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The GDNF family ligands and receptors — implications for neural development
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The GDNF family ligands and receptors — implications for neural development Robert H Baloh*, Hideki Enomoto†, Eugene M Johnson Jr‡ and Jeffrey Milbrandt§ The glial cell line derived neurotrophic factor (GDNF) family has recently been expanded to include four members, and the interactions between these neurotrophic factors and their unique receptor system is now beginning to be understood. Furthermore, analysis of mice lacking the genes for GDNF, neurturin, and their related receptors has confirmed the importance of these factors in neurodevelopment. The results of such analyses reveal numerous similarities and potential overlaps in the way the GDNF and the nerve growth factor (NGF) families regulate development of the peripheral nervous system. Addresses *†§ Departments of Pathology and Internal Medicine, ‡ Department of Neurology and Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA *e-mail: [email protected] † e-mail: [email protected] ‡ e-mail: [email protected] § e-mail: [email protected] Current Opinion in Neurobiology 2000, 10:103–110 0959-4388/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations ARTN artemin DRG dorsal root ganglion E embryonic day GDNF glial cell line derived neurotrophic factor GFL GDNF family ligand α GDNF family receptor α-component GFRα NGF nerve growth factor NRTN neurturin PSPN persephin RET rearranged in transfection (receptor tyrosine kinase) SCG superior cervical ganglion
Introduction Since the discovery of nerve growth factor (NGF) and the establishment of its ability to support neuronal survival [1–3], extensive efforts have been made to identify additional neurotrophic factors that can influence neurons in primary culture, during normal development, or in experimental models of neuronal injury. This work has resulted in the identification of a large and diverse group of proteins that are capable of promoting neuronal survival in various experimental paradigms. Glial cell line derived neurotrophic factor (GDNF) was initially identified as a factor secreted from a glioma cell line capable of supporting embryonic ventral midbrain neuron survival in culture [4]. Our knowledge of the in vitro activities of GDNF expanded rapidly after its discovery to now include survival promotion of additional central neurons (including spinal motor neurons) and at least a subpopulation of all peripheral ganglia yet examined
[5–8,9••]. The discovery of neurturin (NRTN) three years later, which is ~44% identical to GDNF, established the existence of the GDNF family ligands (GFLs) [8]. Furthermore, shortly after the discovery of NRTN, both GDNF and NRTN were found to signal through a multicomponent receptor system comprising a high-affinity ligand-binding coreceptor GFRα (GDNF family receptor α-component) and the RET receptor tyrosine kinase [10–14]. This review briefly describes the recent expansion of the GFLs to include two additional members, persephin (PSPN) and artemin (ARTN), and summarizes the current understanding of ligand–receptor interactions between the four GFLs and GFRα co-receptors. Furthermore, mice with null mutations in the genes encoding GDNF, NRTN and several GDNF family receptors (GFRα1, GFRα2 and GFRα3) have recently provided insight into the critical importance of the GFLs during development, particularly in the peripheral nervous system and in kidney organogenesis. Several excellent reviews of the literature describing the structural biology and therapeutic prospects of the GFLs [15•,16,17•] and the oncogenic role of RET mutations in multiple endocrine neoplasia type 2 (MEN2) can be found elsewhere [18–20].
Expansion of the GDNF family A schematic representation of ligand–receptor interactions of the GFLs characterized by in vitro studies is shown in Figure 1. Shortly after the discovery of the second GFL (NRTN), homology-based PCR screening was used to identify PSPN, and shortly thereafter database searching was used to identify ARTN. As mentioned above, the GFLs signal through a multicomponent receptor complex comprising the RET tyrosine kinase and a high-affinity ligand-binding component (of which there are now GFRα1-GFRα4), that is attached to the cell surface via a glycosyl phosphatidylinositol (GPI) anchor. As RET itself cannot bind the GFLs, both a GFRα and RET are required to form a functional GFL receptor. Extensive receptor activation experiments and receptor binding experiments over the past few years have served to further define the interactions shown in Figure 1. Essentially, each GFL has a preferred co-receptor to which it binds with highest affinity and activates RET most potently. These are GDNF–GFRα1, NRTN–GFRα2, and ARTN–GFRα3 [21–23,24••]. PSPN can bind a protein in the chicken called GFRα4 [25]; however, a mammalian orthologue of this receptor has not yet been identified. The alternative interactions (GDNF–GFRα2, NRTN– GFRα1, ARTN–GFRα1) shown in the figure are clearly functional,
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Figure 1 GDNF
NRTN
GFRα1
ARTN
GFRα2 RET
PSPN
GFRα3 RET
Schematic representation of GDNF family ligand–receptor interactions determined from in vitro studies. The thick arrows represent preferential interactions that are of highest affinity, that activate RET most potently, and that predominate in vivo. The thinner arrows represent alternate interactions that are functional; however, the prevalence of these interactions in vivo is unknown. As indicated in the figure, GFRα4 has been identified only in the chicken.
GFRα4 (avian only) RET Current Opinion in Neurobiology
as they lead to RET activation and neuronal survival in vitro [9••,21,24••,26,27,28••]; however, the prevalence of these interactions in vivo is currently unknown (see below).
The GFLs are critical trophic factors for developing enteric, sympathetic and parasympathetic neurons Although it was not known at the time, the study of RET –/– mice was the first analysis of the function of the GFLs in neurodevelopment. Given the current evidence that RET is a common signaling component for all of the GFLs, mice lacking RET should represent a pan-GFL knockout. However, many aspects of the RET –/– phenotype remain to be explored. As mentioned above, these mice die shortly after birth due to lack of kidneys and enteric neurons distal to the stomach [29]. The critical role of RET signaling in kidney development is interesting, and underscores the difficulty in strictly classifying molecules as neurotrophic factors. However, aside from this major importance of RET in kidney development, there are no other discernable phenotypes outside the nervous system in the RET –/– mice, and expression of RET is largely restricted to nephrogenic and nervous tissue [29,30]. Specific defects in the RET –/– mice are discussed below in terms of the different GFL–GFRα systems. Figure 2 shows the expression of RET during embryogenesis, summarizing the sites of GFL action. A detailed summary of the deficits described below is given in Table 1. α1 system The GDNF–GFRα
Both GDNF and GFRα1-deficient mice fail to develop enteric neurons and kidneys and die perinatally, similar to mice lacking RET [9••,29,31–33,34••]. GDNF is expressed
in the developing metanephrogenic mesenchyme, whereas RET and GFRα1 are expressed in the developing ureteric bud, which grows toward the mesenchyme and branches as it enters [35]. Interestingly, this organization resembles the target-derived trophic interaction observed for neurotrophic factors in neurodevelopment. It is perhaps even more interesting that GDNF appears to be present in limiting quantities even in kidney development, as approximately 30% of GDNF +/– mice lack one kidney, whereas all RET+/– and GFRα1+/– mice have two normal kidneys [9••,29,31–33,34••]. Therefore, even in kidney development, GDNF seems to function analogously to a neurotrophic factor in that it is target-derived and produced in limiting quantities. The GDNF–GFRα1 system is also critical for the development of all enteric neurons and glia distal to the stomach [9••,31–33,34••]. Enteric neurons arise largely from vagal neural crest cells that invade the foregut at around embryonic day (E) 9.5 in the mouse, and then migrate down the length of the bowel [36,37]. While the precise timing and nature of enteric neuron loss in these mice is unknown, several lines of evidence suggest GDNF–GFRα1–RET signaling is critical for maintaining the survival and/or proliferation of enteric precursor cells. RET is expressed by enteric precursors that can differentiate into both neurons and glia [38]. Furthermore, in vitro experiments performed by several groups have demonstrated that GDNF supports the survival and proliferation of enteric progenitors in culture, and have also suggested that GDNF responsiveness diminishes with increasing embryologic age [39–41,42•]. Finally, a large number of apoptotic cells are present in the foregut of RET-deficient mice but not of wild-type mice,
