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Glial cell line-derived neurotrophic family receptor-like: Biological Overview | References

BIOLOGICAL OVERVIEW

Glial cell line-derived neurotrophic factor (GDNF) family ligands are secreted growth factors distantly related to the TGF-β 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, the first invertebrate Gfr-like cDNA (DmGfrl) was cloned and characterized from Drosophila melanogaster, and a DmGfrl mutant allele was generated. It was found that DmGfrl encodes a large GPI-anchored membrane protein with four GFR-like domains. In line with the fact that insects lack GDNF ligands, DmGfrl mediates 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 it was found that both DmGfrl null females and males are 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 did 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 describe 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-Gfrα and Ncam-Gfrα signaling complexes (Kallijarvi, 2012).

There is ample suggestive evidence that neurons in invertebrates require trophic support similarly to vertebrate neurons, although the identification of neurotrophic ligands in e.g., Drosophila has progressed only recently. The first Drosophila homologs of vertebrate neurotrophin family proteins, Drosophila neurotrophin 1 (DNT1), DNT2 and Spä is a tzle, were identified in silico several years ago and recently characterized in detail and shown to possess neurotrophic activity in vivo (Zhu, 2008). Additionally, DmManf, the Drosophila homolog of the novel mammalian CDNF/MANF family of neurotrophic factors, is required for the development of the Drosophila embryonic nervous system (Palgi, 2009; Kallijarvi, 2012 and references therein).

Glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are secreted growth factors distantly related to the TGF-β superfamily. GFLs are crucial for the development and maintenance of distinct populations of central and peripheral neurons, as well as for the organogenesis of the kidney, and spermatogenesis. In mammals, four different GFL-coreceptor pairs exist. They all signal intracellularly through the RET receptor tyrosine kinase (Airaksinen, 2002). Neural cell adhesion molecule (NCAM) is an alternative signaling receptor for GDNF in mammals (Paratcha, 2003). NCAM binds GFRα1 and GDNF and downregulates NCAM-mediated cell adhesion, which activates cytoplasmic protein tyrosine kinase signaling in the absence of RET. Through NCAM, GDNF stimulates Schwann cell migration and axonal growth in hippocampal and cortical neurons in mouse brain (Kallijarvi, 2012).

Mammalian GDNF family alpha receptors (GFRα) contain a conserved arrangement of extracellular cysteine-rich GFRα domains and a C-terminal GPI anchor. Homologs of GFLs, RET and the four mammalian GFRα receptors exist in all vertebrates. RET homologs seem to be present in insects but not in echinoderms. The Drosophila melanogaster RET homolog is expressed in many tissues analogous to the tissues where the gene is expressed in vertebrates, suggesting similar functions in development. GFR-like proteins have been identified in silico in sea urchin, insects and worms, including D. melanogaster and C. elegans. In Drosophila, two partial mRNA sequences encoding fragments of GFR-like proteins have been identified. However, GDNF family ligand genes have not been found in invertebrates by in silico methods. To shed light on the evolutionary origin and function of invertebrate GFR-like proteins, this study set out to characterize the Drosophila melanogaster Gfr-like gene (DmGfrl) gene and protein, to investigate its interaction with the mammalian GDNF receptors and to generate a DmGfrl null allele to investigate the in vivo functions of the receptor (Kallijarvi, 2012).

