Role of neurotrophic factors in development

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reviews of neurotrophic factors in development

Substantially more neurons are gen[ erated in the developing vertebrate RO. e nervous system than survive to maturity, the excess being eliminated in a phase of cell death which begins shortly after target field Alun M. Davies innervation begins (Fig. I). The proposal that developing neurons Neurotrophicfactors are molecules whichpromote and re.late neuronal survival in compete for a supply of a neurothe developing nervous ~y~tem. They are distinsuished from ubiqui~us metabolites trophic factor present in their target necessaryfor cellu~r maintena~e and gro~,tk ~j t~ir specificity: eachneurotrophic field in limiting amounts has received factorpromotes the survival of only certain kinds of neurons during a particular stage considerable support from work on of their development. In addition, it/ms been argued that neurotrophicfactors are nerve growth factor (NGF) TM. involved in many other aspects of neuronal development ranging from axonal This well-characterized protein, guidanee to regulation of neurotransmitter synthesis. Recent developmental studies and the use of specific molecular probes have greatly clarified the role of these consisting of two identical 13200 molecules. dalton polypeptides, promotes the survival of embryonic sensory and sympathetic neurons in culture and prevents loss of these neurons in vivo if administered decades have clearly shown the physiological importance during the period of natural neuronal death. The target of NGF in promoting neuronal survival in the developing fields of sensory and sympathetic neurons contain trace nervous system, the precise regulatory role of this and quantities of NGF in proportion to their iunervation other neurotrophic factors during target field innervation density. The innervating neurons possess specific cell remains unclear. For example, are neurotrophic factors surface receptors which mediate the uptake of NGF in merely involved in adjusting the overall numbers of the target field. Fast axonal transport conveys the neurons, or are they involved in selectively supporting internalized receptor-ligand complex to the cell bodies of neurons on the basis of their fiber terminations in the these neurons where it exerts its survival-promoting target field; that is, do they regulate ne~onal survival action. quantitatively or qualitatively? Central to resolving this Depriving developing NGF-sensitive neurons of their and related issues are the following questions: which cells supply of NGF by the administration of anti-NGF anti- synthesize neurotrophic factors and what is their distribodies during or shortly after the phase of natural bution in the target field; how is synthesis regulated; and neuronal death results in the elimination of these what is the distribution and availability of newly synthesneurons. Likewise, preventing the uptake of NGF from ized factors? The application of sensitive hybridization assays for the target field by destroying adrenergic terminals with 6-hydroxy dopamine or interrupting axonal transport NGF mRNA to the most densely innervated cutaneous with vinblastine or axotomy leads to the death of sym- target field in the mouse embryo (the maxillary process) pathetic neurons; death of these neurons can be pre- has revealed the kinds of cells that synthesize NGF vented by the concomitant administration of exogenous NGF. In recent years, additional molecules have been identified that promote the survival of different kinds of embryonic neurons in cultures-s. Although the physiological importance of these putative neurotrophic factors has not yet been firmly established by use of blocking antibodies in vivo, their discovery has strengthened and extended the trophic factor hypothesis. In particular, two new concepts have emerged. First, that of specificity; the survival-promoting action of each neurotrophic factor is restricted to a defined set of neurons. Second, that of cooperativity; the survival of certain neurons is regulated by more than one neurotrophic factor during target field innervation. In this review, I shall focus first on the role of neurotrophic factors in the regulation of neuronal survival and connectivity. In this respect, I shall discuss the physiological significance of the findings from recent developmental studies of NGF synthesis andNGF receptor expression, and consider the implications of neurotrophic factor specificity and cooperativity. Finally, I shall evaluate the evidence for some of the other roles proposed for neurotrophic factors in the developing Rg. 1. Two early phases in t~hedevelopment of a population nervous system. of neurons in the vertebrate nervous system. (A) The growth

Site, timing and regulation o! neurotrophic factor synthesis Although numerous studies over the past three ~) 1988, Elsevier P~l~calions, Cambridge 0168 - 9525/88/S02.00

of axons from newly differentiated neurons towards their target field. (B) The phase of target field innervation during which a proportion of the neurons die (shown in interrupted lines).

