of downstream signaling pathways including Ras/ERK, phosphoinositol-3 kinase (PI3K)/Akt, phospholipase C-gamma (PLCγ), Janus kinase (JAK)/STAT, and ERK5, several of which have pro-survival effects, most notably the PI3K/Akt pathway (Sariola, 2003; Pascual, 2011). Recent studies of Pink1 and park mutant Drosophila have indicated that PI3K/Akt signaling or components downstream of this pathway rather exacerbates Pink1 and park mutant phenotypes, making it an unlikely candidate for rescue (Klein, 2014).

Additional studies are required to elucidate the details by which Pink1 and Ret regulate complex I activity, and whether this finding is transferrable to mammalian models. In summary, this work shows that Ret signaling can rescue phenotypes of Pink1 mutants by restoring mitochondrial respiration and specifically complex I function, and thereby suggests a potential novel mechanism underlying GDNF‐mediated protection in mammalian PD models. In the future, screening of PD patients for complex I deficiencies and subjecting specifically those individuals to GDNF treatment may provide a new therapeutic strategy (Klein, 2014).

Characterization of Drosophila GDNF receptor-like and evidence for its evolutionarily conserved interaction with neural cell adhesion molecule (NCAM)/FasII

Glial cell line-derived neurotrophic factor (GDNF) family ligands are secreted growth factors distantly related to the TGF-beta superfamily. In mammals, they bind to the GDNF family receptor α (Gfrα) and signal through the Ret receptor tyrosine kinase. In order to gain insight into the evolution of the Ret-Gfr-Gdnf signaling system, this study has cloned and characterized the first invertebrate Gfr-like cDNA [Glial cell line-derived neurotrophic family receptor-like (Gfrl)] from Drosophila melanogaster and generated a Gfrl mutant allele. DmGfrl encodes a large GPI-anchored membrane protein with four GFR-like domains. In line with the fact that insects lack GDNF ligands, DmGfrl mediated neither Drosophila Ret phosphorylation nor mammalian RET phosphorylation. In situ hybridization analysis revealed that DmGfrl is expressed in the central and peripheral nervous systems throughout Drosophila development, but, surprisingly, DmGfrl and DmRet expression patterns were largely non-overlapping. A DmGfrl null allele was generated by genomic FLP deletion, and both DmGfrl null females and males were found to be viable but display fertility defects. The female fertility defect manifested as dorsal appendage malformation, small size and reduced viability of eggs laid by mutant females. In male flies DmGfrl interacted genetically with the Drosophila Ncam (neural cell adhesion molecule) homolog FasII to regulate fertility. These results suggest that Ret and Gfrl do not function as an in cis receptor-coreceptor pair before the emergence of GDNF family ligands, and that the Ncam-Gfr interaction predated the in cis Ret-Gfr interaction in evolution. The fertility defects that were described in DmGfrl null flies suggest that GDNF receptor-like has an evolutionarily ancient role in regulating male fertility and a previously unrecognized role in regulating oogenesis. These results shed light on the evolutionary aspects of the structure, expression and function of Ret-Gfralpha and Ncam-Gfralpha signaling complexes (Kallijarvi, 2012).

ZD6474 suppresses oncogenic RET isoforms in a Drosophila model for type 2 multiple endocrine neoplasia syndromes and papillary thyroid carcinoma

Patients with hereditary medullary thyroid carcinoma (MTC) associated with multiple endocrine neoplasia (MEN) types 2A and 2B and familial MTC (FMTC) have mutations in the RET proto-oncogene. Approximately 40 percent of patients with papillary thyroid carcinoma (PTC) typically have either intrachromosomal or extrachromosomal rearrangements that join the promoter and NH(2)-terminal domains of unrelated genes to the COOH-terminal fragment of RET. The RET point mutations associated with MEN2A, MEN2B, or FMTC, or the chromosomal breakpoints and translocations associated with PTC, typically activate the RET receptor tyrosine kinase (RTK). RET kinase inhibitors are likely to be beneficial for patients with hereditary MTC, where currently there is no effective chemotherapy or radiation therapy. Recently, the low molecular weight tyrosine kinase inhibitor ZD6474 was found to block the enzymatic activity of RET-derived oncoproteins in cultured cell lines. This study developed a Drosophila model for MEN2A and MEN2B diseases by targeting oncogenic forms of RET to the developing Drosophila eye. When fed orally, ZD6474 suppressed RET-mediated phenotypes within the context of this in vivo model. Importantly, ZD6474 showed high efficacy and very low toxicity. This compound failed to significantly suppress an activated form of another RTK, the Drosophila epidermal growth factor receptor, nor did it suppress the activity of downstream components of the RET/Ras pathway. These results support the view that targeting chemical kinase inhibitors such as ZD6474 to tissues with oncogenic forms of RET is a useful treatment strategy for RET-dependent carcinomas (Vidal, 2005).

