The Interactive Fly
Genes involved in tissue and organ development
The stomatogastric nervous system (SNS) consists of several peripheral ganglia that receive input from the brain; these ganglia in turn innervate muscles, pharynx, and gut. Precursors originate from the primordium of the foregut or stomodeum [Images]. The SNS is considered to be derived from the labral segment, the most anterior of the head segments. Early in development several subsets of precursors delaminate from the stomodeal epithelium as individual cells.
Three cells are singled out from within a single proneural cluster, known as the SNS anlage; these cells then initiate a distinct feature of SNS development. Serving as tip cells, they direct a second phase of SNS development wherein an invagination process occurs, forming three distinct epithelial folds with a single proneural-expressing cell at each tip. achaete-scute and neurogenic genes function here in the same manner as they do in the development of the ventral nerve cord, except that three cells are selected, not just one. The homeobox protein Goosecoid marks cells fated to become SNS cells.
Once the invagination process is complete, proneural gene expression appears de novo in all cells contained within the three invaginations; the three invaginations then pinch off from the epithelium to form separate epithelial vesicles. At later stages the cells of the three vesicles migrate to various locations where they differentiate as neurons, organizing into the mature embryonic SNS. Subsequently, these neural cells generate the axonal scaffold. Neurons from the individual ganglia send out pioneer axons that meet up with outgrowing axons from the other ganglia and eventually establish the interconnecting nerves. Other neurons send out axons to establish the nerves that innervate the dorsal pharyngeal muscles, the midgut and the CNS.
The mature embryonic SNS consists of only four ganglia (the frontal ganglion, the esophageal ganglion 1, the esophageal ganglion 2, and the proventricular ganglion) and their associated nerve tracts. The glia of the mature SNS are found as three groups of cells: one group is associated with the frontal ganglion (the frontal ganglion glia), a second group is found in the frontal commissure, at the base of the frontal nerve (commisural glia), and a third group is located at the fork in the recurrent nerve, off of which the two esophageal ganglia extend (esophageal ganglia glia).
The stomatogastric nervous system (SNS) of Drosophila is a simply organized neural circuitry that innervates the anterior enteric system. Unlike the central and the peripheral nervous systems, the SNS derives from a compact epithelial anlage in which three invagination centers, each giving rise to an invagination fold headed by a tip cell, are generated. Tip cell selection involves lateral inhibition, a process in which Wingless (Wg) activity adjusts the range of Notch signaling. RTK signaling mediated by the Epidermal growth factor receptor plays a key role in two consecutive steps during early SNS development. Like Wg, Egfr signaling participates in adjusting the range of Notch-dependent lateral inhibition during tip cell selection. Subsequently, tip cells secrete the Egfr ligand Spitz and trigger local RTK signaling, which initiates morphogenetic movements resulting in the tip cell-directed invaginations within the SNS anlage (González-Gaitán, 2000).
In order to investigate the role of RTK signaling in SNS development, lack-of-function mutants of the Egfr ligand Spitz were examined. In spitz mutants, the formation of the four SNS ganglia is strongly impaired. The SNS anlage, however, forms normally. In addition, the expression domain of wg and proneural AS-C genes is indistinguishable from a wild-type SNS anlage. At the stage when the three ac-expressing cells were singled-out within the wild-type SNS anlage, only one ac positive cell is found in spitz mutants. The same phenotype has been observed in wg mutants or mutants lacking an integral component of the wg pathway. Since no altered wg pattern was found in the spitz mutant SNS anlage, Spitz-dependent RTK signaling may act in parallel or in combination with wg to adjust the proper range of Notch-dependent lateral inhibition. In contrast to wg mutants, however, no invagination fold is observed. This observation indicates that the singled-out ac-expressing cell of spitz mutants has lost the ability to function as a tip cell and possibly fails to induce morphogenetic movements within the SNS anlage (González-Gaitán, 2000).
spitz, like other genes encoding components of the Egfr signaling pathway such as Egfr, Ras, Raf and the cascade of MAP kinases, is ubiquitously expressed. Local activation of Egfr signaling requires the transmembrane protein Star, which is necessary for the secretion of Spitz. Star is expressed in restricted patterns corresponding to the Spitz secreting cells. In the SNS anlage, it was noted that Star becomes restricted to the three tip cells and is maintained in these cells when invagination takes place. As in spitz mutants, the Star mutant SNS anlage is established normally; only one ac-expressing cell is selected and no invagination occurs. Consistently, Star mutants fail to develop the proper set of SNS ganglia and the associated nerves. These observations suggest that tip cells are a Star-dependent source of Spitz activity that triggers Egfr-dependent RTK signaling in the neighboring cells within the SNS anlage. This conclusion is supported by the finding that phosphorylated MAPK, a cellular marker for RTK signaling activity, is indeed activated in cells of the invagination folds, whereas phosphorylated MAPK does not appear in the Star mutant or in the spitz mutant SNS anlage (González-Gaitán, 2000).