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Figure 2 Sites of GFL action during development. As RET is the signaling receptor for all GFLs, the expression pattern of RET presents a comprehensive view of sites of GFL action during development. A representative picture of a parasagittal section of a mouse embryo (E14.5) stained with anti-RET antibodies is shown. Arrows indicate tissues expressing high levels of RET protein. The known GFL–GFRα activating systems involved in the development of each structure are listed underneath. The number of spinal motor neurons is decreased by 20–30% in GDNF–/– and GFRα1–/– newborn mice; however, the identity of this GDNF-dependent subpopulation of neurons is currently unknown. In the trigeminal ganglion (TG), expression of RET begins at around E12.5. By in situ hybridization analysis, GDNF–/– and NRTN–/– animals have been shown to lack GFRα1- and GFRα2-expressing neurons, respectively. Newborn GDNF–/– mice are missing 23% of dorsal root ganglion (DRG) neurons. By contrast, no significant neuron loss was observed in GFRα1–/– mice, suggesting that GDNF may signal through GFRα2 in this system. ‘GFRα?’ indicates that it is not clear which GFRα is being utilized by GDNF for signaling. In adult NRTN–/– mice, 45% of GFRα2-expressing neurons are missing from the DRG. Therefore, in both these sensory ganglia (TG and DRG), GDNF and NRTN are critical for the development of a subpopulation of neurons. Development of the superior cervical ganglion (SGC) is also influenced by GDNF, as GDNF–/– mice are missing 35% of SCG neurons at birth. However, it is not clear through which GFRα receptor GDNF is signaling (GFRα?), as neither GFRα1–/– nor GFRα2–/– mice have a significant loss of SCG neurons. Rostral migration of the developing SCG is severely affected in GFRα3–/– mice, revealing a potential role for ARTN in SCG migration (indicated by ‘ARTN?’ in the figure). Enteric neurons alternate their GFL dependency
during development. While GDNF is a critical factor for early migrating enteric precursors, NRTN is important for the proper functioning and maintenance of mature enteric neurons. Target innervation by the sphenopalatine ganglion (SPG) is dramatically reduced in NRTN–/– and GFRα2–/– mice. All the cranial parasympathetic ganglia exhibit deficits in NRTN-deficient mice, establishing NRTN as a crucial neurotrophic factor for the development
suggesting that the enteric progenitors die due to lack of GDNF–GFRα1–RET signaling shortly after entering the foregut [42•]. In addition to enteric neurons, subpopulations of cutaneous sensory neurons in the DRG and visceral sensory neurons in the nodose ganglion also depend on the GDNF–GFRα1 system for survival. However, the precise population of cells that is missing in these ganglia is unknown. In the DRG, it was recently demonstrated that a distinct subpopulation of nociceptive neurons express RET and shift from NGF to GDNF responsiveness postnatally [43,44•]. Unfortunately, the death of GDNF and GFRα1-deficient mice at birth precludes analysis of the postnatal GDNF dependence by these neurons. In the trigeminal system, GDNF is expressed in the developing
of parasympathetic neurons. Although RET is highly expressed in motor neurons of the facial nucleus (FN) and in midbrain dopaminergic neurons, and GDNF and NRTN are potent survival factors for these neurons in vitro and after injury, GDNF and NRTN are not required for the survival of these neurons during development. The potential biological role of GFL signaling in these neurons is unclear, as represented by the ‘?’ in the figure.
whisker follicle whereas GFRα1 and RET are expressed in the trigeminal ganglion [45•,46,47]. Interestingly, GFRα1-expressing neurons are absent in the trigeminal ganglia of GDNF-deficient mice [45•], and adult GDNF+/– mice have a dramatic reduction in the number of Aβcaliber myelinated sensory nerve endings in the whisker follicle [46]; this suggests that GDNF functions as a targetderived trophic factor for these sensory neurons, present in limiting quantity. Similarly to mice lacking members of the NGF family, the GFL knockouts generated thus far have little or no observable deficit in neuronal survival in the CNS. Although GDNF was initially reported to be the most potent known trophic factor for motor neurons [48], GDNF- and GFRα1deficient mice have only minor deficits in motor neuron
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Table 1 Summary of developmental deficits in RET–/–, GFL–/– and GFRα–/– mice. RET–/–
GFRα1–/–
GDNF–/–
GFRα2–/–
NRTN–/–
GFRα3–/–
CNS Dopaminergic Substantia nigra
ND
ns
ns
ncd
ncd
ND
Motor Trigeminal Spinal motor
ND ND
22% loss 24% loss
19% loss 22—31% loss
ND ND
ND ND
ND ND
PNS Sensory Trigeminal Nodose DRG
ND ND ND
ns 0—15% loss ns
ns† 40% loss 23% loss
Responsiveness to NRTN ↓‡ ns ns
GFRα2 neuron ↓# ns GFRα2 neuron ↓#
ns ND ns
Autonomic Sympathetic SCG
100% loss
ns
35% loss
ns
ns
ND ND ND ND
ND ND ND ND
ND ND ND ND
Parasympathetic Ciliary Submandibular Sphenopalatine Otic ENS
100% loss in the gut distal to the stomach
100% loss in the gut distal to the stomach
Other deficits
Agenesis or dysgenesis of the kidney
Agenesis or dysgenesis of the kidney
20—40% (P1) 100% (P60) loss*
ncd 50% loss 81% loss 45% loss Loss of nerve fibers to the target tissues
ND ncd ND ND
Reduced AChE fiber density and impaired contractile activity of the gut
ncd
Growth retardation
(Normal growth)
(Normal growth)
Viability
Die
Die
Die
Live
Live
Live
References
[29]
[9••,34••]
[31—33,45•]
[28••]
[50••]
[53••]
Reduction of neuron number is expressed as % value. † Although neuronal loss is not significant by counting neuron profile numbers, there are significantly fewer GFRα1-expressing neurons in GDNF–/– mice. ‡ Explant and dissociated culture of the trigeminal ganglion of GFRα2-deficient mice exhibited impaired neurite outgrowth and poor survival in response to NRTN, respectively. #In newborn NRTN–/– mice, the number of GFRα2-expressing trigeminal neurons is significantly reduced. In adult NRTN–/– mice, 45% of GFRα2-expressing neurons are absent from the DRG. Whether these observations reflect actual loss of neurons or loss of
GFRα2 expression requires further investigation. *The SCG in GFRα3–/– mice lacks about 20–40% of neurons at postnatal day 1 (P1), and is almost completely absent by P60. The developing SCG of GFRα3–/– mice exhibits a migratory defect that is accompanied by loss of target innervation, which potentially leads to a secondary loss of target-derived trophic support. AChE, acetylcholine esterase; ENS, enteric nervous system; ncd, no clear deficit on visual inspection (not counted); ND, not determined; ns, difference not statistically significant by neuronal count; PNS, peripheral nervous system.
number at birth (20–30% loss) [9••,31,33]. It was recently shown that transgenic mice overexpressing GDNF under a muscle-specific promotor have hyperinnervation of their neuromuscular junction (NMJ), suggesting a role for GDNF in regulating motor neuron innervation density rather than survival [49•]. However it remains to be seen whether there is a deficit in NMJ innervation in GDNFdeficient mice. Furthermore, while GDNF is a potent survival-promoting agent for midbrain dopaminergic neurons, the GDNF– GFRα1 system does not appear to be critical for survival of these central neurons before birth [9••,31–33,34••].
