Novel roles for neurotrophins are suggested by BDNF and NT3 mRNA expression in developing neurons

Neuron, Vol. 9,449-463, September, 1992, Copyright 0 1992 by Cell Press

Novel Roles for Neurotrophins Are S by BDNF and NT-3 mRNk in Developing Neurons Leslayann C. Schecterson and Mark Bothwell Department of Physiology and Biophysics SJ-40 University of Washington Seattle, Washington 98195

Summary

The results of our in situ hybridization experiments demonstrate that sensory neurons, sympathetic neurons, and motoneurons express brain-derived neurotrophic factor and/or neurotrophin-3 mRNAs during development in mouse. In accordance with previous data, we also find neurotrophins in the targets of sensory neurons (skin) and motoneurons (muscle) and the neurotrophin receptors ~75, t&A, and t&B in sensory and sympathetic ganglia. These results suggest that neurotrophins have roles other than being target-derived factors that support neuron survival during developmental cell death (neurotrophic hypothesis), but may be transported in an ortho- grade fashion in neurons and released from axon terminals. We discuss several novel roles for neurotrophins, including autocrine/paracrine regulation of neuron survival, regulation of Schwann cell activity, and neuron to target signaling.

Introduction

During development of the central and peripheral nervous systems in vertebrates many more neurons are generated than ultimately survive and make functional connections with their targets. Neuronal survival is assumed to be regulated by target-derived factors, a concept referred to as the neurotrophic hypothesis. This theory predicts that as neuronal pop ulations undergo developmental cell death, the neurons compete for a limited quantity of a neurotrophic factor or factors produced exclusively by their target tissues. Nerve growth factor (NGF) was the first neurotrophic factor demonstrated to be required for normal development. Anti-NGF antibodies injected into newborn rodents specifically destroy the peripheral sympathetic nervous system (Cohen, 1960), while prenatal exposure to NGF antibodies also causes the death of sensory neurons of spinal ganglia (johnson et al., 1983). Subsequently, brain-derived neurotrophic factor (BDNF) was isolated (Barde et al., 1982), and molecular clones were obtained (Leibrock et al., 1989), re- vealing that the amino acid sequence of BDNF was about 50% identical to that of NGF. More recently, with the use of polymerase chain reaction (PCR) cloning techniques, additional NGF-like neurotrophic factors have been identified: neurotrophin3 (NT-3) (Mai- sonpierre et al., 1990; Hohn et al., 1990; Rosenthal et al., 1990) and NT-4/5 (Hallbook et al., 1991; Berkemeier et al., 1991; Ip et al., 1992). These five factors are collec- tively referred to as neurotrophins.

Expression

The various neurotrophins differ functionally in their abilities to promote survival of distinct neuronal populations in vivo and in vitro. Explants of chick peripheral ganglia have been used to distinguish the biological activities of NGF, BDNF (Davies et al., 1986), and NT-3 (Maisonpierre et al., 1990; Hohn et al., 1990). Both NGF and BDNF promote neuronal survival and differentiation in cultures of dissociated ganglionic neurons (Lindsay and Rohrer, 1985) and prevent natu- rallyoccurringcell death in specific peripheral ganglia when administered to developing avian embryos (Hofer and Barde, 1988). NGF and BDNF support the survival of neural crest-derived dorsal root ganglion (DRG) cells, but only BDNF and NT-3 can promote survival of placode-derived sensory neurons of the nodoseganglion (Lindsay and Rohrer, 1985; Rosenthal et al., 1990), and only NGF and NT-3 can promote neuronal survival in paravertebral chain sympathetic ganglion explants (Barde et al., 1982; Maisonpierre et al., 1990). BDNF supports survival and neurite outgrowth in about 50% of the neurons in dissociated cultures of embryonic DRGs. If BDNF and NGF are added to- gether, approximately 100% of the neurons survive, indicating that BDNF and NGF most likely support different subpopulations of DRG neurons. NT-3 also supports survival of 500/o-60% of the neurons in dissociated cultures of embryonic chick DRGs (Maison- Pierre et al., 1990; Rosenthal et al., 1990), suggesting that the neuronal subpopulations that respond to NT-3 also respond to either BDNF or NGF. Although NT-3 can supportthe survival of neuronal populations in DRG, nodose ganglion, and sympathetic ganglion explants, the maximum response induced by NT-3 in a specific ganglion differs from the maximum response induced by either NGF alone or BDNF alone (Maison- Pierre et al., 1990; Hohn et al., 1990; Rosenthal et al., 1990). This suggests that subpopulations of neurons with differing neurotrophin sensitivities may exist within each of these types of ganglia.

Two classes of neurotrophin receptors have been identified. Initially, molecular cloning studies characterized a 75-80 kd NGF receptor protein (Johnson et al., 1986; Radeke et al., 1987) that binds several (perhaps all) neurotrophins with equivalent affinity (Rod- riguez-Tebar et al., 1990; Squint0 et al., 1991). How- ever, since this protein by itself is not capable of mediating cellular responses to neurotrophins, the name NGF receptor is not entirely appropriate. Con- sequentlythis protein will be referred to simply as p75 in reference to its molecular mass. A second class of neurotrophin receptors is encoded by the trk gene family (trk, trkB, and trkC). In the following text, trk will be referred to as trkA, while trk will denote the family of related molecules. The trk genes encode tyrosine kinase receptors, which, in the absence of p75 expression, are capable of mediating cellular responses to neurotrophins (Cordon-Card0 et al., 1991;

Glass et al., 1991; Lamballe et al., 1991). However, expression of p75 appears to enhance the sensitivity to neurotrophins of cells expressing trkA receptors (and by analogy, perhaps trkB and trkC also), perhaps in part by increasing the binding affinity of the trk receptors (Kaplan et al., 1991; Hempstead et al., 1991). Since neurotrophin-responsive neurons appear generally to express p75 as well as one of the trk genes, it appears likely that p75 is a functional component of neurotrophin receptors in most or perhaps all neurotrophin-responsive neurons.