To examine whether activated Spitz is sufficient to induce cell movements within the SNS anlage, use was made of the GAL4/UAS system to misexpress secreted Spitz in an ectopic pattern. This was achieved through the expression of activated Spitz from a UAS promotor driven transgenethat was activated by Gal4 under the control of the actin promotor. Under the conditions applied, scattered UAS-dependent transgene expression is observed throughout the early embryo, including the SNS anlage. When activated Spitz is expressed in such a pattern, a variable number of supernumerary infoldings within the SNS anlagen are observed, indicating that activated Spitz is sufficient to initiate cell movements. This result, in conjunction with the observation that the invaginated cells express phosphorylated MAPK, provides evidence that tip cell-derived activated Spitz triggers RTK signaling to initiate the invagination process. This proposal was tested by blocking Egfr signaling in the anterior most region of the SNS anlage that gives rise to the first invagination fold. For this, a GAL4 driver (SNS1-Gal4) was used that causes UAS-dependent gene expression in the corresponding region of the SNS anlage. SNS1-Gal4-mediated expression of a dominant-negative Egfr mutant form from a UAS-controlled transgene causes a specific suppression of the anterior most invagination fold without affecting the others (González-Gaitán, 2000).
The results demonstrate that RTK signaling participates in the selection of tip-cell-dependent invagination centers in the SNS anlage and is subsequently required to initiate morphogenetic movements resulting in invagination folds. This study does not focus on how RTK signaling ties into the wg-modulated Notch signaling process previously shown to be necessary for the selection of the three SNS invagination centers. The data indicate, however, that RTK signaling acts either in parallel or in combination with wg signaling to adjust the proper range of Notch-dependent lateral inhibition. Although in both wg and Egfr signaling mutants, only one ac-expressing cell is singled-out, the selected cells differ with respect to whether they function as tip cells or not. In wg mutants, the single cell causes an invagination, whereas in Egfr signaling mutants, the selected cell fails to provide this feature of SNS invagination centers. The results, therefore, consistently argue that tip cell-derived Spitz triggers local RTK signaling and thereby initiates the formation of invagination folds each headed by the Spitz-secreting tip cell. Thus, Egfr-dependent RTK signaling in Drosophila does not only participate in cell fate decisions and cell proliferation, but also triggers morphogenetic movements within an epithelium, as has been recently demonstrated for fibroblast growth factor (FGF) signaling. It will be interesting to see whether the role of the EGF pathway in cell migration differs at the cellular level from cell migration events triggered by activated FGF receptors (González-Gaitán, 2000).
The stomatogastric nervous system (SNS) associated with the foregut was studied in 3rd instar larvae of Drosophila melanogaster and Calliphora vicina (blowfly). In both species, the foregut comprises pharynx, esophagus, and proventriculus. Only in Calliphora does the esophagus form a crop. The position of nerves and neurons was investigated with neuronal tracers in both species and GFP expression in Drosophila. The SNS is nearly identical in both species. Neurons are located in the proventricular and the hypocerebral ganglion (HCG), which are connected to each other by the proventricular nerve. Motor neurons for pharyngeal muscles are located in the brain not, as in other insect groups, in the frontal ganglion. The position of the frontal ganglion is taken by a nerve junction devoid of neurons. The junction is composed of four nerves: the frontal connectives that fuse with the antennal nerves (ANs), the frontal nerve innervating the cibarial dilator muscles and the recurrent nerve that innervates the esophagus and projects to the HCG. Differences in the SNS are restricted to a crop nerve only present in Calliphora and an esophageal ganglion that only exists in Drosophila. The ganglia of the dorsal organs give rise to the ANs, which project to the brain. The extensive conformity of the SNS of both species suggests functional parallels. Future electrophysiological studies of the motor circuits in the SNS of Drosophila will profit from parallel studies of the homologous but more accessible structures in Calliphora (Spoess, 2008).
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).
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Genes involved in organ development
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