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Fig. 2. Localization of NGF mRNA in the most densely innervated cutaneous target field of the mouse embryo (the snout region in which whiskers develop) by in situ hybridization. Panels (a) and (b) are, respectively, phase-contrast and dark-field micrographs of the autoradiograph. The distribution of labelling (panel (b)) accords with the density of sensory endings in the target field after the phase of neuronal death. Both the surface epithelium, the thickness of which is shown in panel (a) by a small piece that has become detached (large arrow), and the epithelial components of developing whisker follicles (small arrows) are densely labelled. The presumptive dermis, the mesenchyme which lies just beneath the surface epithelium, is more densely labelled than the poorly innervated deep mesenchyme. Reproducedfrom Ref. 9, with permission.

during development 9. Quantitativenorthern blotanalysis of NGF mRNA in the enzymatically separated epithelial and mesenchymal components of the target field showed that it is present at about five-fold higher concentrations in the epithelium. In situ hybridization supported this result and showed in addition that NGF mRNA expression in the mesenchyme is higher in the region adjacent to the epithelium (Fig. 2). This distribution of NGF mRNA accords with the density of sensory endings in the target field after the phase of neuronal death: the epidermis (derived from the epithelium) is the most densely innervated, the dermis (derived from the subjacent mesenchyme) has a lower innervation density and the subcutaneous tissue (derived from the deep mesenchyme) possesses fewest endings. These findings suggest that the distribution of NGF synthesis governs the spatial organization of nerve endings in the target field. This could be accomplished not only by the selective maintenance of neurons that innervate regions rich in NGF, but also by the modification of axonal terminal branches in the target field after the phase of neuronal death. Campenot I° has shown that the extent of sympathetic neurite branches in vitro can be modified by the local availability of NGF; neurite branches that are exposed to NGF are maintained whereas those that are deprived of NGF are eliminated. The finding that NGF is synthesized in epithelium discounts the view, based on imnqu~.3histochemical

studies of the denervated iris, that NGF is synthesized exclusively by Schwann cellsxx. The demonstration that transection of the adult sciatic nerve triggers NGF synthesis4 and NGF receptor expression Iz in Schwann cells suggests that the appearance of NGF irnmunoreactivity in Schwann cells of the denervated iris may be the consequence of the response of these cells to nerve injury. The recent finding, however, that the sciatic nerve normally contains appreciable levels of NGF mRNA during the first post-natal week, falling to ~dult levels by the third week4, suggests that at least some NGF synthesis does occur in Schwann cells during development. Determination of the stage at which NGF synthesis commences in the nerve, in particular whether it precedes or follows the onset of neuronal death, will provide valuable information for understanding its physiological significance. It is likely, however, that NGF synthesis in this location is a consequence of the normal occurrence of axonal degeneration during development (comparable to the effects of nerve injury in the adult) and does not play an important role in regulating neuronal survival. Although studies of NGF mRNA localization in the maxillary process indicate the kinds of cells that syn~.hesize NGF during development, they provide no direct information on the distribution of newly synthesized NGF in the target field and its availability 0 the innervating nerve fibers. This information is essential for under-