Throughout embryogenesis, p75 is expressed in primary sensory neurons, sympathetic neurons, and motoneurons (Yan and Johnson, 1988; Ernfors et al., 1989; Heuer et al., 1990). Primary sensory neurons also express both trkA and trkB, while only expression of trkB has been reported in sympathetic neurons, and neither is expressed in motoneurons (Martin-Zanca et al., 1990; Klein et al., 1990b). The pattern of expression of trkC in the peripheral nervous system has not been reported. While the neurotrophin responsiveness of sensory neurons and sympathetic neurons has been extensively documented, the functional significance of p75 expression in motoneurons is less clear. Devel- oping motoneurons expressing p75 bind and retro- gradely transport NGF (Yan et al., 1988), yet several

investigators have reported that motoneurons do not respond to NGF or other neurotrophins (Yan et al., 1988; Arakawa et al., 1990). In contrast, Wayne and Heaton (1990) have reported that NGF weakly pro- motes neurite extension in embryonic motoneurons in culture.

While the spatiotemporal patterns of expression of NGF in targets of innervation by the peripheral nervous system in rodents have been extensivelycharac- terized (Shelton and Reichardt, 1984; Heumann et al., 1984; Bandtlow et al., 1987; Davies et al., 1987; Wheeler and Bothwell, 1992), less detailed information is avail- able for other neurotrophins. To characterize system- atically the possible physiological sources of neurotrophins for the peripheral nervous system, we have performed in situ hybridization using antisense riboprobes directed against the neurotrophins NCF, BDNF, and NT-3, examining mouse embryos ranging in age from embryonic day (E) 8.5 to postnatal day (P) 1. Contrary to the prediction of the neurotrophic hypothesis, according to which neurotrophic factors are derived exclusively from innervated targets, we found that several neuronal populationsand their target tissues express neurotrophins simultaneously. In addition, transcripts encoding the ~75, trkA, and trkB neurotrophin receptors were found to be expressed

Table 1. Expression of NGF, BDNF, NT-3, and p75 mRNAs in Mouse Embryos from 11.5 Days of Gestation (E11.5) to PI

E11.5 E12.5 E14.5 E15.5 El 7.5 PI

DRG BDNF NT-3

P75 Trigeminal

BDNF

P75 Sympathetic ganglia

BDNF NT-3

P75 Motoneurons

NT-3

P75

+++ + +++

++ - +++

++ - +++

- +++

- +++

- +++

++ ++

- +++

- +++

f +++

+ ++

++ + +++

++ + +++

++ ++ +++

++ ++ +++

-

-

Nonneuronal Expression Cochlea

BDNF NT-3

Vestibular BDNF

-

+++ +++

+++ +++

+++ +++

+++ +++

ii+ +++

+++ +++

++ ii+

++ +++

ND ND

+ ++

++ ++ ++ ++ i Skin (face, digits)

NCF BDNF NT-3

Hair follicles (mesenchyme) NT-3

Muscle NT-3

P75

+ ++ ++

++ ++ +++

+++ ++ +++

+ - ++

++ 1 -

+++ ++ +

++ ++

++ ++

++ +++

++ +++

Degrees of expression were as follows: (-) none, (+) weak, (+ +) moderate, and (+ + +) strong. ND, not determined.

BDNF and NT-3 in Neurons 451

Figure 1. Neurotrophin Expression in Targets of Sensory Neurons in Mouse Embryos of 15.5 Days Gestation

In situ hybridization using either NCF, BDNF, or NT-3 antisense riboprobes on sagittal sections at E15.5. (A) Bright-field view of snout with vibrissae (v). (B) Dark-field view of (A), hybridized with NT-3 antisense riboprobe. (C) Bright-field view of tongue (t) and mandible (md). (D) Dark-field view of mandible area in right-hand box in (C), hybridized with BDNF antisense riboprobe. (E) Section adjacent to (D), dark-field view of mandible area in right-hand box in (C), hybridized with NT-3 antisense riboprobe. (F) Dark-field view of lower left-hand boxed area in (C) hybridized with NGF antisense riboprobe. (C) Adjacent section to (F), hybridized with sense probe. BDNF and NT-3 mRNA expression are primarily confined to the mesenchyme in the mandible as shown in (D) and (E), whereas NGF mRNA expression is primarily in the epithelium (F). Bar, 100 urn (A-C); 50 urn (D-C).

in the same neuronal populations expressing neurotrophin mRNAs. The possible significance of these findings is discussed.

Results

Neurotrophins Are Expressed in the Target Fields of Sensory and Motor Neurons Somatosensory The cutaneous targets of the trigeminal ganglion in mouse include the whisker pad and the mandible. The whisker pad is innervated by the maxillary branch of the trigeminal and is the most densely innervated cutaneous target field in the mouse. The mandible is innervated by the mandibular branch of the trigeminal ganglion.

NGF has been detected in the cutaneous targets of trigeminal nerves beginning at around El1 in mouse (Davies et al., 1987). We have detected the expression of NGF, BDNF, and NT-3 mRNAs in epidermal and dermal regions of the skin from E11.5 to E17.5 (Table 1). NGF transcripts were found mostly in epidermal cells overlying the dermis and in a few cells in the dermal layer, in agreement with previous results (Da- vies et al., 1987; Heuer et al., 1998), whereas BDNF and NT-3 transcripts were expressed primarily in dermal mesenchyme. This pattern of expression was observed very clearly in the mandible (Figure 1) and digits (Table 1) at E15.5.