NRTN, like GDNF, can influence developing enteric neurons in vitro [40,42•]. Examination of the enteric nervous system in NRTN and GFRα2-deficient mice revealed that there is a decrease in myenteric plexus density, and that this correlated with a functional deficit in contractile rhythm and activity [28••,50••]. Interestingly, the embryonic gut expresses high levels of GFRα1 that decrease after birth, whereas GFRα2 expression is weak in the embryonic bowel and peaks several weeks after birth [21,28••,45•,51]. Therefore, though the GDNF–GFRα1 system is critical for early events in enteric nervous system development, resulting in its more dramatic phenotype, the NRTN–GFRα2 system appears to be important for later developmental events, and/or for the maintenance of the mature enteric nervous system.
α2 system The NRTN–GFRα
In contrast to the striking phenotype of the GDNF and GFRα1-deficient mice, mice lacking NRTN or GFRα2 display a relatively mild phenotype. The mice are born, breed normally, and have no gross developmental defects in any organ system [28••,50••].
GFRα2 is expressed in sensory ganglia, and NRTN supports the survival of a subpopulation of DRG and trigeminal ganglion neurons in culture [8,44•,45•]. While
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no decrease in the number of sensory neurons was detected in NRTN or GFRα2-deficient mice, GFRα2-expressing neurons are fewer, or absent, in the sensory ganglia of NRTN-deficient mice [50••]. This indicates that either this small subpopulation of neurons has died in the absence of NRTN, or that they have lost expression of GFRα2. Further work is required to determine the function of GFRα2-expressing sensory neurons, and whether they require NRTN for survival support, or for maintenance of proper expression of neuronal markers (including GFRα2). While members of the NGF family are well-established trophic factors for most parts of the peripheral nervous system, their absence does not affect the development of parasympathetic neurons, and they do not influence these neurons in vitro. Interestingly, the only grossly observable phenotype in the NRTN- and GFRα2-deficient mice is apparent ptosis (i.e. drooping of the eyelids), which results from the complete absence of parasympathetic innervation to the lacrimal gland [28••,50••]. Furthermore, both NRTNand GFRα2-deficient mice also lack parasympathetic innervation to the submandibular gland, and have a significant loss of neurons in the submandibular ganglion [28••,50••]. As NRTN is expressed in the developing lacrimal and submandibular glands, and GFRα2 and RET are expressed in developing parasympathetic ganglion neurons [28••,50••,52], it appears that NRTN is functioning as a true target-derived neurotrophic factor that supports the survival of these developing parasympathetic neurons. This was the first demonstration of a neurotrophic factor that is critical for parasympathetic neuron development. α3 system The ARTN–GFRα
Although the phenotype of mice lacking ARTN is unknown, the phenotype of mice lacking GFRα3 was recently described [53••]. Interestingly, like the NRTN– GFRα2-deficient mice, mice lacking GFRα3 also exhibit ptosis. However, the cause of ptosis in these two cases is quite different — in NRTN–GFRα2-deficient mice, ptosis appears secondary to the loss of lacrimal gland secretion, due of lack of parasympathetic innervation. By contrast, GFRα3-deficient mice lack sympathetic innervation to the superior tarsus muscle, and therefore have a true inability to keep the eyelid raised. This lack of sympathetic innervation is due to a severe defect in superior cervical ganglion (SCG) development. In GFRα3-deficient mice, neurons of the SCG do not properly migrate during embryogenesis and the ganglion is found at a more caudal position than normal [53••]. Sympathetic innervation of the superior tarsus and submandibular gland is absent, suggesting that the target projections of the SCG are lost. The cells of this SCG rudiment die with a time course that resembles that of the NGF/TrkA knockouts [54,55], suggesting the possibility that in the absence of GFRα3 signaling (for early migratory events), the SCG does not properly innervate its targets and the neurons die due to lack of target-derived NGF. Somewhat surprisingly, no deficits in any other peripheral ganglia were detected in
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GFRα3-deficient mice, despite high-level GFRα3 expression in other sensory and sympathetic ganglia during development [56]. α–RET knockouts Discrepancies in the GFL–GFRα
Receptor binding experiments in vitro demonstrated that while GDNF preferentially binds and activates GFRα1– RET, it can also activate GFRα2–RET [21,22,26,27]. Analysis of the GFL and GFRα knockouts generated thus far indicates that the preferential interactions are the most prominent, and the importance of the alternative ligandreceptor interactions (i.e. GDNF–GFRα2) in vivo is unknown. However, GDNF-deficient mice have greater neuron loss in the DRG than GFRα1-deficient mice, suggesting that GDNF may utilize another receptor to support the survival of these neurons, presumably GFRα2–RET [9••,34••]. Therefore, it is possible that the ‘non-preferred’ GDNF ligand–receptor interactions do occur in vivo, but the prevalence of these interactions may vary in different biological systems. An initial report of RET-deficient mice indicated that the animals completely lack the SCG at birth [57]. Neither the GDNF–GFRα1 nor the NRTN–GFRα2 systems are responsible for this deficit, as mice missing these components have little (GDNF–/–) or no (NRTN–/–) reduction in the number of SCG neurons [9••,28••,31–33,34••,50••]. As mentioned above, GFRα3 (presumably mediating ARTN signaling) is critical for the development of the SCG; however, at birth the SCG is present in GFRα3 knockouts, but was reported absent in RET knockouts. Therefore, either some as yet unknown RET-activating system is critical for prenatal events in SCG development, or perhaps the altered location of the SCG caused it to be mistaken for being absent in the first reports of RET-deficient mice.
Parallels and intersections in the developmental roles of the NGF and the GDNF families Analysis of knockout mice has revealed extensive parallels in the mechanisms by which the NGF family and GDNF families influence neurodevelopment. First, while receptors for both families are present on central neurons and they can both potently influence central neurons in vitro, they appear to not be critical (at least individually) for the survival of CNS neurons during development. This adds further support to the suggestion that CNS neurons may derive redundant trophic support from many neurotrophic factors [58]. Second, members of both families are critically important for the development of distinct subsets of peripheral neurons. Together, the GDNF and NGF families probably account for the trophic support of the vast majority of peripheral neurons at some stage of their development. Third, members of both families function as target-derived trophic factors in some systems, and as paracrine-derived trophic factors in others before target innervation has occurred. Finally, there is evidence that particularly in the target-derived systems, members of both the NGF and GDNF families are present in limiting
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quantities, as evidenced by deficits in mice lacking one copy of the trophic factor gene. It is also clear that the NGF and GDNF families do not function simply as parallel neurotrophic systems; rather, it is likely that they overlap and alternate in their influence of peripheral neuron development. For example, it is clear that a subset of nociceptive neurons in the DRG, that initially are dependent on NGF for prenatal trophic support, switch such that they begin to express RET and can be supported by GDNF in vitro and after injury [43,44•]. Additional scenarios where peripheral neurons alternate their dependence on NGF or GDNF family members are sure to be discovered in the near future.