Drosophila RET contains an active tyrosine kinase and elicits neurotrophic activities in mammalian cells

The RET receptor tyrosine kinase controls kidney organogenesis and development of subpopulations of enteric and sensory neurons in different vertebrate species, including humans, rodents, chicken and zebrafish. RET is activated by binding to a ligand complex formed by a member of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors bound to its cognate GFRα GPI-linked co-receptor. Despite the absence of GDNF or GFRα molecules in the Drosophila genome, a RET orthologue (dRET) has recently been described in this organism and shown to be expressed in subpopulations of cells of the excretory, digestive and nervous systems, thus resembling the expression pattern of RET in vertebrates. This study reports on the initial biochemical and functional characterization of the dRET protein in cell culture systems. Full-length dRET could be produced in mammalian and insect cells. Similar to its human counterpart (hRET), overexpression of dRET resulted in its ligand-independent tyrosine phosphorylation, indicating that it bears an active tyrosine kinase. Unlike hRET, however, the extracellular domain of dRET was unable to interact with mammalian GDNF and GFRα1. Self association between dRET molecules could neither be detected, indicating that dRET is incapable of mediating cell adhesion by homophilic interactions. A chimeric molecule comprising the extracellular domain of hRET and the kinase domain of dRET was constructed and used to probe ligand-mediated downstream activities of the dRET kinase in PC12 cells. GDNF stimulation of cells transfected with the hRET/dRET chimera resulted in neurite outgrowth comparable to that obtained after transfection of wild-type hRET. These results indicate significant conservation between the biological effects elicited by the human and Drosophila RET kinases, and suggest functions for dRET in neuronal differentiation in the fly (Abrescia, 2005).

Expression pattern of Drosophila ret suggests a common ancestral origin between the metamorphosis precursors in insect endoderm and the vertebrate enteric neurons

The RET gene, encoding a receptor tyrosine kinase, is unusual among human protooncogenes in that its mutant alleles are implicated in a developmental defect involving enteric neurons as well as in tumorigenesis. The cells affected in both types of disorders are derived from the neural crest. Targeted disruption of mouse ret has revealed an additional role in kidney development. This study reports the analysis of a ret homolog in Drosophila melanogaster, an arthropod with no neural crest. Drosophila ret (D-ret) encodes a protein of 1,235 amino acids that has all of the domains identified in the vertebrate ret, including a cadherin motif. During embryogenesis, D-ret mRNA is first detected in the yolk sac at the late gastrula stage. In the postgastrula, D-ret is expressed in the foregut neurons, excretory system, peripheral ganglia, and the central nervous system. Thus, despite the wide divergence of early embryonic fate maps between vertebrates and invertebrates, D-ret is expressed in cells that are presumed to be the functional equivalents of the ret-expressing cells in vertebrates. Unexpectedly, D-ret is also expressed in the imaginal islands of the endodermal gut. These cells are proliferation-competent precursors for adult midgut that are diffusely embedded in the growth-arrested juvenile gut. These ret-expressing nonneuronal cells are strikingly analogous to vertebrate enteric neurons in their topography, but not in their cell fate. These finding suggests a previously unrecognized phylogenetic relationship between the ret-expressing cells in vertebrates and the precursor reserves of metamorphosing insects (Hahn, 2001).

The Drosophila Ret gene is transcribed in multiple alternatively spliced forms

The 5' and 3' ends of the Drosophila homolog of the vertebrate c-ret gene, Ret, were cloned, and its predicted protein sequence was derived. The extracellular domain of Ret is very widely diverged from that of its vertebrate counterparts but the cadherin motif present in vertebrate c-ret proteins can also be discerned in Ret. As with the vertebrate gene, multiple splice variants were detected at the 5'-end of Ret, one of which inserts an exon with a protein-terminating frameshift into the cDNA. In contrast to human c-ret, which may vary its signalling specificity by using splicing-derived, alternative C-terminal sequences, Ret cDNAs showed no variation at their 3'-ends (Huen, 2000).