NGF and NT-3 mRNA expression was highest in the developing skin around E15.5. In contrast, BDNF mRNA expression was strong at E11.5 in the dermal

Neuron 452

Figure 2. NT-3 mRNA Is Expressed in Muscle, theTargetof Moto- neurons (E15.5)

(A) Bright field of sagittal section of upper back muscle. (8) Dark field of (A), hybridized with NT-3 antisense riboprobe. (C) Section adjacent to (B), hybridized with control NT-3 sense riboprobe. Bars, 100 Wm.

mesenchyme surrounding the entire embryo and decreased significantly by E15.5, becoming confined to limited areas. Interestingly, those areas expressing BDNF at E15.5 are regions that are sensitive to touch, such as the digits, lips, tongue, and mandible, and contain specialized mechanoreceptors, Merkel disks, located at the junction of dermis and epidermis. NT-3 mRNA was highly expressed in the mesenchyme surrounding the developing vibrissae in the whisker pad at E14.5 and E15.5, with expression decreasing by E17.5 (Figure 1; Table 1). Muscle Developing skeletal muscle receives innervation by both motor and sensory neurons, and axons from these neurons reach their targets during the period

when myoblasts are fusing to form myotubes. We detected substantial expression of NT-3 in developing muscle during this period (Figure 2). NT-3 mRNA was present in almost every cell in the muscle from E11.5 to E15.5. Presumablythesecellsare myoblasts. Expres- sion of NT-3 mRNA was very weak or nonexistent by E17.5. Remarkably, the period of expression of NT-3 correlates with the timing of p75 expression in developing rat myoblasts (Wheeler and Bothwell, 1992). We observed a similar pattern of p75expression in mouse myoblasts (data not shown).

Developing Sensory Neurons Express Neurotrophin mRNAs While the expression of neurotrophins in various innervated tissues was generally in accord with our ex- pectations, we were surprised to observe extensive expression of neurotrophins in the neurons themselves. Specifically, BDNF was expressed in three types of sensory ganglia that originate from different embryonic sources. Sensory neurons of spinal ganglia (DRGs) derive exclusively from neural crest. The trigeminal ganglia contain neurons of both neural crest and epibranchial placode origin, and the neurons of the geniculate ganglia derive exclusively from the epibranchial placode. As discussed above, sensory neurons of neural crest origin are functionally responsive to NGF, BDNF, and NT-3 to varying degrees during development, while neurons deriving from epibranchial placode respond to BDNF and NT-3, but not NGF.

In mouse, the trigeminal ganglia become recogniz- able at E8.5. BDNF transcripts were detectable in the trigeminal ganglion in a few cells beginning at E14.5 and appeared in more neurons in theganglion at E15.5 and E17.5 (Figures 3A-3C). Large cells, lightly stained by methyl green in the ganglia, were identified as neurons. Most of the silver grains appeared over neurons and not the adjacent smaller, darker stained glial cells. The gangliacontained many of these smaller cells, but silver grains were never seen clustered over them. Neurons of the geniculate ganglia also expressed BDNF mRNAs beginning at E14.5 (Figure 3D). Expres- sion of other neurotrophins, NGF and NT-3, was not observed in either the trigeminal or geniculate ganglia.

In neurons of DRGs we have observed expression of NT-3 transcripts as well as BDNF transcripts. Begin- ning at E15.5 some, but not all, of the neurons in the DRGs express BDNF mRNA, and an even fewer number of neurons express NT-3 mRNA (Figure 4). On sections hybridized with sense probe, O-3 grains appeared over the large, lightly stained cells identified as neurons. On sections hybridized with antisense riboprobes, positively labeled cells were identified as those cells containing at least 8 grains per cell. Usu- ally, positive cells contained 15-25 grains clustered over them. The percentage of neurons expressing neurotrophins was determined by comparing the number of positive neurons with the total number of


Figure 3. BDNF mRNA Localization in Cranial Sensory Ganglia by In Situ Hybridization Using BDNF Antisense Riboprobe and Control Sense Riboprobe to Sagittal Sections of E15.5 Embryos

(A) Trigeminal ganglion (TC), hybridized with BDNF antisense riboprobe and (6) closely adjacent section hybridized with control BDNF sense riboprobe. (CT Higher magnification of TG using Nomarski optics, demonstrating silver grains over neurons in the TC. Higher magnification of boxed cells is shown in inset. (D) Ceniculate ganglion (gem hybridized with BDNF antisense riboprobe. Bar, 100 urn (A, B, and D); IO pm (CT.

neurons in several ganglia. Neurons were identified by their large size and lack of intense staining. At E15.5, 40%-50% of all neurons in DRCs expressed BDNF, and IO%-15% of the neurons in DRGs expressed NT-3.

Adjacent paraffin sections, 8 pm thick, were com- pared to determine whether the same cells expressing NT-3 transcripts were also expressing BDNF mRNA. Oftentimes the same neuron could be identified in

adjacent sections, but expression of both BDNF and NT-3 mRNA was never seen in such cells. The expression of NT-3 mRNA transcripts by a few neurons was seen at E15.5 in three separate experiments but expression appears to be transient (Table I), since expression of NT-3 mRNA in DRGs was not observed at E17.5 or PI. In contrast, BDNF mRNA expression was present in many neurons in DRGs at E15.5, E17.5, and PI (Table 1).