Conclusions The recent addition of PSPN and ARTN to the GDNF family ligands, along with confirmation from gene knockout studies of the importance of GDNF and NRTN in neurodevelopment, has established the GFLs as a second family of neurotrophic factors (the other being the NGF family) where different members influence distinct events and support the survival of distinct classes of peripheral neurons during development. While there is some in vitro cross-talk between the different GFLs and GFRαs, the preferred interactions (GDNF–GFRα1 and NRTN– GFRα2) appear most prevalent in vivo. Elucidating the developmental roles of the GFLs has significantly expanded our understanding of peripheral neuron development, as we now know that GFLs are critical for the development of enteric and parasympathetic neurons, two classes of neurons whose trophic dependencies were previously unknown. In addition, the GFLs also influence the development of sensory and sympathetic neurons, and therefore have a broader spectrum of developmental action than the NGF family.
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Cacalano G, Farinas I, Wang L-C, Hagler K, Forgie A, Moore M, α1 is an Armanini M, Phillips H, Ryan AM, Reichardt LF et al.: GFRα essential receptor component for GDNF in the developing nervous system and kidney. Neuron 1998, 21:53-62. This study, together with [34••], established the necessary role of GFRα1 as a GDNF co-receptor in vivo. These authors also provided the first demonstration that NRTN–GFRα2 signaling supports parasympathetic submandibular neuron survival in culture. 10. Trupp M, Arenas E, Fainzilber M, Nilsson A-S, Sieber B-A, Grigoriou M, Kilkenny C, Salazar-Grueso E, Pachnis V, Arumae U et al.: Functional receptor for GDNF encoded by the c-ret proto-oncogene. Nature 1996, 381:785-789. 11. Durbec P, Marcos-Gutierrez CV, Kilkenny C, Grigoriou M, Wartiowaara K, Suvanto P, Smith D, Ponder B, Costantini F, Saarma M et al.: GDNF signalling through the Ret receptor tyrosine kinase. Nature 1996, 381:789-793. 12. Jing S, Wen D, Yu Y, Holst PL, Luo Y, Fang M, Tamir R, Antonio L, Hu Z, Cupples R et al.: GDNF-induced activation of the ret protein α, a novel receptor for tyrosine kinase is mediated by GDNFR-α GDNF. Cell 1996, 85:1113-1124. 13. Treanor JJS, Goodman L, Sauvage F, Stone DM, Poulsen KT, Beck CD, Gray C, Armanini MP, Pollock RA, Hefti F et al.: Characterization of a multicomponent receptor for GDNF. Nature 1996, 382:80-83.
Some of the remaining challenges are obvious — for instance, the generation and study of mice lacking ARTN and PSPN, and the identification and confirmation of a mammalian GFRα4 that functions as a receptor for PSPN. Furthermore, continued detailed study of the timing and the mechanism of neuronal loss in GFL–GFRα–RET deficient mice, and comparisons with the influences of NGF family members, should allow an understanding of how the trophic demands of most peripheral neurons are met at each stage of development.
14. Creedon DJ, Tansey MG, Baloh RH, Osborne PA, Lampe PA, Fahrner TJ, Heuckeroth RO, Milbrandt J, Johnson EM Jr: Neurturin shares receptors and signal transduction pathways with glial cell linederived neurotrophic factor in sympathetic neurons. Proc Natl Acad Sci USA 1997, 94:7018-7023.
Acknowledgements
17. •
We thank all members of the Milbrandt and Johnson labs for invaluable discussions regarding the ideas and issues presented here. The authors are supported by National Institutes of Health grants R01 AG13729 and R01 AG13730.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
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39. Hearn CJ, Murphy M, Newgreen D: GDNF and ET-3 differentially modulate the numbers of avian enteric neural crest cells and enteric neurons in vitro. Dev Biol 1998, 197:93-105. 40. Heuckeroth RO, Lampe PA, Johnson EM, Milbrandt J: Neurturin and GDNF promote proliferation and survival of enteric neuron and glial progenitors in vitro. Dev Biol 1998, 200:116-129.
23. Klein RD, Sherman D, Ho WH, Stone D, Bennett Gl, Moffat B, Vandlen R, Simmons L, Gu Q, Hongo JA et al.: A GPI-linked protein that interacts with Ret to form a candidate neurturin receptor. Nature 1997, 387:717-721.
41. Chalazonitis A, Rothman TP, Chen J, Gershon MD: Age-dependent differences in the effects of GDNF and NT-3 on the development of neurons and glia from neural crest-derived precursors α-1 in immunoselected from the fetal rat gut: expression of GFRα vitro and in vivo. Dev Biol 1998, 204:385-406.
24. Baloh RH, Tansey MG, Lampe PA, Fahrner TJ, Enomoto H, Simburger K, •• Leitner ML, Araki T, Johnson EMJ, Milbrandt J: Artemin, a novel member of the GDNF ligand family supports peripheral and α3-RET receptor central neurons and signals through the GFRα complex. Neuron 1998, 21:1291-1302. This paper described the identification of ARTN, the most recently discovered GFL, and established that ARTN binds and signals through the former orphan receptor GFRα3–RET.
42. Taraviras S, Marcos-Gutierrez CV, Durbec P, Jani H, Grigoriou M, • Sukumaran M, Wang LC, Hynes M, Raisman G, Pachnis V: Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system. Development 1999, 126:2785-2797. This paper demonstrated that RET is a necessary receptor element for GDNF and NRTN to support neuronal survival in vitro, confirming that both RET and a GFRα are required for effective GFL signaling.
25. Enokido Y, de Sauvage F, Hongo J-A, Ninkina N, Rosenthal A, α4 and the tyrosine kinase Ret form Buchman VL, Davies AM: GFRα a functional receptor complex for persephin. Curr Biol 1998, 8:1019-1022.
43. Molliver DC, Wright DE, Leitner ML, Parsadanian AS, Doster K, Wen D, Yan Q, Snider WD: IB4-binding DRG neurons switch from NGF to GDNF dependence in early postnatal life. Neuron 1997, 19:849-861.
26. Suvanto P, Wartiovaara K, Lindahl M, Arumae U, Moshnyakov M, Horelli-Kuitunen N, Airaksinen MS, Palotie A, Sariola H, Saarma M: Cloning, mRNA distribution and chromosomal localisation of the β, a gene for glial cell line-derived neurotrophic factor receptor-β α. Hum Molec Genet 1997, 6:1267-1273. homologue to GDNFR-α
44. Bennett DL, Michael GJ, Ramachandran N, Munson JB, Averill S, Yan Q, • McMahon SB, Priestley JV: A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons after nerve injury. J Neurosci 1998, 18:3059-3072. This paper and [43] established that the IB4-binding non-TrkA expressing subset of nociceptive neurons in the DRG express RET, and respond to GDNF in vitro and after nerve injury.
27.
Sanicola M, Hession C, Worley D, Carmillo P, Ehrenfels C, Walus L, Robinson S, Jaworski G, Wei H, Tizard R et al.: Glial cell line-derived neurotrophic factor-dependent RET activation can be mediated by two different cell-surface accessory proteins. Proc Natl Acad Sci USA 1997, 94:6238-6243.
