InteractiveFly: GeneBrief

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

Gene name - Glial cell line-derived neurotrophic family receptor-like

Synonyms - munin

Cytological map position - 92E8-92E12

Function - receptor

Keywords - oogenesis, spermatogenesis, CNS, PNS, FasII receptor

Symbol - Gfrl

FlyBase ID: FBgn0262869

Genetic map position - chr3R:16204718-16275315

Classification - GDNF: GDNF/GAS1 domain

Cellular location - GPI-anchored membrane protein

NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Myers, L., Perera, H., Alvarado, M. G. and Kidd, T. (2017). The Drosophila Ret gene functions in the stomatogastric nervous system with the Maverick TGFbeta ligand and the Gfrl co-receptor. Development [Epub ahead of print]. PubMed ID: 29361562
The RET receptor tyrosine kinase is critical 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 homologue (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 (SNS) 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 to respond to a ligand. This study identified the TGFbeta 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.

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 SpƤtzle, 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 FasIIspin allele has been shown to disrupt the looping of the male genitalia and spermiduct. Interestingly, in FasIIspin 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 FasIIe76;;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).


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

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

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

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

Biological Overview

date revised: 8 August 2013

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