Neuron 454

Figure 4. BDNF and NT-3 mRNA Localization in Peripheral Sensory Ganglia of E15.5 Embryos by In Situ Hybridization

(A) Bright-field illumination of DRCs from a sagittal section. (B) Dark field of (A), hybridized with BDNF antisense riboprobe. (C) Section adjacent to (B), hybridized with NT-3 antisense riboprobe. (D-F) Higher magnification view with Nomarski optics. Sections hybridized with control BDNF sense probe (D), BDNF antisense probe (E), and NT-3 antisense probe (F). The results demonstrate expression of BDNF transcripts in many neurons and NT-3 transcripts in a few neurons. Bar, 100 urn (A-C); 10 urn (D-F).


Figure 5. BDNF and NT-3 mRNA Expression in Sympathetic Ganglia in El55 Mouse Embryos

(A) Bright field of sympathetic ganglia in sagittal section. (B) Dark field of (A), hybridized with BDNF antisense riboprobe. adjacent to (B), hybridized with NT-3 antisense riboprobe. Bar, 100 pm.

(C) Set

Sections from developmental stages E8.5-PI were also examined by in situ hybridization for NGF expression. Despite an 8 week autoradiographic exposure period (see Experimental Procedures), no expression of NGF mRNA was detected in cranial or DRG sensory neurons.

Some Neurons in Sympathetic Ganglia Express Neurotrophins Like many sensory neurons, the neurons in the sympathetic ganglia derive from the neural crest. In culture, these neurons are responsive to NGF and NT-3, but not BDNF (Maisonpierre et al., 1990; Rosenthal et al., 1990). At E14.5, 40%-45% of the neurons in several sympathetic ganglia expressed BDNF mRNA. In these same ganglia, a smaller number of neurons, IO%-15%, expressed NT-3 mRNAs (Figure 5). Expression of mRNAsencoding BDNFand NT-3continued insympa- thetic neurons at E15.5, E17.5, and PI. At these later embryonic stages and in newborn animals, BDNF and NT-3 were both expressed in some, but not all sympathetic neurons. However, we have not yet determined whether the populations of cells expressing BDNF and NT-3 are separate or overlapping.

Motoneurons Express NT-3 mRNA Transcripts throughout EmbryoBenesis In spinal cord motoneurons, developmental cell death takes place during the period from El3 until birth. We detected strong expression of NT-3 mRNA in motoneurons from E11.5 to PI (Figure 6; Table 1). From E13.5, we identified motoneurons by their large size and location in the spinal cord ventral horn, whereas at E11.5, presumptive motoneurons could only be tentatively identified based on their ventral location in the neural tube (Figures 6A and 6B). AS shown in Figures 6D and 6E, most, if not all, motoneu-

rons express NT-3 transcripts at E15.5. The appearance of the hybridization signal is less pronounced at PI, because the motoneurons .are fewer in number and more dispersed. Nevertheless, a substantial level of NT-3 mRNA was detected in all the motoneurons in the newborn animal also (Table 1).

P75 Expression in Neurons Developing sensory neurons and sympathetic neurons are known to express p75 and to respond to neurotrophic factors. To compare more closely the developmental pattern of p75 and neurotrophin expression, in situ hybridization was performed on nearly adjacent sections in the same experiments described above. Indeed, p75 transcripts were present at the time of neurotrophin expression in trigeminal and geniculate cranial sensory ganglia, sympathetic ganglia, DRGs, and motoneurons (Figure 7). In some cases neuronal expression of p75 commenced at embryonic stages prior to the beginning of neurotrophin expression (Table 1). Trigeminal gangliaexpressed p75 mRNAs beginning at E11.5, while BDNF mRNAexpres- sion was not present until E14.5 in a few neurons and in most neurons by E15.5. All sympathetic ganglia examined at E14.5 expressed p75 mRNA, but only a few ganglia expressed BDNF and NT-3 mRNA transcripts, and only a few neurons in the ganglia expressed NT-3 mRNA at this early stage. Transcripts encoding BDNF and NT-3 were not present in DRGs until E15.5; however, p75 mRNAwas strongly expressed in the DRGs at E11.5-PI. Motoneurons strongly expressed both NT-3 and p75 mRNAs at every stage analyzed, from E11.5-PI.

mRNAs Encoding trkA and trkB Are Expressed in the Same Populations of Neurons as ~75 mRNAs Since neuronal responses to neurotrophins probably involve receptors of the trk gene family acting in con-

456

Figure 6. In Situ Hybridization, Demonstrating That NT-3 mRNA Is Abundantly Expressed in Motoneurons of E11.5 and E15.5 Mouse Embryos

(A) Transverse section of E11.5 spinal cord shown under bright-field illumination. (B) Dark field of (A), hybridized with NT-3 antisense riboprobe, demonstrating NT-3 mRNA localization in motoneurons (mn). (C)Bright-field illumination of spinal cord from sagittal section of E15.5 embryo and (D) dark field of the same section hybridized with NT-3 antisense riboprobe. Silver grains are present over the neurons in the ventral portion of the spinal cord only, the location of the developing motoneurons. The boxed area is shown at higher magnification using Nomarski optics in (E). Bar, 100 urn (A-D); 10 urn (E).

cert with ~75, we also examined the spatial pattern of specifically in trigeminal ganglia and DRGs, not the expression of trkAand trkB mRNA in neuronal popula- geniculate ganglia, and our results are generally in tions expressing neurotrophins using in situ hybrid- agreement (Figure 8). In our studies, trkA mRNA was ization. Martin-Zanca et al. (1990) reported that trkA also expressed strongly in sympathetic ganglia, also was expressed in neurons of neural crest origin only, of neural crest origin. trkB transcripts were also pres-


Figure 7. Expression of p75 mRNA Is Abun- dant in Neurons That Also Express Neuro- trophins