A Drosophila homolog of human proto-oncogene ret transiently expressed in embryonic neuronal precursor cells including neuroblasts and CNS cells

A Drosophila gene encoding a putative receptor tyrosine kinase has been identified by screening a genomic DNA library with a DNA probe for a Drosophila homolog of fibroblast growth factor receptors. The newly isolated gene codes for a transmembrane protein most similar in sequence to a mammalian proto-oncogene ret; thus, the gene was termed Dret. Dret mRNA is transcribed in very small amounts in the embryonic, larval, and pupal stages. Whole mount in situ hybridization experiments revealed that the mRNA is transiently expressed in neuroblasts in early embryos. In late embryos, Dret mRNA was detected in subpopulations of differentiating CNS and PNS cells. In addition, Dret expression was affected in neurogenic mutants. These results suggest that Dret can be considered as a functional homolog of mammalian ret and should play important roles in neurogenesis (Sugaya, 1994).

Functions of Ret orthologs in other species

RET functions as a Dual-Specificity Kinase that requires allosteric inputs from juxtamembrane elements

Receptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. This study shows that the juxtamembrane (JM) segment enhances RET (see Drosophila Ret oncogene) catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. This phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. These findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery (Plaza-Menacho, 2016).

Search PubMed for articles about Drosophila Ret

Abrescia, C., Sjostrand, D., Kjaer, S. and Ibanez, C. F. (2005). Drosophila RET contains an active tyrosine kinase and elicits neurotrophic activities in mammalian cells. FEBS Lett 579: 3789-3796. PubMed ID: 15978587

Airaksinen, M. S., Holm, L. and Hatinen, T. (2006). Evolution of the GDNF family ligands and receptors. Brain Behav Evol 68: 181-190. PubMed ID: 16912471

Almeida, M. Q. and Hoff, A. O. (2012). Recent advances in the molecular pathogenesis and targeted therapies of medullary thyroid carcinoma. Curr Opin Oncol 24: 229-234. PubMed ID: 22343387

Amiel, J., et al. (2008). Hirschsprung disease, associated syndromes and genetics: a review. J Med Genet 45: 1-14. PubMed ID: 17965226

Bonanomi, D., Chivatakarn, O., Bai, G., Abdesselem, H., Lettieri, K., Marquardt, T., Pierchala, B. A. and Pfaff, S. L. (2012). Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals. Cell 148: 568-582. PubMed ID: 22304922

Bourane, S., Garces, A., Venteo, S., Pattyn, A., Hubert, T., Fichard, A., Puech, S., Boukhaddaoui, H., Baudet, C., Takahashi, S., Valmier, J. and Carroll, P. (2009). Low-threshold mechanoreceptor subtypes selectively express MafA and are specified by Ret signaling. Neuron 64: 857-870. PubMed ID: 20064392

Buchon N, Broderick NA, Kuraishi T, Lemaitre B (2010). Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection. BMC Biol 8: 152. PubMed ID: 21176204

Cockburn, J. G., Richardson, D. S., Gujral, T. S. and Mulligan, L. M. (2010). RET-mediated cell adhesion and migration require multiple integrin subunits. J Clin Endocrinol Metab 95: E342-346. PubMed ID: 20702524

Dar, A. C., Das, T. K., Shokat, K. M. and Cagan, R. L. (2012). Chemical genetic discovery of targets and anti-targets for cancer polypharmacology. Nature 486: 80-84. PubMed ID: 22678283

Fukuda, T., Kiuchi, K. and Takahashi, M. (2002). Novel mechanism of regulation of Rac activity and lamellipodia formation by RET tyrosine kinase. J Biol Chem 277: 19114-19121. PubMed ID: 11886862

Hahn, M. and Bishop, J. (2001). Expression pattern of Drosophila ret suggests a common ancestral origin between the metamorphosis precursors in insect endoderm and the vertebrate enteric neurons. Proc Natl Acad Sci U S A 98: 1053-1058. PubMed ID: 11158593