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T I G - May 1988, Vol. 4, no. 5 standing the nature of the competition of neurons for NGF. There are two basic alternatives: either NGF is diffusible within the target field or its distribution and availability are restricted to the cells that make it. Diffusion of newly synthesized NGF would result in its being freely available throughout the target field. This implies quantitative regulation of neuronal survival, the number of neurons supported being governed by the available concentration d NGF. On the other hand, restriction of NGF synthesis and availability to certain target field cells is an attractive mechanism not only for regulating neuronal number but also for selectively supporting neurons on the basis of their axon terminations in the target field. In this model, NGF-producing cells can be regarded as specific target cells that support the survival of only those NGF-sensitive neurons that encounter them. In the case d NGF-dependent sensory neurons, it is possible that the NGF-producing target cells are the progenitors of specialized receptor cells that are associated with sensory nerve endings in the mature target field (e.g. Merkel cells in the epidermis). In the case d other kinds of neurons, it is possible that the neurotrophic factor-producing target cells are either the appropriate post-synaptic neurons or effector cells. One way in which NGF might be restricted to the cells that synthesize it is if these cells also express low-~ffinity NGF receptors 2 (Kd -- 1.7 × 10-9M). Newly synthesized NGF could then be held on the cell surface until it was encountered by an NGF-sensitive nerve fiber and was sequestered by high-~ffinity receptors (Kd = 2.3 × 10-11M). The finding that Schwann cells both synthesize NGF and express low-~nity NGF receptors 4"19-after axotomy in adult rats suggests that a similar mechanism may operate in regeneration. In this situation, however, it would have a different function: stim,dation of axon regrowth by presenting NGF to the regenerating nerve fibers. Recognition of the importance of NGF in controlling neuronal number and innervation density has directed attention to the regulation of NGF synthesis. Although nothing is known of the factors which control the onset, distribution and magnitude of NGF synthesis in development, recent work on the regenerating sciatic nerve in adult mice has shown that interleukin 1 released by macrophages plays an important role in stimulating NGF synthesis in Schwann cells~3.

Duration and regulation of neurotrophic factor dependence Knowledge of the onset, duration and regulation of neurotrophic factor dependence in developing neurons is a further important element in understanding the involvement of these factors in establishing specific patterns of innervation. As with most aspects of neurotrophic factor research, the most extensive data come from work on NGF. Detailed developmental studies have shown that the survival and growth of cultured sensory neurons is independent of NGF before the cells innervate their targets, and that these neurons express NGF receptors and become responsive to NGF when their axons reach their targets3'9. The finding that early sensory neurons cultured before target field innervation express NGF receptors at about the time they normally make contact with their targets in vivo suggests that receptor expression occurs as part of an intrinsic developr~ental program~.

This does not, however, exclude the possibility that the " - - , , , , / target field may 'fine tune' receptor expression. Like sensory neurons, sympathetic neurons also pass through a phase of NGF-independent survival early in their development 14. Whether transition to NGF dependence in these neurons is also related to target field innervation has yet to be determined. In vivo studies of the effects of anti-NGF antibodies and in vitro studies of the influence of NGF on neuronal survival have provided evidence that sensory and sympathetic neurons lose their dependence on NGF shortly after the phase of neuronal death has taken place in the corresponding gangiia 2.3. The loss of NGF dependence in developing sensory neurons is associated with a marked fall in the level of NGF receptor mRNA and in the number of NGF receptors per neuron Is. In contrast, the number of these molecules in sympathetic neurons increases throughout development, reaching levels in the adult which are severalfold higher than at birth 15. The presence of large numbers of NGF receptors on mature sympathetic neurons is related to the importance of NGF in regulating neurotransmitter synthesis in these cells (see below). Are the conclusions regarding the developmental timecourse of NGF dependence applicable to other neurotrophic factors and other neurons? Data available from work on brain-derived neurotrophic factor (BDNF) are consistent with these conclusions. Like NGF, BDNF is a small basic protein of molecular mass 12 300 daltons5. Its receptor-binding kinetics are also very similar to those of NGFle; Scatchard analysis indicates that BDNFresponsive sensory neurons possess two classes of receptors with differing affinities (Kd of 1.3 × 10-9M and 1.7 × 10-11M). BDNF promotes the survival of sensory neurons during the same period as NGF 17"~s, suggesting that it too regulates survival during the same phase of neuronal development. Its importance during this phase of development in vivo is demonstrated by the increased survival of sensory neurons that follows the administration of BDNF to quail embryos TM. Which second messenger system(s) is (are) activated by NGF receptor occupancy? Although numerous studies have provided some evidence for rap~ changes in Na +K 4- ATPase activity and cAMP, Ca2 ion, inositol trisphosphate, protein phosphorylation and protein methylation levels2°, no clear picture has emerged. The finding that the cytoplasmic domain of the NGF receptor has no significant sequence homology with the corresponding domains of other cell-surface receptors 2~ raises the exciting prospect that a novel series of events is involved.