At the start of this project, two Drosophila melanogaster cDNA fragments predicting amino acid sequence with similarity to the GFRα domains of mammalian GDNF receptor proteins had been annotated in Genbank. Starting from these cDNA fragments, RACE, RT-PCR and in silico sequence analysis was used to assemble what was presumed to be the full genomic structure of the gene, and altogether six transcripts produced from this locus were identified. Based on previously suggested nomenclature, this gene was named Drosophila melanogaster Glial cell line-derived neurotrophic factor family receptor-like, or DmGfrl. The two major DmGfrl transcripts (A and B) detectable on Northern blots were found to differ only in their 5' untranslated regions and the 5' coding sequence preceding the first GFRα-like domain, including the translation initiation site and a predicted signal sequence. The exons harboring the translation start sites for transcript A and B are separated in the genome by ~27 kb, which indicates that the two main transcripts are very likely to have separate promoter regions. Such differential promoter usage may serve to allow regulation of the same gene product by separate sets of transcription factors in different developmental and/or physiological contexts. Indeed, DmGfrl transcript A is predominant in embryos. Both major DmGfrl transcripts encode a protein with four cysteine-rich GFRα-like domains, which is in line with previous in silico predictions. Similarity to the mammalian GFRα receptors is restricted to these domains, which have a characteristic arrangement of 10 cysteine residues in each domain. Interestingly, a Gfr-like gene in C. elegans predicts a similarly large protein of >1000 amino acids with four GFRα-like domains. Based on gene structures a common origin has been proposed for the exons encoding D1 to D3 in insect and sea urchin Gfr-like proteins and vertebrate GFRα genes, which suggests that a protoGFRα receptor evolved before the protostome-deuterostome divergence (Kallijarvi, 2012).

Insects lack GDNF family ligands, but having cloned the Drosophila receptor homologs it was asked whether they might respond to mammalian GDNF and whether DmGfrl could mediate mammalian RET phosphorylation. Both experiments suggested that DmRet and DmGfrl are not structurally sufficiently conserved to bind to mammalian GDNF or interact with the mammalian receptor homologs. It is interesting to speculate that one of the seven Drosophila TGF-β ligands could function as a soluble ligand ('protoGDNF') for DmRet and/or DmGfrl (Kallijarvi, 2012).

During Drosophila embryogenesis, DmRet is expressed in many tissues that are functionally analogous to those in which mammalian RET is expressed, including foregut neurons, the excretory system, peripheral ganglia and the central nervous system (Hahn, 2001). DmGfrl and DmRet expression in the embryonic nervous system and in the larval and adult brain was compared using in situ hybridization. The expression pattern of DmGfrl was generally concordant with the neuronal cell expression of GFRα1 and GFRα2 in mice, in which expression at both the mRNAs and proteins has been reported in several brain areas, the spinal cord and various peripheral ganglia. Interestingly, however, DmGfrl expression was detected in the Malpighian tubules, the Drosophila analog of mammalian kidney. In line with previously published in situ hybridization data (Hahn, 2001; Sugaya, 2004), DmRet was found to be first expressed in the yolk sac and subsequently in the ventral neuroectoderm starting from embryonic stage 13. DmRet and DmGfrl expression coincided temporally but not spatially during embryogenesis. In the larval and adult brain, DmGfrl and DmRet expression patterns were also completely non-overlapping. Thus, it is concluded that DmRet and DmGfrl likely do not function as an in cis receptor-co-receptor pair as do mammalian RET and GFRα receptors. However, the data do not rule out the possibility that DmRet and DmGfrl could interact via an alternative mode, for example in trans (cell-to-cell) or by cleavage and diffusion of soluble DmGfrl. In the absence of a DmRet null allele or a suitable hypomorphic allele, a genetic interaction between DmRet and DmGfrl was sought in misexpression experiments. No evidence was found that DmGfrl coexpression could modify ectopic DmRet-induced phenotype in the eye. The ectopic expression experiment is, however, inconclusive, and progress in this direction will require the generation of a DmRet allele suitable for genetic interaction experiments (Kallijarvi, 2012).