Han, C., Wang, D., Soba, P., Zhu, S., Lin, X., Jan, L. Y. and Jan, Y. N. (2012). Integrins regulate repulsion-mediated dendritic patterning of drosophila sensory neurons by restricting dendrites in a 2D space. Neuron 73: 64-78. PubMed ID: 22243747

Hernandez, K., Myers, L. G., Bowser, M. and Kidd, T. (2015). Genetic tools for the analysis of Drosophila stomatogastric nervous system development. PLoS One 10(6): e0128290. PubMed ID: 26053861

Huen, D. S., Elsdon, M. and Ponder, B. A. (2000). The Drosophila Ret gene is transcribed in multiple alternatively spliced forms. Mol Gen Genet 264: 335-340. PubMed ID: 11085274

Kallijarvi, J., Stratoulias, V., Virtanen, K., Hietakangas, V., Heino, T. I. and Saarma, M. (2012). Characterization of Drosophila GDNF receptor-like and evidence for its evolutionarily conserved interaction with neural cell adhesion molecule (NCAM)/FasII. PLoS One 7: e51997. PubMed ID: 23284846

Kim, M. E., Shrestha, B. R., Blazeski, R., Mason, C. A. and Grueber, W. B. (2012). Integrins establish dendrite-substrate relationships that promote dendritic self-avoidance and patterning in drosophila sensory neurons. Neuron 73: 79-91. PubMed ID: 22243748

Klein, P., Muller-Rischart, A. K., Motori, E., Schonbauer, C., Schnorrer, F., Winklhofer, K. F. and Klein, R. (2014). Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants. EMBO J 33: 341-355. PubMed ID: 24473149

Koike-Kumagai, M., Yasunaga, K., Morikawa, R., Kanamori, T. and Emoto, K. (2009). The target of rapamycin complex 2 controls dendritic tiling of Drosophila sensory neurons through the Tricornered kinase signalling pathway. EMBO J 28: 3879-3892. PubMed ID: 19875983

Kowsky, S., Poppelmeyer, C., Kramer, E. R., Falkenburger, B. H., Kruse, A., Klein, R. and Schulz, J. B. (2007). RET signaling does not modulate MPTP toxicity but is required for regeneration of dopaminergic axon terminals. Proc Natl Acad Sci U S A 104: 20049-20054. PubMed ID: 18056810

Kramer, E. R., Knott, L., Su, F., Dessaud, E., Krull, C. E., Helmbacher, F. and Klein, R. (2006). Cooperation between GDNF/Ret and ephrinA/EphA4 signals for motor-axon pathway selection in the limb. Neuron 50: 35-47. PubMed ID: 16600854

Kramer, E. R., Aron, L., Ramakers, G. M., Seitz, S., Zhuang, X., Beyer, K., Smidt, M. P. and Klein, R. (2007). Absence of Ret signaling in mice causes progressive and late degeneration of the nigrostriatal system. PLoS Biol 5: e39. PubMed ID: 17298183

Lairmore, T. C., Dou, S., Howe, J. R., Chi, D., Carlson, K., Veile, R., Mishra, S. K., Wells, S. A., Jr. and Donis-Keller, H. (1993). A 1.5-megabase yeast artificial chromosome contig from human chromosome 10q11.2 connecting three genetic loci (RET, D10S94, and D10S102) closely linked to the MEN2A locus. Proc Natl Acad Sci U S A 90: 492-496. PubMed ID: 8093642

Luo, W., Enomoto, H., Rice, F. L., Milbrandt, J. and Ginty, D. D. (2009). Molecular identification of rapidly adapting mechanoreceptors and their developmental dependence on ret signaling. Neuron 64: 841-856. PubMed ID: 20064391

Macagno, J. P., Diaz Vera, J., Yu, Y., MacPherson, I., Sandilands, E., Palmer, R., Norman, J. C., Frame, M. and Vidal, M. (2014). FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells. PLoS Genet 10(3): e1004262. PubMed ID: 24676055

Markham, A., Cameron, I., Bains, R., Franklin, P., Kiss, J. P., Schwendimann, L., Gressens, P. and Spedding, M. (2012). Brain-derived neurotrophic factor-mediated effects on mitochondrial respiratory coupling and neuroprotection share the same molecular signalling pathways. Eur J Neurosci 35: 366-374. PubMed ID: 22288477