Neurotrophic factor specificity The specificity of neurotrophic factors has been studied most extensively in the peripheral nervous system (PNS), where it is relatively easy to obtain homogeneous preparations of functionally distinct neurons. The spectrum of activity of several different factors for sensory, parasympathetic and sympathetic neurons is summarized in Table 1. With the exception of ciliary neurotrophic factor (CNTF), each factor has a unique and circumscribed specificity. What is the physiological significance of specificity? Its most likely purpose is to enable a given target field to regulate its innervation by different classes of neurons independently of each other. For example, sympathetic

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Table 1. The survival-promoting effects of different neurotrophic factors in the peripheral nervous system Neurotrophic factor

Sympathetic neurons

Parasympathetic neurons

Sensory neurons

NGF

Yes

No

BDNF

No

No

Partially purified factor(s) from pig lung CNTF

No

Yes

Subset (predominantly BDNF-insensitive) Subset (predominantly NGF-insensitive) No

8

Yes

Yes

Yes

6

and parasympathetic neurons both innervate the iris, but depend on different neurotrophic factors for survival. When a target field is innervated by different classes of neurons during separate periods of development, however, differences in neurotrophic factor dependence are not required for regulating their relative innervation densities. For example, both sensory and sympathetic neurons depend on NGF for survival, but innervate their common peripheral target tissues during separate periods of development. In addition to its apparent lack of specificity, CNTF differs from other neurotrophic factors in a number of respect~ 5. Unlike NGF and BDNF, which are present in neuronal target fields in very low quantities4's, CNTF is a very abundant protehl that nonetheless has similar potency to NGF and BDNF in bioassays. Whereas NGF and BDNF promote the survival of sensory neurons from the earliest stages of target field innervation, CNTF does not influence the survival of cultured sensory neurons until the phase of cell death is almost over. Could these findings mean that CNTF is a general neuronal maintenance factor in late development? This seems unlikely in the case of neonatal sympathetic neurons which are eliminated by anti-NGF despite the presence of large amounts of CNTF in vivo at this stage and neuronal responsiveness to this factor. Clearly, much work, including in vivo studies using blocking antibodies, is required to determine the importance of CNTF in neural development. The difficult problem of elucidating neurotrophic factor specificity in the central nervous system (CNS) will require improved methods for recognizing and isolating defined neuronal populations for in vitro study. Where the necessary techniques have developed, for example in the case of the retina, it has been shown that BDNF is specific for retinal ganglion cells~2. Various approaches have also provided considerable indirect evidence that the cholinergic neurons of the basal forebrain are dependent on NGF for survival23. Both these cholinergic neurons and retinal ganglion cells are similar to PNS neurons in that they possess long axons that innervate distant target fields. It will be of considerable interest to ascertain whether neurotrophic factors play a role in the development of interneurons which only make connections in their immediate vicinity.

Cooperation of neurotrophic factors Is the survival of neurons that innervate more than one target field regulated by neurotrophic factors from each of their target fields? Instrumental in resolving this issue were neurons of the embryonic chick trigeminal mesencephalic nucleus (TMN). These NGF-insensitive sensory neurons innervate two separate target fields:

Refs 1-3 5.17.18

motoneurons in the brainstem and stretch receptors in jaw musculature. In pure neuronal cultures, TMN neurons die unless supplemented with either BDNF or an extract of embryonic skeletal muscle (SM_x), which contains a neurotrophic factor distinct from BDNF24. Over 70% of TMN neurons survive in the presence of saturating levels of either factor alone and there is no additional survival with both factors together, indicating that each neuron responds to both factors. The trophic action of each factor on developing TMN neurons is maximal during the period of cell death in the TMN. This finding, together with the observation that these factors potentiate each other at very low concentrations, suggests that distinct neurotrophic factors from the CNS and periphery cooperate in regulating the survival of sensory neurons during development. This forms the basis of a mechanism for selectively supporting the survival of neurons that make appropriate terminations in both target fields. If there are limiting amounts of the factors in the respective central and peripheral target fields, then a neuron needs to make appropriate terminations in both target fields to procure sufficient trophic support to survive. It remains to be investigated whether the above principle holds true for other populations of neurons that innervate multiple target fields. In addition, given the evidence that the size of certain populations of neurons is regulated not only by the target fields they innervate but also by the afferent neurons they receive, it will be interesting to ascertain whether the survival of these neurons is promoted by different neurotrophic factors from their targets and afferent neurons.