28. Rossi J, Luukko K, Poteryaev D, Laurikainen A, Sun YF, Laakso T, •• Eerikainen S, Tuominen R, Lakso M, Rauvala H et al.: Retarded growth and deficits in the enteric and parasympathetic nervous α2, a functional neurturin receptor. system in mice lacking GFRα Neuron 1999, 22:243-252. •• This paper, together with [50 ], demonstrated the critical role of NRTNGFRα2 signaling in the development of parasympathetic neurons, and established that GFRα2-RET functions predominantly as a receptor for NRTN in vivo. 29. Schuchardt A, D’Agati V, Larsson-Blomberg L, Constantini F, Pachnis V: Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 1994, 367:380-383. 30. Pachnis V, Mankoo B, Constantini F: Expression of the c-ret protooncogene during mouse embryogenesis. Development 1993, 119:1005-1017. 31. Moore MW, Klein RD, Farinas I, Sauer H, Armanini M, Phillips H, Reichart LF, Ryan AM, Carver-Moore K, Rosenthal A: Renal and neuronal abnormalities in mice lacking GDNF. Nature 1996, 382:76-79. 32. Pichel JG, Shen L, Hui SZ, Granholm A-C, Drago J, Grinberg A, Lee EJ, Huang SP, Saarma M, Hoffer BJ et al.: Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 1996, 382:73-76. 33. Sanchez MP, Silos-Santiago I, Frisen J, He B, Lira SA, Barbacid M: Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 1996, 382:70-73. 34. Enomoto H, Araki T, Jackman A, Heuckeroth RO, Snider WD, α1-deficient mice have deficits in •• Johnson EM Jr, Milbrandt J: GFRα the enteric nervous system and kidneys. Neuron 1998, 21:317-324. See annotation [9••]. 35. Sariola H, Sainio K: The tip-top branching ureter. Curr Opin Cell Biol 1997, 9:877-884. 36. Le Douarin NM, Teillet MA: Experimental analysis of the migration and differentiation of neuroblasts of the autonomic nervous system and of neuroectodermal mesenchymal derivatives, using a biological cell marking technique. Dev Biol 1974, 41:162-184. 37.
Gershon MD: Genes and lineages in the formation of the enteric nervous system. Curr Opin Neurobiol 1997, 7:101-109.
38. Lo L, Anderson DJ: Postmigratory neural crest cells expressing cRET display restricted developmental and proliferative capacities. Neuron 1995, 15:527-539.
45. Naveilhan P, Baudet C, Mikaels A, Shen L, Westphal H, Ernfors P: α3, a glial cell line-derived • Expression and regulation of GFRα neurotrophic factor family receptor. Proc Natl Acad Sci USA 1998, 95:1295-1300. This study provided the first evidence of GDNF–GFRα1 pairing in vivo, showing that GDNF–/– mice were selectively deficient in GFRα1-expressing neurons in the trigeminal ganglion. 46. Fundin BT, Mikaels A, Westphal H, Ernfors P: A rapid and dynamic regulation of GDNF-family ligands and receptors correlate with the developmental dependency of cutaneous sensory innervation. Development 1999, 126:2597-2610. 47.
Golden JP, DeMaro JA, Osborne PA, Milbrandt J, Johnson EM Jr: Expression of Neurturin, GDNF, and GDNF family-receptor mRNA in the developing and mature mouse. Exp Neurol 1999, 158:504-528.
48. Henderson CE, Phillips HS, Pollock RA, Davies AM, Lemeulle C, Armanini M, Simpson LC, Moffet B, Vandlen RA, Koliatsos VE, Rosenthal A: GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science 1994, 266:1062-1064. 49. Nguyen QT, Parsadanian AS, Snider WD, Lichtman JW: • Hyperinnervation of neuromuscular junctions caused by GDNF overexpression in muscle. Science 1998, 279:1725-1729. This report demonstrates an interesting example of GDNF functioning in a non-canonical manner for a neurotrophic factor, using transgenic mice to demonstrate that GDNF, but not other neurotrophic factors, can regulate motor endplate innervation density. 50. Heuckeroth RO, Enomoto H, Grider JR, Golden JP, Hanke JA, •• Jackman A, Molliver DC, Bardgett ME, Snider WD, Johnson EM Jr et al.: Gene targeting reveals a critical role for neurturin in the development and maintenance of enteric, sensory, and parasympathetic neurons. Neuron 1999, 22:253-263. This paper, together with [28••], demonstrates the importance of NRTN as a target-derived trophic factor critical for the development of parasympathetic neurons signaling through the GFRα2–RET receptor. 51. Widenfalk J, Nosrat C, Tomac A, Westphal H, Hoffer B, Olson L: β Neurturin and glial cell line-derived neurotrophic factor receptor-β β), novel proteins related to GDNF and GDNFR-α α with (GDNFR-β specific cellular patterns of expression suggesting roles in the developing and adult nervous system and in peripheral organs. J Neurosci 1997, 17:8506-8519. 52. Nosrat CA, Tomac A, Hoffer BJ, Olson L: Cellular and developmental patterns of expression of Ret and glial cell linederived neurotrophic factor receptor alpha mRNAs. Exp Brain Res 1997, 115:410-422.
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53. Nishino J, Mochida K, Ohfuji Y, Shimazaki T, Meno C, Ohishi S, α3, a component of the •• Matsuda Y, Fujii H, Saijoh Y, Hamada H: GFRα artemin receptor, is required for migration and survival of the superior cervical ganglion. Neuron 1999, 23:725-736. This paper provides the first insight into the developmental importance of ARTN-GFRα3 signaling, indicating that one of the major roles of GFRα3RET appears to be for proper migration of sympathetic neurons of the superior cervical ganglion. 54. Smeyne RJ, Klein R, Schnapp A, Long LK, Bryant S, Lewin A, Lira SA, Barbacid M: Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 1994, 368:246-249. 55. Crowley C, Spencer SD, Nishimura MC, Chen KS, Pitts-Meek S, Armanini MP, Ling LH, McMahon SB, Shelton DL, Levinson AD et al.:
Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. Cell 1994, 76:1001-1012. 56. Baloh RH, Gorodinsky A, Golden JP, Tansey MG, Keck CL, Popescu NC, α3 is an orphan member of the Johnson EM Jr, Milbrandt J: GFRα GDNF/neurturin/persephin receptor family. Proc Natl Acad Sci USA 1998, 95:5801-5806. 57.
Durbec PL, Larsson-Blomberg LB, Schuchardt A, Costantini F, Pachnis V: Common origin and developmental dependence on cret of subsets of enteric and sympathetic neuroblasts. Development 1996, 122:349-358.
58. Snider WD: Functions of the neurotrophins during nervous system development: What the knockouts are teaching us. Cell 1994, 77:627-638.