Sections in (A)-(E)areElS.S sagittal sections hybridized with either p75 antisense riboprobe or control, p75 Sense riboprobe. (A) Section containing trigeminal ganglion CrC) hybridized with ip75 antisense riboprobe, closely adjacen? to section of TC in Figure 2A and (B) adjgcent section to (A) hybridized with contjol, p75 sense riboprobe. (Cl Geniculate

t anglion (gen) in sec-

tion closely adjacent t that shown in Fig- ure 3D. (D) Sympatheti/c ganglia hybridized with p75 antisense ribprobe in section closely adjacent to se@ions in Figure 6. (E) Sagittal section contaihing both DRCs and motoneurons (mn), ckjsely adjacent to section in Figure 5C. (F) Tkansverse section of spinal cord from E11.5 embryo, demonstrating that p75 rnRNA expression is present in DRGs earlier than nburotrophins are expressed (refer to Figure 4 and Table 1). Sec- tion is closely adjacent to section in Figure 4A. Bars, 100 Wm.

ent in neurons of neural crest origin, but in contrast to t&A, trkf3 mRNA labeled only a small subpopulation of neurons in dorsal root and sympathetic ganglia, in amannersimilartothatdescribed byKleinetal.(1989). trkA transcripts were present in all neurons in dorsal root and sympathetic ganglia, whereas trk6 was present in only 130/o-17% of the neurons in DRGs and about 20% of the neurons in sympathetic ganglia. These results indicate that, at E15.5, sensory and sympathetic neurons express the neurotrophin receptors ~75, trkA, and trkB, as well as the neurotrophins BDNF and NT-3. It remains to be determined whether or not neurotrophins and neurotrophin receptors are coexpressed within individual neurons. Motoneu- rons express NT-3 and p75 mRNAs throughout embryogenesis. trkA is not expressed in motoneurons;

however, trkB transcripts were expressed at a low level throughout the spinal cord, and some of those weakly expressing cells may be motoneurons. It re-

mains to be determined whether motoneurons express trkC, or perhaps a novel trk gene.

Discussion

The data described here concerning sites of expression of ~75, trkA, and trkB are generally in good agreement with previous studies. However, one novel aspect of our findings is that we have detected expression of trkA in neurons of sympathetic ganglia. Previ- ous studies failed to report this site of expression (Martin-Zanca et al., 1990); however, our result is in accordance with physiological data, since sympa-

NCYJKNl 458

Figure 8. Expression of trk mRNAs in Neurons in E15.5 Embryos

Sagittal sectionsdemonstrate results of in situ hybridization using trkAantisense riboprobe, control trkA sense riboprobe, frkB antisense riboprobe, and trkB control sense riboprobe. (A) Trigeminal ganglion hybridized with trkA antisense riboprobe and (B) an adjacent section hybridized with control trkA sense riboprobe. (C) DRGs and (D) sympathetic ganglia hybridized with trkA antisense riboprobe. (E) DRGs in a section adjacent to(C), hybridized with trkB antisense riboprobe and (F) section adjacent to (E) hybridized with trkB control sense riboprobe. (G) Sympathetic ganglia hybridized with trkB antisense riboprobe. Bars, 100 urn.

thetic neurons are known to respond to NGF and trkA is the only known neurotrophin receptor that has been shown to mediate responses to NGF.

It is of some interest that weobserve trkAexpression in virtually all neurons of trigeminal ganglia. In avians, a minority of trigeminal ganglion neurons derive from the trigeminal placode rather than neural crest (No- den, 1978; D’Amico-Martel and Noden, 19801, but the relative contribution of neural crest and placode to development of the mammalian trigeminal ganglion is unknown. Our observation that trkA is expressed in all trigeminal ganglion neurons suggests that all

neurons in the mouse trigeminal ganglion derive from neural crest or, alternatively, that trkA expression is not entirely limited to neural crest derivatives.

Previous studies have interpreted data pertaining to tissue sites of expression of neurotrophins in the context of the conventional neurotrophic hypothesis, which predicts that neurotrophic factor expression should be limited to cell populations that receive innervation by neurotrophin-responsive neurons. In agreement with these earlier studies, we have documented expression of neurotrophins in targets of the peripheral nervous system. Ourdata permit more pre-


cise localization of the sites of expression than was possible from earlier studies, particularly with regard to targets of the sensory nervous system. In addition, however, our results demonstrate that the situation is much more complex than previously recognized, because the same neurotrophins expressed within targets of innervation are also expressed by the in- nervating neurons.

The pattern of expression of BDNF and NT-3 mRNA in targets of sensory innervation differs markedly from that of NCF. Whereas NGF isactivelytranscribed mainly by the epithelial cells of skin and hair follicles (Davies et al., 1987; Wheeler and Bothwell, 1992), NT-3 and BDNF are transcribed mainly in the adjacent dermal mesenchymal cells. Perhaps this difference re- flects the differing sites of projections of various sensory neuronal subpopulations, and the identity of these subpopulations has not been determined. Skel- etal muscle receives innervation by proprioceptive sensory neurons, and it is possible that the expression of NT-3 by presumptive myoblasts provides trophic support to these sensory neurons during development. However, other possible functions for NT-3 expressed by myoblasts must also be considered, as discussed in detail below.