Myers, L., Perera, H., Alvarado, M. G. and Kidd, T. (2018). The Drosophila Ret gene functions in the stomatogastric nervous system with the Maverick TGFbeta ligand and the Gfrl co-receptor. Development 145(3). PubMed ID: 29361562

Pachnis, V., Mankoo, B. and Costantini, F. (1993). Expression of the c-ret proto-oncogene during mouse embryogenesis. Development 119: 1005-1017. PubMed ID: 8306871

Pascual, A., Hidalgo-Figueroa, M., Gomez-Diaz, R. and Lopez-Barneo, J. (2011). GDNF and protection of adult central catecholaminergic neurons. J Mol Endocrinol 46: R83-92. PubMed ID: 21357726

Perea, D., Guiu, J., Hudry, B., Konstantinidou, C., Milona, A., Hadjieconomou, D., Carroll, T., Hoyer, N., Natarajan, D., Kallijarvi, J., Walker, J. A., Soba, P., Thapar, N., Burns, A. J., Jensen, K. B. and Miguel-Aliaga, I. (2017). Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia. EMBO J 36(20):3029-3045. PubMed ID: 28899900

Plaza-Menacho, I., Barnouin, K., Barry, R., Borg, A., Orme, M., Chauhan, R., Mouilleron, S., Martinez-Torres, R. J., Meier, P. and McDonald, N. Q. (2016). RET functions as a Dual-Specificity Kinase that requires allosteric inputs from juxtamembrane elements. Cell Rep 17(12): 3319-3332. PubMed ID: 28009299

Read, R. D., Goodfellow, P. J., Mardis, E. R., Novak, N., Armstrong, J. R. and Cagan, R. L. (2005). A Drosophila model of multiple endocrine neoplasia type 2. Genetics 171: 1057-1081. PubMed ID: 15965261

Runeberg-Roos, P. and Saarma, M. (2007). Neurotrophic factor receptor RET: structure, cell biology, and inherited diseases. Ann Med 39: 572-580. PubMed ID: 17934909

Sariola, H. and Saarma, M. (2003). Novel functions and signalling pathways for GDNF. J Cell Sci 116: 3855-3862. PubMed ID: 12953054

Schuchardt, A., D'Agati, V., Larsson-Blomberg, L., Costantini, F. and Pachnis, V. (1994). Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 367: 380-383. PubMed ID: 8114940

Soba, P., Han, C., Zheng, Y., Perea, D., Miguel-Aliaga, I., Jan, L. Y. and Jan, Y. N. (2015). The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion. Elife 4. PubMed ID: 25764303

Sugaya, R., Ishimaru, S., Hosoya, T., Saigo, K. and Emori, Y. (1994). A Drosophila homolog of human proto-oncogene ret transiently expressed in embryonic neuronal precursor cells including neuroblasts and CNS cells. Mech Dev 45: 139-145. PubMed ID: 8199050

Tian, A., Benchabane, H., Wang, Z. and Ahmed, Y. (2016). Regulation of stem cell proliferation and cell fate specification by wingless/Wnt signaling gradients enriched at adult intestinal compartment boundaries. PLoS Genet 12: e1005822. PubMed ID: 26845150

Vidal, M., Wells, S., Ryan, A. and Cagan, R. (2005). ZD6474 suppresses oncogenic RET isoforms in a Drosophila model for type 2 multiple endocrine neoplasia syndromes and papillary thyroid carcinoma. Cancer Res 65: 3538-3541. PubMed ID: 15867345

Yan, K.S., Gevaert, O., Zheng, G.X.Y., Anchang, B., Probert, C.S., Larkin, K.A., Davies, P.S., Cheng, Z.F., Kaddis, J.S., Han, A., Roelf, K., Calderon, R.I., Cynn, E., Hu, X., Mandleywala, K., Wilhelmy, J., Grimes, S.M., Corney, D.C., Boutet, S.C., Terry, J.M., et al., (2017) Intestinal enteroendocrine lineage cells possess homeostatic and injury-inducible stem cell activity. Cell Stem Cell 21: 78-90. PubMed ID: 28686870

date revised: 23 June 2023

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