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The GDNF family ligands and receptors — implications for neural development Robert H Baloh*, Hideki Enomoto†, Eugene M Johnson Jr‡ and Jeffrey Milbrandt§ The glial cell line derived neurotrophic factor (GDNF) family has recently been expanded to include four members, and the interactions between these neurotrophic factors and their unique receptor system is now beginning to be understood. Furthermore, analysis of mice lacking the genes for GDNF, neurturin, and their related receptors has confirmed the importance of these factors in neurodevelopment. The results of such analyses reveal numerous similarities and potential overlaps in the way the GDNF and the nerve growth factor (NGF) families regulate development of the peripheral nervous system. Addresses *†§ Departments of Pathology and Internal Medicine, ‡ Department of Neurology and Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA *e-mail: [email protected] † e-mail: [email protected] ‡ e-mail: [email protected] § e-mail: [email protected] Current Opinion in Neurobiology 2000, 10:103–110 0959-4388/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations ARTN artemin DRG dorsal root ganglion E embryonic day GDNF glial cell line derived neurotrophic factor GFL GDNF family ligand α GDNF family receptor α-component GFRα NGF nerve growth factor NRTN neurturin PSPN persephin RET rearranged in transfection (receptor tyrosine kinase) SCG superior cervical ganglion
Introduction Since the discovery of nerve growth factor (NGF) and the establishment of its ability to support neuronal survival [1–3], extensive efforts have been made to identify additional neurotrophic factors that can influence neurons in primary culture, during normal development, or in experimental models of neuronal injury. This work has resulted in the identification of a large and diverse group of proteins that are capable of promoting neuronal survival in various experimental paradigms. Glial cell line derived neurotrophic factor (GDNF) was initially identified as a factor secreted from a glioma cell line capable of supporting embryonic ventral midbrain neuron survival in culture [4]. Our knowledge of the in vitro activities of GDNF expanded rapidly after its discovery to now include survival promotion of additional central neurons (including spinal motor neurons) and at least a subpopulation of all peripheral ganglia yet examined
[5–8,9••]. The discovery of neurturin (NRTN) three years later, which is ~44% identical to GDNF, established the existence of the GDNF family ligands (GFLs) [8]. Furthermore, shortly after the discovery of NRTN, both GDNF and NRTN were found to signal through a multicomponent receptor system comprising a high-affinity ligand-binding coreceptor GFRα (GDNF family receptor α-component) and the RET receptor tyrosine kinase [10–14]. This review briefly describes the recent expansion of the GFLs to include two additional members, persephin (PSPN) and artemin (ARTN), and summarizes the current understanding of ligand–receptor interactions between the four GFLs and GFRα co-receptors. Furthermore, mice with null mutations in the genes encoding GDNF, NRTN and several GDNF family receptors (GFRα1, GFRα2 and GFRα3) have recently provided insight into the critical importance of the GFLs during development, particularly in the peripheral nervous system and in kidney organogenesis. Several excellent reviews of the literature describing the structural biology and therapeutic prospects of the GFLs [15•,16,17•] and the oncogenic role of RET mutations in multiple endocrine neoplasia type 2 (MEN2) can be found elsewhere [18–20].
Expansion of the GDNF family A schematic representation of ligand–receptor interactions of the GFLs characterized by in vitro studies is shown in Figure 1. Shortly after the discovery of the second GFL (NRTN), homology-based PCR screening was used to identify PSPN, and shortly thereafter database searching was used to identify ARTN. As mentioned above, the GFLs signal through a multicomponent receptor complex comprising the RET tyrosine kinase and a high-affinity ligand-binding component (of which there are now GFRα1-GFRα4), that is attached to the cell surface via a glycosyl phosphatidylinositol (GPI) anchor. As RET itself cannot bind the GFLs, both a GFRα and RET are required to form a functional GFL receptor. Extensive receptor activation experiments and receptor binding experiments over the past few years have served to further define the interactions shown in Figure 1. Essentially, each GFL has a preferred co-receptor to which it binds with highest affinity and activates RET most potently. These are GDNF–GFRα1, NRTN–GFRα2, and ARTN–GFRα3 [21–23,24••]. PSPN can bind a protein in the chicken called GFRα4 [25]; however, a mammalian orthologue of this receptor has not yet been identified. The alternative interactions (GDNF–GFRα2, NRTN– GFRα1, ARTN–GFRα1) shown in the figure are clearly functional,
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Figure 1 GDNF
NRTN
GFRα1
ARTN
GFRα2 RET
PSPN
GFRα3 RET
Schematic representation of GDNF family ligand–receptor interactions determined from in vitro studies. The thick arrows represent preferential interactions that are of highest affinity, that activate RET most potently, and that predominate in vivo. The thinner arrows represent alternate interactions that are functional; however, the prevalence of these interactions in vivo is unknown. As indicated in the figure, GFRα4 has been identified only in the chicken.
GFRα4 (avian only) RET Current Opinion in Neurobiology
as they lead to RET activation and neuronal survival in vitro [9••,21,24••,26,27,28••]; however, the prevalence of these interactions in vivo is currently unknown (see below).
The GFLs are critical trophic factors for developing enteric, sympathetic and parasympathetic neurons Although it was not known at the time, the study of RET –/– mice was the first analysis of the function of the GFLs in neurodevelopment. Given the current evidence that RET is a common signaling component for all of the GFLs, mice lacking RET should represent a pan-GFL knockout. However, many aspects of the RET –/– phenotype remain to be explored. As mentioned above, these mice die shortly after birth due to lack of kidneys and enteric neurons distal to the stomach [29]. The critical role of RET signaling in kidney development is interesting, and underscores the difficulty in strictly classifying molecules as neurotrophic factors. However, aside from this major importance of RET in kidney development, there are no other discernable phenotypes outside the nervous system in the RET –/– mice, and expression of RET is largely restricted to nephrogenic and nervous tissue [29,30]. Specific defects in the RET –/– mice are discussed below in terms of the different GFL–GFRα systems. Figure 2 shows the expression of RET during embryogenesis, summarizing the sites of GFL action. A detailed summary of the deficits described below is given in Table 1. α1 system The GDNF–GFRα
Both GDNF and GFRα1-deficient mice fail to develop enteric neurons and kidneys and die perinatally, similar to mice lacking RET [9••,29,31–33,34••]. GDNF is expressed
in the developing metanephrogenic mesenchyme, whereas RET and GFRα1 are expressed in the developing ureteric bud, which grows toward the mesenchyme and branches as it enters [35]. Interestingly, this organization resembles the target-derived trophic interaction observed for neurotrophic factors in neurodevelopment. It is perhaps even more interesting that GDNF appears to be present in limiting quantities even in kidney development, as approximately 30% of GDNF +/– mice lack one kidney, whereas all RET+/– and GFRα1+/– mice have two normal kidneys [9••,29,31–33,34••]. Therefore, even in kidney development, GDNF seems to function analogously to a neurotrophic factor in that it is target-derived and produced in limiting quantities. The GDNF–GFRα1 system is also critical for the development of all enteric neurons and glia distal to the stomach [9••,31–33,34••]. Enteric neurons arise largely from vagal neural crest cells that invade the foregut at around embryonic day (E) 9.5 in the mouse, and then migrate down the length of the bowel [36,37]. While the precise timing and nature of enteric neuron loss in these mice is unknown, several lines of evidence suggest GDNF–GFRα1–RET signaling is critical for maintaining the survival and/or proliferation of enteric precursor cells. RET is expressed by enteric precursors that can differentiate into both neurons and glia [38]. Furthermore, in vitro experiments performed by several groups have demonstrated that GDNF supports the survival and proliferation of enteric progenitors in culture, and have also suggested that GDNF responsiveness diminishes with increasing embryologic age [39–41,42•]. Finally, a large number of apoptotic cells are present in the foregut of RET-deficient mice but not of wild-type mice,
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Figure 2 Sites of GFL action during development. As RET is the signaling receptor for all GFLs, the expression pattern of RET presents a comprehensive view of sites of GFL action during development. A representative picture of a parasagittal section of a mouse embryo (E14.5) stained with anti-RET antibodies is shown. Arrows indicate tissues expressing high levels of RET protein. The known GFL–GFRα activating systems involved in the development of each structure are listed underneath. The number of spinal motor neurons is decreased by 20–30% in GDNF–/– and GFRα1–/– newborn mice; however, the identity of this GDNF-dependent subpopulation of neurons is currently unknown. In the trigeminal ganglion (TG), expression of RET begins at around E12.5. By in situ hybridization analysis, GDNF–/– and NRTN–/– animals have been shown to lack GFRα1- and GFRα2-expressing neurons, respectively. Newborn GDNF–/– mice are missing 23% of dorsal root ganglion (DRG) neurons. By contrast, no significant neuron loss was observed in GFRα1–/– mice, suggesting that GDNF may signal through GFRα2 in this system. ‘GFRα?’ indicates that it is not clear which GFRα is being utilized by GDNF for signaling. In adult NRTN–/– mice, 45% of GFRα2-expressing neurons are missing from the DRG. Therefore, in both these sensory ganglia (TG and DRG), GDNF and NRTN are critical for the development of a subpopulation of neurons. Development of the superior cervical ganglion (SGC) is also influenced by GDNF, as GDNF–/– mice are missing 35% of SCG neurons at birth. However, it is not clear through which GFRα receptor GDNF is signaling (GFRα?), as neither GFRα1–/– nor GFRα2–/– mice have a significant loss of SCG neurons. Rostral migration of the developing SCG is severely affected in GFRα3–/– mice, revealing a potential role for ARTN in SCG migration (indicated by ‘ARTN?’ in the figure). Enteric neurons alternate their GFL dependency
during development. While GDNF is a critical factor for early migrating enteric precursors, NRTN is important for the proper functioning and maintenance of mature enteric neurons. Target innervation by the sphenopalatine ganglion (SPG) is dramatically reduced in NRTN–/– and GFRα2–/– mice. All the cranial parasympathetic ganglia exhibit deficits in NRTN-deficient mice, establishing NRTN as a crucial neurotrophic factor for the development
suggesting that the enteric progenitors die due to lack of GDNF–GFRα1–RET signaling shortly after entering the foregut [42•]. In addition to enteric neurons, subpopulations of cutaneous sensory neurons in the DRG and visceral sensory neurons in the nodose ganglion also depend on the GDNF–GFRα1 system for survival. However, the precise population of cells that is missing in these ganglia is unknown. In the DRG, it was recently demonstrated that a distinct subpopulation of nociceptive neurons express RET and shift from NGF to GDNF responsiveness postnatally [43,44•]. Unfortunately, the death of GDNF and GFRα1-deficient mice at birth precludes analysis of the postnatal GDNF dependence by these neurons. In the trigeminal system, GDNF is expressed in the developing
of parasympathetic neurons. Although RET is highly expressed in motor neurons of the facial nucleus (FN) and in midbrain dopaminergic neurons, and GDNF and NRTN are potent survival factors for these neurons in vitro and after injury, GDNF and NRTN are not required for the survival of these neurons during development. The potential biological role of GFL signaling in these neurons is unclear, as represented by the ‘?’ in the figure.