While the functional implications of expression of neurotrophins in innervated tissues are readily appar- ent, the function of neurotrophin expression within the neurons themselves is less clear. Expression of neurotrophins in CNS neurons has previously been interpreted according to the conventional neurotrophic model. For example, it has been suggested that hippocampal neuronsexpress neurotrophins because they receive axonal afferents from basal forebrain cholinergic neurons and other neuronal populations that are functionally regulated by neurotrophins (Ernfors et al., 1990). However, sympathetic neurons are innervated only by preganglionic sympathetic neurons of spinal cord, for which neurotrophin responsiveness has not been reported, and most motoneurons are innervated only by spinal cord interneur- ons, which are not known to be responsive to neurotrophins. The situation is even more clear for dorsal rootganglionicsensoryneurons,which receive little afferent axonal input and consequently cannot be providing trophic support to other neurons via axonal contacts on sensory neuronal soma. Clearly, for sensory neurons, the neurotrophins expressed must fill some unconventional role.

The timing of expression of the neurotrophins in sensory targets and in sensory neurons themselves may provide some clues about respective functional roles. Davies et al. (1987) demonstrated that NGF is not produced in maxillary pad targets of trigeminal sensory neurons until after axons reach their target, beginning at Ell. In agreement with these findings, we observe an increase in NGF mRNA levels in the maxillary pad from E11.5 until E14.5, and we find that NT-3 transcripts appear with a similar time course, but with a different distribution, being expressed in

dermal rather than epithelial components of the maxillary pad. In contrast, while BDNF resembles NT-3 in being expressed in dermal sites, BDNF transcripts reach maximal levels several days earlier (Ell.S), coin- cident with the arrival of the first sensory axons, and BDNF mRNA levels decline by E14.5. Thus, BDNF may be particularly important (relative to NGF and NT-3) in providing trophic support to sensory neurons during the earliest phases of target innervation. At E11.5 these neurons are already expressing p75 (our data) and trkA mRNAs (our data; Martin-Zanca et al., 1990; Klein et al., 1990b) and are therefore presumably capable of responding to neurotrophins. Sensory neurons of trigeminal ganglia do not begin expressing BDNF until E14.5, several days after BDNF transcripts appear in the maxillary pad, and expression within neurons in- creases subsequently while expression in the target decreases. In DRG neurons also, neurotrophin transcripts (for both BDNF and NT-3 in this case) do not appear in neurons until several days after expression of these neurotrophins commences in the target tissue.

The coexpression of neurotrophins and neurotrophin receptors in the same cell population is not without precedent. VkB and trkC transcripts are expressed in neurons of the pyramidal layer and dentate gyrus in the hippocampus(Klein et al., 1990a; Lamballe et al., 1991). The same population of neurons expresses BDNF and NT-3, the effecters of the trkB and trkC receptors (Phillips et al., 1990). Thus, it is possible that study of sensory neurons may illuminate principles that will apply to CNS neurons as well.

We can envision four possible explanations for the expression of neurotrophins by sensory neurons. The same principles may apply to sympathetic and motoneurons, as well as hippocampal neurons.

Autocrine Action It is possible that some neurons may provide their own trophic support by autostimulation. Obviously, if this pattern of action were effective and widespread, sensory neurons might not require exogenous neurotrophins as they are known to do. However, since neurotrophin expression by sensory neurons does not begin until after their axons contact neurotrophins produced by the target tissue, it is conceivable that autocrine expression of neurotrophins is induced by target-derived neurotrophins. A similar scenario is possible for sympathetic neurons, since the visceral targets of sympathetic ganglia are producing abundant amounts of transcripts for NGF, BDNF, and NT-3 at E11.5 (data not shown) and sympathetic ganglia express ~75, trkA, and trkB prior to the time of com- mencement of neurotrophin expression by these neurons. Since neurotrophins and neurotrophin receptors are expressed only within subpopulations of sensory neurons, the potential for autocrine action depends upon whether or not particular neurotrophins and neurotrophin receptors are coexpressed. For example, coexpression in a neuron of BDNF and trkB,

which is a BDNF receptor, would suggest an autocrine mode of action. However, it must be noted that cellular compartmentalization of the BDNF and trkB proteins might prevent autocrine response even if they are coexpressed. On the other hand, if BDNF is expressed in a subpopulation of neurons that expresses only trkA, an NGFINT3 receptor, then autocrine action is not possible. Generally speaking, we do not have sufficient data to determine whether individual neurons coexpress neurotrophins with cognate receptors. However, since trkA, an NT-3 receptor, is expressed in nearly all DRG neurons while NT-3 is expressed in a subpopulation of neurons, the oppor- tunity for autocrine regulation appears to exist.

Paracrine Action In the latter situation noted above, one might posit that BDNF released from the soma of one sensory neuron might provide trophic support to an adjacent neuron. Our results suggest that BDNF and NT-3 are expressed in different sensory neuronal subgroups. Thus, BDNF might be expressed in neurons with NT-3 receptors, while NT-3 is expressed in neurons with BDNF receptors.

Activity on Schwann Cells The two scenarios described above assume that neurons expressing neurotrophins release them from the cell soma, or from dendrites. However, there is no strong justification for making this assumption. Pro- teins released in vesicular form by epithelial cells can travel within cells by two pathways generally referred to as the constitutive pathway and the regulated pathway (Kelly, 1985). NGF is apparently released from epithelial cells by the regulated pathway (Edwards et al., 1988), and it is reasonable to expect that other neurotrophins also usethis pathway. The regulated pathway of epithelial and endocrine cells closely resembles the pathway for axonal release of neuropeptides and neurotransmitters (Burgess and Kelly, 1987). Conse- quently, proteins such as NGF and, by analogy, perhaps other neurotrophins are likely to be released from axonal sites when expressed in neurons.