Regulation of neurotransmitter and neuropeptide synthesis NGF specifically induces the rate-limiting enzymes in noradrenaline synthesis - tyrosine hydroxylase and dcpamine beta hydroxylase - in sympathetic neurons ~. It induces the rate-limiting enzyme in acetylcholine synthesis (choline acetyl transferase) in basal forebrain cholinergic neurons ~. NGF also regulates the level of the neuropeptide substance P in dorsal root ganglia25. All these effects occur in developing and mature neurons. This may provide an explanation for the continued production of NGF by neuronal target fields in the adu!t. by which time neurons no longer require it for survival. Neurotrophic factors may regulate the level of neurotransmitters and neuropeptides in neurons in accordance with the extent of their terminal branches. Do neurotrophic factors guide nerve fibers to their target fields in development? Several in vivo and in vitro studies of the effects of

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NGF on the growth of post-embryonic and regenerating sensory and sympathetic nerve fibers have led to the widely held view that NGF attracts these classes of nerve fibers to their target fields by chemotropism during development2e. Intracranial injection of NGF in neonatal rodents results in the growth of sympathetic nerve fibers into the vertebral column and along the spinal cord towards the brain. Sensory neurites show a preferred orientation up a concentration gradient of NGF and turn towards a source of NGF in their immediate vicinity. Quite apart from the implausibility of concentration gradients of a single molecule accounting for the numerous specific routes taken by different sets of sympathetic and sensory nerve fibers in development, it has recently been shown that NGF synthesis in developing cutaneous target fields does not occur before the arrival of sensory nerve fibers and that these fibers do not possess NGF receptors until they reach their targets 9. Thus, the view that NGF guides early nerve fibers to their target fields must be abandoned. The apparent chemotropic effects of NGF observed in the above studies are easily explained in terms of the trophic effects of NGF and its physical properties at the massively high concentrations used in these studies. These issues are discussed in detail elsewhere 3.