whisker follicle whereas GFRα1 and RET are expressed in the trigeminal ganglion [45•,46,47]. Interestingly, GFRα1-expressing neurons are absent in the trigeminal ganglia of GDNF-deficient mice [45•], and adult GDNF+/– mice have a dramatic reduction in the number of Aβcaliber myelinated sensory nerve endings in the whisker follicle [46]; this suggests that GDNF functions as a targetderived trophic factor for these sensory neurons, present in limiting quantity. Similarly to mice lacking members of the NGF family, the GFL knockouts generated thus far have little or no observable deficit in neuronal survival in the CNS. Although GDNF was initially reported to be the most potent known trophic factor for motor neurons [48], GDNF- and GFRα1deficient mice have only minor deficits in motor neuron
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Table 1 Summary of developmental deficits in RET–/–, GFL–/– and GFRα–/– mice. RET–/–
GFRα1–/–
GDNF–/–
GFRα2–/–
NRTN–/–
GFRα3–/–
CNS Dopaminergic Substantia nigra
ND
ns
ns
ncd
ncd
ND
Motor Trigeminal Spinal motor
ND ND
22% loss 24% loss
19% loss 22—31% loss
ND ND
ND ND
ND ND
PNS Sensory Trigeminal Nodose DRG
ND ND ND
ns 0—15% loss ns
ns† 40% loss 23% loss
Responsiveness to NRTN ↓‡ ns ns
GFRα2 neuron ↓# ns GFRα2 neuron ↓#
ns ND ns
Autonomic Sympathetic SCG
100% loss
ns
35% loss
ns
ns
ND ND ND ND
ND ND ND ND
ND ND ND ND
Parasympathetic Ciliary Submandibular Sphenopalatine Otic ENS
100% loss in the gut distal to the stomach
100% loss in the gut distal to the stomach
Other deficits
Agenesis or dysgenesis of the kidney
Agenesis or dysgenesis of the kidney
20—40% (P1) 100% (P60) loss*
ncd 50% loss 81% loss 45% loss Loss of nerve fibers to the target tissues
ND ncd ND ND
Reduced AChE fiber density and impaired contractile activity of the gut
ncd
Growth retardation
(Normal growth)
(Normal growth)
Viability
Die
Die
Die
Live
Live
Live
References
[29]
[9••,34••]
[31—33,45•]
[28••]
[50••]
[53••]
Reduction of neuron number is expressed as % value. † Although neuronal loss is not significant by counting neuron profile numbers, there are significantly fewer GFRα1-expressing neurons in GDNF–/– mice. ‡ Explant and dissociated culture of the trigeminal ganglion of GFRα2-deficient mice exhibited impaired neurite outgrowth and poor survival in response to NRTN, respectively. #In newborn NRTN–/– mice, the number of GFRα2-expressing trigeminal neurons is significantly reduced. In adult NRTN–/– mice, 45% of GFRα2-expressing neurons are absent from the DRG. Whether these observations reflect actual loss of neurons or loss of
GFRα2 expression requires further investigation. *The SCG in GFRα3–/– mice lacks about 20–40% of neurons at postnatal day 1 (P1), and is almost completely absent by P60. The developing SCG of GFRα3–/– mice exhibits a migratory defect that is accompanied by loss of target innervation, which potentially leads to a secondary loss of target-derived trophic support. AChE, acetylcholine esterase; ENS, enteric nervous system; ncd, no clear deficit on visual inspection (not counted); ND, not determined; ns, difference not statistically significant by neuronal count; PNS, peripheral nervous system.
number at birth (20–30% loss) [9••,31,33]. It was recently shown that transgenic mice overexpressing GDNF under a muscle-specific promotor have hyperinnervation of their neuromuscular junction (NMJ), suggesting a role for GDNF in regulating motor neuron innervation density rather than survival [49•]. However it remains to be seen whether there is a deficit in NMJ innervation in GDNFdeficient mice. Furthermore, while GDNF is a potent survival-promoting agent for midbrain dopaminergic neurons, the GDNF– GFRα1 system does not appear to be critical for survival of these central neurons before birth [9••,31–33,34••].
NRTN, like GDNF, can influence developing enteric neurons in vitro [40,42•]. Examination of the enteric nervous system in NRTN and GFRα2-deficient mice revealed that there is a decrease in myenteric plexus density, and that this correlated with a functional deficit in contractile rhythm and activity [28••,50••]. Interestingly, the embryonic gut expresses high levels of GFRα1 that decrease after birth, whereas GFRα2 expression is weak in the embryonic bowel and peaks several weeks after birth [21,28••,45•,51]. Therefore, though the GDNF–GFRα1 system is critical for early events in enteric nervous system development, resulting in its more dramatic phenotype, the NRTN–GFRα2 system appears to be important for later developmental events, and/or for the maintenance of the mature enteric nervous system.
α2 system The NRTN–GFRα
In contrast to the striking phenotype of the GDNF and GFRα1-deficient mice, mice lacking NRTN or GFRα2 display a relatively mild phenotype. The mice are born, breed normally, and have no gross developmental defects in any organ system [28••,50••].