If sensory, sympathetic, and motoneurons release neurotrophins from axonal sites, there are two possible targets of neurotrophin action: Schwann cells and target organs. During the period of embryonic development under consideration here, p75 is abundantly expressed on Schwann cells associated with the axons of sensory, sympathetic, and motoneurons (Schatte- man et al., 1988; Heuer et al., 1990). Furthermore, t&B mRNA was found in cells along axonal tracts, which appear to be Schwann cells (Klein et al., 1990b; Martin-Zanca et al., 1990). Consequently, Schwann cells may have functional receptors for BDNF and NT-3. Some previous studies have failed to note any response of Schwann cells to NGF, from which it was concluded that the p75 receptors fulfilled some other function (Taniuchi et al., 1988). However, p75 may be a component of BDNF and NT-3 receptors, as well

as NGF receptors. Seilheimer and Schachner (1987), employing relatively high concentrations of NGF, observed modest induction of the cell adhesion protein Ll, and these concentrations of NGF may be acting via BDNF receptors (Rodriguez-Tebar et al., 1990). Simi- larly, the observation of these investigators that polyclonal antibodies against NGF decreased the endoge- nous expression of Ll in cultured Schwann cells must be interpreted with caution in view of the recent discovery that polyclonal antibodies against NGF may cross-react with BDNF and NT-3 and the discovery that Schwann cells in culture secrete BDNF (Acheson et al., 1991). It seems possiblethat Ll expression in Schwann cells in culture may be promoted by endogenously produced BDNF acting upon trkB BDNF receptors. Thus the result of Seilheimer and Schachner (1987) is consistent with a possible role of axonally released BDNF (and/or NT-3) in regulating Ll-mediated axon/ Schwann cell adhesion.

Neuron to Target Signaling Vesicular release of proteins from axons occurs pre- dominantly at the axon terminus. Thus, it may be of considerable significance that p75 receptors are expressed abundantly on the embryonic target tissues of sensory, sympathetic, and motoneurons. As we and others have observed, p75 is expressed abundantly on the diverse terminal targets of sensory neurons. For example, the dermal mesenchymal cells express p75 transiently in development during the period of initial contact with sensory axons (Heuer et al., 1990), and there is abundant p75 expression on fibroblastic cells of the densely innervated adult rat tooth pulp, the capsular cells of adult Meissner and Pacianian sensory end organs, and the epithelial cells of taste buds (G. Schatteman, unpublished data). As described here and elsewhere, p75 is expressed abundantly on myoblasts, and the time of expression coincides with the period during which motor axons first contact the developing muscle mass. Similarly, p75 is expressed abundantly on embryonic mesenchymal cells of tissues receiving sympathetic innervation, such as vas- cular tissue (Schatteman et al., 1988; Heuer et al., 1990) and lung bronchioles (Wheeler and Bothwell, 1992). We have tentatively identified these p75-positive cells as smooth muscle precursor cells, and as for skeletal muscle myoblasts, the period of p75 expression coincides both with the period of muscle cell differentiation and with the period of initial sympathetic axonal contact.

Thus, we are prompted to consider whether neurotrophins might be released from sensory, sympathetic, and motoneurons as a mechanism for regulating the differentiation of the target tissue. While p75 may be an important component of neurotrophin receptors, current models indicate that functional responses of cells cannot occur unless trk-type proteins are also expressed. Previous studies describing localization of trkAand trkB concluded that expression was entirely restricted to neural tissue. However, for trkB,

Neuron 462

conditions are sufficiently stringent, unlabeled NGF and NT-3 should not hybridize to BDNF mRNA in the sample. When all three factors were tested in this manner, no evidence of cross- hybridization was found. Adjacent sections of embryos demonstrated specific labeling in different cells with the different probes. Also, specific labeling of each radioactive probe was displaced with a lOOO-fold excess of the same unlabeled antisense message. Radiolabeled sense probes were used as a general measure of tissue background in adjacent or closely adjacent sections.

Acknowledgments

We wish to thank Phyllis Harbor for excellent technical assis- tance. We are grateful to Drs. Karen Deyerle, Gina Schatteman, and Christopher Von Bartheld for helpful discussion of the data and critically reading the manuscript. We thank Drs. Susan Mea- kin and Eric Shooter for providing the t&A cDNA clone and Dr. David Middlemas for providing us with the trkB cDNA clone.

This research was supported by National Institutes of Health grants NS-23343 and NS-07332.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertiserned in accordance with 18 USC Sec- tion 1734 solely to indicate this fact.

Received November 26, 1991; revised June 16, 1992.

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this conclusion was arrived at by defining “neural tissue” in an exceedingly general way to include mesenchymal tissues through which nerves course, including cranial mesenchyme and the wall of the dorsal aorta (Klein et al., 1990b). Our preliminary results (data not shown) demonstrate extensive expression of trkl3 mRNA in the mesenchymeof theembryonic maxillary pad and tongue as well as in other nonneural tissues. Our results are still fragmentary, as we have not care- fully examined the coexpression of p75 and trk mRNAs in nonneuronal tissues, and we have not yet characterized the localization of trkC. Also, the riboprobe used in these experiments codes for an extra- cellular region of the trkB protein, and therefore trkB transcripts present in nonneural tissues may not code for receptors containing the kinase domain or receptors capable of transducing a signal. Nevertheless, it seems likely that at least some terminal targets of the peripheral nervous system express the necessary components of functional neurotrophin receptors. There is extensive literature documenting the trophic effects of sensory, sympathetic, and motor innervation on terminal targets, and it is interesting to consider that neurotrophins might play some role in such neuron to target trophic interactions. Because of the potential importance of such postulated roles of neu- ronally derived neurotrophins on Schwann cells and on innervated tissues, it is essential that the site of storage and release of neurotrophin proteins in neurons be characterized in future studies.