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2 Thoenen, H. and Barde, Y. A. (1980) Annu. Rev. Physiol. 60, 28t-335 3 Davies, A. M. (1987)Development 101, 185-208 4 Heumann, R. (1987)J. Exp. Biol. 132, 133-150 5 Barde, Y. A. Edgar, D. and Thoenen, H. (1982)EMBO .L 1, 549-553 6 Barbin, G., Manthorp,M. and Varon, S. (1984)]. Neurochem. 43, 1468-1478 7 Gurney, M. E., Heinrich, S. P., Lee, M. R. and Yin, H. (1986) Science234, 566-574 8 Wallace, T. L. and Johnson, E. M. (1987)Brain Res. 411, 351363 9 Davies, A. M. et al. (1987)Nature 326, 353-358 I0 Campenot, R. B. (1982)Dev. Biol. 93, 13-21 I1 Rush, R. A. (1984)Nature312, 364-367 12 Taniuchi,M., Clarke, H. B. andJohnson, E. M. (1986)Proc. Natl Acad. Sci. USA 83, 4094-4098 13 Lindholm,D., Heumann, R., Meyer, M. and Thoenen, H. (1987) Nature 330, 658-659 14 Coughlin, M. D. and Collins, M. B. (1985)Dev. Biol. 110, 392401 15 Buck, C. R., Martinez, H. J., Black, I. B. and Chao, M. V. (1987) Proc. Natl Acad. Sd. USA 84, 3060-3063 16 Rodriguez-Tebar,A. and Barde, Y. A. ]. Neurosd. (in press) 17 Lindsay, R. M., Thoenen, H. and Barde, Y. A. (1985)Dev. Biol. 112, 319-328 18 Davies, A. M., Thoenen, H. and Barde, Y. ~ (1986)]. Neurosci. 6, 1897-1904 19 Hofer, M. M. and Barde, 1'. A. (1988)Noture331, 261-262 20 Acheson, A. Vogl, W., Huttner, W. B. 3_-.dThoenen, H. (1986) EMBO ]. 5, 2799-2803 21 Redeke, M.J., Misko, T. P., Hsu, C., Herzenberg, L. A. and Shooter, E. M. (1987)Noture 325, 593-597 O t h e r r o l e s of n e u r o t r o p h i c f a c t o r s Certain neurons whose survival is not influenced by 22 Johnson,J. E., Barde, Y. A., Schwab, M. and Thoenen, H. (1986) ]. Neurosci. 6, 3031-3038 NGF (e.g. ~ neurons 27, TMN neurons 2s and 23 Korsching, S. (1986) Trends Neurosd. 9, 570-573 motoneurons ) express NGF receptors during 24 Davies, A. M., Thoenen, H. and Barde, Y. A. (1986)Nature 319, embryonic development. This raises the possibility that 497-499 NGF serves some other function in the development of 25 Adler,J. E., Kessler,J. A. and Black, I. B. (1984)Dev. Biol. 102, 417-425 these cells. 26 Levi-Montalcini,R. (1982)Annu. Rev. Neurosd. 5, 341-362 It has recently been proposed that the mitogen basic 27 Raivich, G., Zimmermann,A. and Sutter, A. (1985)EMBO]. 4, fibroblast growth factor CoFGF) is ~. neurotrophic factor 637-644 for hippocampal and cortical neurons 29. However, certain 28 Davies, A.M., Lumsden, A. G. S. and Rohrer, H. (1987) N e u r o s ~ e 20, 37--46 technical problems associated with CNS neuronal Morrison, R. S. (1987)J. Neurosci. Res. 17, 99-101 cultures call for some caution. In particular, there is the 29 30 Gensburger, C., Labourdette, G. and Sensenbrenner, M. (1987) difficulty of removing all glial cells and neuronal progeni,FEBS Lett. 217, 1-5 tor cells from these cultures. Because of the production 31 Faik, P., Walker, J. I. H., Redmill, A. A. M. and Morgan, M. J. Nature (in press) of neurotrophic factors by cultured glia, it is difficult to exclude the possibility that bFGF exerts its apparent trophic effects on neurons indirectly. Where it is possible A. M. Davies is in the Department of Anatomy, St George's Hospital Medical S c l ~ l , Cranmer Terrace, Tooting, London to remove all gila from CNS cultures, for example in the SW17 ORE, UK. case of TMN neurons, bFGF has no effect on neuronal survival. But when glial cells are not removed, bFGF Pool your u s e f u l h i n t s through Technical Tips appreciably increases the proportion of TMN neurons that survive (A. M. Davies, unpublished). Furthermore, Technical Tips is a place where readers can exchange information about new experimental techniques. To make it has recently been shown that bFGF is mitogenic for this section really useful to experimental geneticists and cortical neuronal progenitor cells3°. Thus, increased developmental biologists we need the active participation proliferation and differentiation of these cells in cortical of our readers. If you have information about methods cultures could account for the increased number of developed in your lab or elsewhere why not share it with neurons in the presence of bFGF. your colleagues through the Technical Tips section of It has been reported that neuroleukin is both a Trends in Genetics? neurotrophic factor and a lymphokine7. However, it has Each month Technical Tips draws attention to such recently been shown that the published sequence of methods by presenting very brief articles. These do not neuroleuldn is identical to that of the ubiquitous intracelattempt to provide all the information required to use the method but rather a clear outline of the method's claimed lular enzyme glucose-6-phosphate isomerase 3z. Thus, as advantages and present and potential applications; with several other putative neurotrophic factors, here is readers can then look to the reference for complete a clear case where detailed in vivo atudies are required to details. The only exception to this general policy concerns determine the physiological relevance of this protein in descriptions of unpublished methods where more precise neuronal development. experimental details should be provided. Please send all information to: Trends in Genetics, Elsevier Publications, 68 Hills Road, Cambridge CB2 1LA, References UK, or call (0223) 315961. I Levi-Montalcini,R. and Angeletti, P. U. (1968)Physiol. Rev. 48, r ,, i L534-569

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