To gain insight into the in vivo function of the DmGfrl receptor a DmGfrl null allele by was generated by FLP-mediated genomic deletion. DmGfrl null flies were grossly normal and viable. However, they displayed a severe defect in both male and female fertility. The reduced female fertility results from an oogenesis defect as the mutant females laid fewer eggs than normally and a large fraction of those were small and had abnormal dorsal appendages. The egg morphology defect was efficiently rescued by transgene expression under the widely active daughterless and actin drivers, indicating that the phenotype is specific to loss of DmGfrl expression, and likely dependent on the somatic tissue of the ovary. However, the transgene did not rescue the reduced viability of the eggs or the reduced fecundity of the females. This suggests that the reduced egg viability is either dependent on germline cells, in which this transgene should not be expressed, or is not rescued by the DmGfrlA isoform used in these experiments. Similarly to females, in DmGfrl null males a fertility defect was observed that was not fully penetrant. Because the fecundity of DmGfrl null males was much more reduced than their absolute fertility, it was reasoned that a defect in spermatogenesis is a likely cause. Dissection of the testis histology and function in the mutant flies, as well as further rescue experiments will likely clarify the mechanism of the fertility defect in DmGfrl null males. Interestingly, on the basis of a proteomics study DmRet protein is present in adult spermatozoa, which warrants studies of the putative conserved function of DmRet in spermatogenesis (Kallijarvi, 2012).

Finally, on the basis of molecular evidence from mammals, it was of interest to look if DmGfrl might interact with the Drosophila NCAM homolog FasII. In mammals NCAM binds GDNF and GFRα1 and functions as an alternative signaling receptor for GDNF, mediating neuronal migration and axonal growth (Paratcha, 2003). FasII is widely expressed in the embryonic VNC, making it likely that it is also expressed in the DmGfrl-expressing neurons. WA hypomorphic FasII allele was combined with the delDmGfrl allele and whether the former could modify the male fertility phenotype of DmGfrl null flies was investigated. Strikingly, the double homozygous males were completely infertile, indicating a strong genetic interaction between DmGfrl and the FasII allele. There is currently little data linking NCAM/FasII function to reproduction. Nevertheless, on the basis of in silo data both DmGfrl and FasII are expressed at low levels in the testis and ovary. There is evidence for a role of FasII in the hormonal control of the development of Drosophila male genitalia, as a FasII^spin allele has been shown to disrupt the looping of the male genitalia and spermiduct. Interestingly, in FasII^spin flies, the innervation of corpora allata in the ring gland is disrupted, which the authors suggest may lead to elevated level of juvenile hormone and eventually to the looping defect. In preliminary inspection, no rotation defect was observed in the external male genitalia in FasII^e76;;del/Df males, but this lead will be worth further study. No gross embryonic or adult neuronal phenotypes was observed in the DmGfrl null flies. As subtle developmental or behavioral phenotypes may be present this question will require careful further studies (Kallijarvi, 2012).

The strong genetic interaction between DmGfrl and FasII that was described is corroborated by data showing biochemical interaction between the ectopically expressed receptors. These data are the first to suggest that the GFRα1-NCAM interaction described in mammalian systems is evolutionarily conserved. Together with the results suggesting that DmRet and DmGfrl do not function in cis in Drosophila, which lacks GDNF ligands, these data imply that DmGfrl may be an evolutionarily ancient binding partner for NCAM/FasII. Whether or not a soluble ligand exists in Drosophila and is needed to activate the putative FasII-DmGfrl signaling complex needs to be tackled in future studies (Kallijarvi, 2012).

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).

Search PubMed for articles about Drosophila Gfrl

Airaksinen, M. S. and Saarma, M. (2002). The GDNF family: signalling, biological functions and therapeutic value. Nat Rev Neurosci 3: 383-394. PubMed ID: 11988777

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

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

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

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

Palgi, M., Lindstrom, R., Peranen, J., Piepponen, T. P., Saarma, M. and Heino, T. I. (2009). Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons. Proc Natl Acad Sci U S A 106: 2429-2434. PubMed ID: 19164766

Paratcha, G., Ledda, F. and Ibanez, C. F. (2003). The neural cell adhesion molecule NCAM is an alternative signaling receptor for GDNF family ligands. Cell 113: 867-879. PubMed ID: 12837245

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

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

Zhu, B., Pennack, J. A., McQuilton, P., Forero, M. G., Mizuguchi, K., Sutcliffe, B., Gu, C. J., Fenton, J. C. and Hidalgo, A. (2008). Drosophila neurotrophins reveal a common mechanism for nervous system formation. PLoS Biol 6: e284. PubMed ID: 19018662

date revised: 25 April 2018

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