Additionally, proper interpretation of the phenom- ena we have described requires a detailed under- standing of the selectivity of particular neurotrophin receptors for various neurotrophins. Studies implicat- ing trkA as a receptor for NGF and NT-3, trkB as a receptor for BDNF and NT-3, and trkC as a receptor for NT-3 have been performed with these receptors transfected into fibroblasts. The possibility remains that cellular environment may influence the neurotrophin specificity of the trk proteins, in which case results obtained in fibroblasts may not be strictly ap- plicable to neurons. That this might be the case is suggested by the finding that sympathetic neurons express trkB, a putative BDNF receptor (Squintoet al., 1991; our results), yet BDNF does not promote the survival of sympathetic neurons (Lindsay and Rohrer, 1985).

Experimental Procedures

Animals Embryos were obtained from time-mated BALB/c mice. The morning of the vaginal plug is considered E0.5. Pregnant mice were sacrificed by cervical dislocation, the uterus was removed, and embryos were dissected. Embryos were fixed in 10% forma- lin overnight at room temperature, placed in 50% ethanol at 4OC overnight, and then placed in 70% ethanol at 4OC until processing. Tissues were processed, embedded in paraffin, sec- tioned at 8 pm, and placed on silane-coated slides.

Probes Mouse NCF, BDNF, and NT-3 cDNAs wereobtained by PCRusing mouse genomic DNA as template, a 3’ oligonucleotide directed

against a sequence conserved among the three factors (WIRID), and factor-specific 5’sequences: VPEAHW for NCF, VNCPRA for BDNF,and DSLNSLfor NT-3.The3’primercontainedarestriction siteforEcoRI,andtheNCFandBDNFprimerscontained arestric- tion site for Hindlll. The mouse NT-3 contains a Hindlll site 53 bases downstream of the primer sequence. Genomic DNA was used preferentially over cDNA prepared from mRNA, since the neurotrophins are not abundantly expressed in many cells and theentirecodingsequenceofthepropeptideiscontained within one exon. cDNAs obtained by PCR were digested with EcoRl and Hindlll, cloned into the plasmid pGEM 32, and then sequenced to confirm their identity as the mouse neurotrophic factors. A 550 bp mouse p75 cDNA was obtained through PCR using oligo- nocleotides directed against 5’ and 3’ conserved sequences, HSGECC and ATSPV, respectively, and pooled E16, E18, and P2 mouse brain cDNA libraries prepared by Dr. A. Lanahan (Johns Hopkins University, Baltimore, MD). The resulting PCR product was approximately 1.2 kb. The oligonucleotide primers contained a Sal1 site, and cDNAs were cloned into pGEM 32. The mouse cDNA contains an internal Sall site; therefore, a clone containing 550 bp of 3’coding sequence was obtained. Plasmids encoding a 600 bp rat trkA cDNA in vectors suitable for transcribing antisense and sense RNA were kindly provided by Dr. E. Shooter (Stanford University, Stanford, CA). A full-length cDNA encoding frkBwas kindly provided by Dr. D. Middlemas (Univer- sityof California, LaJolla, CA).A306 bp Pst-Hindlll fragmentwas removed and subcloned into pGEM 32. This probe is capable of recognizing several species of trkt3 mRNA described by others (Klein et al., 1991; Middlemas et al., 1991).

The plasmid pCEM 32 contains promoter sequences for SP6 and T7 polymerases. Plasmids containing either NGF, BDNF, or NT-3 cDNAs were linearized with Hindlll, and [35S]UTP labeled single-stranded RNA probes synthesized using T7 polymerase. Sense probes used as controls for background hybridization were made by linearizing plasmids with EcoRl and transcribing with SP6 enzyme. Plasmid containing the p75 cDNA was linearized with EcoRl and transcribed with SP6 polymerase to synthesize antisense probe and linearized with Pst and transcribed with T7forsenseprobe.TwoplasmidscontainingtrkAwereobtained, with the 600 bp t&A cDNA in opposite orientations in plasmids containing the SP6 polymerase promoter site. Each plasmid was linearized with Xhol, and [35S]UTP labeled single-stranded RNA probes synthesized. trkB pCEM 32 was linearized with Hindlll and transcribed with SP6 to synthesize antisense probe and linearized with Pstl and transcribed with T7 to make sense probe. After the labeling reaction, all probes underwent alkaline hydro- lysis to an approximate length of 150 nt.

In Situ iiybridization The in situ hybridization procedure employed in our laboratory is the method of Cox et al. (1984) as modified by Heuer et al. (1990). Paraffin-embedded tissue sections were hybridized with radiolabeled antisense or sense probes at 50°C for 16 hr. Follow- ing hybridization, the sections were treated with RNAase A to digest the unhybridized, single-stranded probe and coated with emulsion for autoradiography. The length of exposure for the emulsion-coated slides is typically 3-4 weeks for the p75 and trk probes and 5-6 weeks for the neurotrophic factor probes. After slides were developed the tissuewas counterstained with methyl green.

The neurotrophic factors NGF, BDNF, and NT-3 contain several regions of identity in the amino acid sequence as well as the nucleotide sequence. Therefore, it was neccessary to establish that hybridization conditions were sufficiently stringent such that no cross-hybridization was being detected among the NCF, BDNF, and NT-3 mRNAs. Specificity was determined bycompeti- tion experiments. A lOO@fold greater concentration of a cRNA encoding a factor different from the radiolabeled cRNA was added to the hybridization mix. For example, radiolabeled BDNF antisense RNA was hybridized to tissue sections in the presence or absence of a lOO@fold greater concentration of unlabeled NCF or NT-3 antisense RNA. The result should be identical to the result obtained with labeled BDNF alone. If the hybridization


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Novel roles for neurotrophins are suggested by BDNF and NT3 mRNA expression in developing neurons

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