GFRα2 is expressed in sensory ganglia, and NRTN supports the survival of a subpopulation of DRG and trigeminal ganglion neurons in culture [8,44•,45•]. While
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no decrease in the number of sensory neurons was detected in NRTN or GFRα2-deficient mice, GFRα2-expressing neurons are fewer, or absent, in the sensory ganglia of NRTN-deficient mice [50••]. This indicates that either this small subpopulation of neurons has died in the absence of NRTN, or that they have lost expression of GFRα2. Further work is required to determine the function of GFRα2-expressing sensory neurons, and whether they require NRTN for survival support, or for maintenance of proper expression of neuronal markers (including GFRα2). While members of the NGF family are well-established trophic factors for most parts of the peripheral nervous system, their absence does not affect the development of parasympathetic neurons, and they do not influence these neurons in vitro. Interestingly, the only grossly observable phenotype in the NRTN- and GFRα2-deficient mice is apparent ptosis (i.e. drooping of the eyelids), which results from the complete absence of parasympathetic innervation to the lacrimal gland [28••,50••]. Furthermore, both NRTNand GFRα2-deficient mice also lack parasympathetic innervation to the submandibular gland, and have a significant loss of neurons in the submandibular ganglion [28••,50••]. As NRTN is expressed in the developing lacrimal and submandibular glands, and GFRα2 and RET are expressed in developing parasympathetic ganglion neurons [28••,50••,52], it appears that NRTN is functioning as a true target-derived neurotrophic factor that supports the survival of these developing parasympathetic neurons. This was the first demonstration of a neurotrophic factor that is critical for parasympathetic neuron development. α3 system The ARTN–GFRα
Although the phenotype of mice lacking ARTN is unknown, the phenotype of mice lacking GFRα3 was recently described [53••]. Interestingly, like the NRTN– GFRα2-deficient mice, mice lacking GFRα3 also exhibit ptosis. However, the cause of ptosis in these two cases is quite different — in NRTN–GFRα2-deficient mice, ptosis appears secondary to the loss of lacrimal gland secretion, due of lack of parasympathetic innervation. By contrast, GFRα3-deficient mice lack sympathetic innervation to the superior tarsus muscle, and therefore have a true inability to keep the eyelid raised. This lack of sympathetic innervation is due to a severe defect in superior cervical ganglion (SCG) development. In GFRα3-deficient mice, neurons of the SCG do not properly migrate during embryogenesis and the ganglion is found at a more caudal position than normal [53••]. Sympathetic innervation of the superior tarsus and submandibular gland is absent, suggesting that the target projections of the SCG are lost. The cells of this SCG rudiment die with a time course that resembles that of the NGF/TrkA knockouts [54,55], suggesting the possibility that in the absence of GFRα3 signaling (for early migratory events), the SCG does not properly innervate its targets and the neurons die due to lack of target-derived NGF. Somewhat surprisingly, no deficits in any other peripheral ganglia were detected in
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GFRα3-deficient mice, despite high-level GFRα3 expression in other sensory and sympathetic ganglia during development [56]. α–RET knockouts Discrepancies in the GFL–GFRα
Receptor binding experiments in vitro demonstrated that while GDNF preferentially binds and activates GFRα1– RET, it can also activate GFRα2–RET [21,22,26,27]. Analysis of the GFL and GFRα knockouts generated thus far indicates that the preferential interactions are the most prominent, and the importance of the alternative ligandreceptor interactions (i.e. GDNF–GFRα2) in vivo is unknown. However, GDNF-deficient mice have greater neuron loss in the DRG than GFRα1-deficient mice, suggesting that GDNF may utilize another receptor to support the survival of these neurons, presumably GFRα2–RET [9••,34••]. Therefore, it is possible that the ‘non-preferred’ GDNF ligand–receptor interactions do occur in vivo, but the prevalence of these interactions may vary in different biological systems. An initial report of RET-deficient mice indicated that the animals completely lack the SCG at birth [57]. Neither the GDNF–GFRα1 nor the NRTN–GFRα2 systems are responsible for this deficit, as mice missing these components have little (GDNF–/–) or no (NRTN–/–) reduction in the number of SCG neurons [9••,28••,31–33,34••,50••]. As mentioned above, GFRα3 (presumably mediating ARTN signaling) is critical for the development of the SCG; however, at birth the SCG is present in GFRα3 knockouts, but was reported absent in RET knockouts. Therefore, either some as yet unknown RET-activating system is critical for prenatal events in SCG development, or perhaps the altered location of the SCG caused it to be mistaken for being absent in the first reports of RET-deficient mice.
Parallels and intersections in the developmental roles of the NGF and the GDNF families Analysis of knockout mice has revealed extensive parallels in the mechanisms by which the NGF family and GDNF families influence neurodevelopment. First, while receptors for both families are present on central neurons and they can both potently influence central neurons in vitro, they appear to not be critical (at least individually) for the survival of CNS neurons during development. This adds further support to the suggestion that CNS neurons may derive redundant trophic support from many neurotrophic factors [58]. Second, members of both families are critically important for the development of distinct subsets of peripheral neurons. Together, the GDNF and NGF families probably account for the trophic support of the vast majority of peripheral neurons at some stage of their development. Third, members of both families function as target-derived trophic factors in some systems, and as paracrine-derived trophic factors in others before target innervation has occurred. Finally, there is evidence that particularly in the target-derived systems, members of both the NGF and GDNF families are present in limiting
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quantities, as evidenced by deficits in mice lacking one copy of the trophic factor gene. It is also clear that the NGF and GDNF families do not function simply as parallel neurotrophic systems; rather, it is likely that they overlap and alternate in their influence of peripheral neuron development. For example, it is clear that a subset of nociceptive neurons in the DRG, that initially are dependent on NGF for prenatal trophic support, switch such that they begin to express RET and can be supported by GDNF in vitro and after injury [43,44•]. Additional scenarios where peripheral neurons alternate their dependence on NGF or GDNF family members are sure to be discovered in the near future.
Conclusions The recent addition of PSPN and ARTN to the GDNF family ligands, along with confirmation from gene knockout studies of the importance of GDNF and NRTN in neurodevelopment, has established the GFLs as a second family of neurotrophic factors (the other being the NGF family) where different members influence distinct events and support the survival of distinct classes of peripheral neurons during development. While there is some in vitro cross-talk between the different GFLs and GFRαs, the preferred interactions (GDNF–GFRα1 and NRTN– GFRα2) appear most prevalent in vivo. Elucidating the developmental roles of the GFLs has significantly expanded our understanding of peripheral neuron development, as we now know that GFLs are critical for the development of enteric and parasympathetic neurons, two classes of neurons whose trophic dependencies were previously unknown. In addition, the GFLs also influence the development of sensory and sympathetic neurons, and therefore have a broader spectrum of developmental action than the NGF family.
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Some of the remaining challenges are obvious — for instance, the generation and study of mice lacking ARTN and PSPN, and the identification and confirmation of a mammalian GFRα4 that functions as a receptor for PSPN. Furthermore, continued detailed study of the timing and the mechanism of neuronal loss in GFL–GFRα–RET deficient mice, and comparisons with the influences of NGF family members, should allow an understanding of how the trophic demands of most peripheral neurons are met at each stage of development.
14. Creedon DJ, Tansey MG, Baloh RH, Osborne PA, Lampe PA, Fahrner TJ, Heuckeroth RO, Milbrandt J, Johnson EM Jr: Neurturin shares receptors and signal transduction pathways with glial cell linederived neurotrophic factor in sympathetic neurons. Proc Natl Acad Sci USA 1997, 94:7018-7023.
Acknowledgements
17. •
We thank all members of the Milbrandt and Johnson labs for invaluable discussions regarding the ideas and issues presented here. The authors are supported by National Institutes of Health grants R01 AG13729 and R01 AG13730.
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