NT-3 Is Expressed in nn Cells Surrounding Sympathetic Ganglia
To determine the site of NT-3 expression relative to sympathetic ganglia in vivo, we examined sections of fetal mice harboring a lacZ
gene inserted into the NT-3
locus by homologous recombination (Fariñas et al., 1994
). The expression pattern of β-galactosidase in these mice reflects the expression of endogenous NT-3 (I. F., unpublished data). Strikingly, β-galactosidase-positive cells in these mice surrounded but excluded sympathetic ganglia primordia as revealed by counter-staining with antibody to peripherin (, arrows). The distribution of the β-galactosidase positive cells at E13.5 overlapped that of cells expressing p75LNGFR
, a marker of peripheral glia and their neural crest progenitors (Stemple and Anderson, 1992
) (, arrow). p75+
nn cells isolated from rat E14.5 sympathetic ganglia by preparative flow cytometry were labeled by an antibody to NT-3 (see Experimental Procedures; ). The expression of NT-3
mRNA in these cells was independently confirmed by reverse transcription–polymerase chain reaction (RT–PCR) analysis, which also indicated that NT-3
mRNA was undetectable in neuroblasts isolated with monoclonal antibody B2 (see below; data not shown). Thus, both the antibody staining and the mRNA analysis confirm that the pattern of β-galactosidase expression seen in sections likely reflects that of endogenous NT-3 and demonstrates that this neurotrophin is not expressed by ganglionic neuroblasts at this stage, but rather by surrounding nn cells, at least some of which are p75+
Expression of NT-3 in Sympathetic Ganglionic Nonneuronal Cells In Vivo
Expression of NT-3 in Isolated p75+ Ganglionic Nonneuronal Cells
Growth Factors Enhance NT-3 mRNA Expression and NT-3-Mediated Bioactivities in Cultured Nonneuronal Cells
We next examined the regulation of NT-3 production in nn cells isolated from ganglia by negative selection with monoclonal antibody (MAb) B2 (Birren et al., 1993
). The B2−
cell population excludes the sympathetic neuroblasts and contains the p75+
population as well as other nonneuronal cells (see Experimental Procedures). Because of the larger cell numbers obtained, it was more convenient to work with B2−
cells than p75+
cells as a source of immunoisolated nn cells. These nonneuronal cells were cultured in an insulin-free, serum-free defined medium in the presence of various growth factors, including epidermal growth factor (EGF), insulin-like growth factor (IGF)-1 and IGF-2, fibroblast growth factor (FGF), NGF, brain-derived neurotrophic factor (BDNF), retinoic acid, and members of the transforming growth factor-β (TGFβ) superfamily. Of these factors, CNTF, rhGGF2, and PDGF all up-regulated of NT-3
mRNA by greater than 5-fold after 24 hr (). No significant effect was observed with IGF-1, NGF (), or the other factors tested (data not shown). By 48 hr, NT-3
mRNA levels were up-regulated over 40-fold (normalized to actin) in rhGGF2- or CNTF-treated cultures (). Cell number was also dramatically increased in rhGGF2-treated cultures (not shown); however, the normalization to actin indicates that there is a significant induction of NT-3 on a per cell basis, independent of proliferation.
Relative Levels of NT-3 mRNA in Cultures of B2− Ganglionic Nonneuronal Cells Treated with Various Factors
We next asked whether the increase in NT-3
mRNA caused by growth factor treatment was correlated with an enhanced secretion of functional NT-3, as measured by bioassays using conditioned medium (CM) from the nn cells. It has been demonstrated previously that recombinant NT-3 has at least two biological activities on isolated B2+
sympathetic neuroblasts: at low concentrations (0.3–1.0 ng/ml), it can support their survival; at higher concentrations (30–50 ng/ml), it can promote mitotic arrest and consequent induction of TrkA (Verdi and Anderson, 1994
). We therefore asked whether these bioactivities were present in nn cell CM and if so whether they could be inhibited by function-blocking antibodies to NT-3 (design illustrated in ).
Schematic Diagram Illustrating Design of Conditioned Medium Experiments
We first assayed survival in cultures of isolated E14.5 B2+
cells. CM from untreated B2−
nn cells enhanced neuroblast survival 2-fold (, top, no addition, versus , bottom, CM [untreated B2−
cells]). By 48 hr, the percentage of surviving neurons in the presence of CM was at least double that seen in controls (; see ). This survival-enhancing effect was abolished by adding a function-blocking anti-NT-3 antibody (Ghosh et al., 1994
; Ghosh and Greenberg, 1995
) to the CM (compare , bottom, CM [B2−
cells] plus anti-NT-3 with , top, no addition). The anti-NT-3 antibody had no effect on neuroblast survival in basal medium, nor did it reduce neuroblast survival in the presence of insulin (, top), another survival factor for sympathetic neuroblasts (DiCicco-Bloom and Black, 1988
; DiCicco-Bloom et al., 1993
; Zackenfels et al., 1995
). These biological controls indicated that the anti-NT-3 antibody does not inhibit neuroblast survival nonspecifically. These data imply that nn cells constitutively secrete some NT-3, which is able to enhance neuroblast survival. This conclusion is consistent with the results of the RT–PCR and immunostaining analyses, in which NT-3
mRNA was clearly detectable in untreated nn cells (; see ).
Neuroblast Survival at 48 hr Promoted by Growth Factors and nn Cell–Conditioned Media
Neuroblast Enhancement of Secretion of NT-3-Mediated Neurotrophic Activity from Nonneuronal Cells Is Dependent upon Endogenous Neuregulins
rhGGF2, CNTF, and PDGF all enhanced the neuroblast survival-promoting activity of B2− nn cell–CM (prepared from 24 hr cultures) by, on average, ~50% (, bottom). Importantly, these factors had no neuroblast survival–promoting activity on their own (, top). In general, rhGGF2 was most effective, consistent with the RT–PCR data indicating that this growth factor produced the strongest up-regulation of NT-3 mRNA in the nn cells (see ). Importantly, all the survival-enhancing activity of these growth factor–treated CMs could be blocked by the anti-NT-3 antibody (, bottom, CM [B2− cells plus GGF] plus anti-NT-3; data not shown). These data suggest that rhGGF2, PDGF, and CNTF all enhance neuroblast survival indirectly, by increasing the secretion of NT-3 from nn cells. The apparent difference between the extent of NT-3 mRNA induction by GGF at 24 hr (about 6-fold; ), and the extent of the increase in survival-promoting activity with GGF-treated CM (50%) likely reflects the fact that the amount of additional NT-3 in the CM is saturating for the survival assay, as indicated by the fact that equivalent survival was obtained with this CM as with saturating amounts of recombinant NT-3 (). Alternatively, it could reflect posttranscriptional regulation of NT-3 synthesis or secretion.
In these experiments, we consistently observed a population of neuroblasts that survived independently of NT-3. In two experiments, this basal NT-3-independent survival was approximately 50% at 24 hr and 23% at 48 hr (, top, experiments I and II, no addition; data not shown). In a third experiment, in which the neuroblast plating density was reduced by a factor of 3, basal survival was 32% at 24 hr and 15% at 48 hr (, top, experiment III, no addition; data not shown). These data are consistent with the observation of a population of NT-3-independent sympathetic neurons in NT-3 −/−
mice (Ernfors et al., 1994
; Fariñas et al., 1994
). The size of this NT-3-independent population in the superior cervical ganglion in vivo is approximately 50% of the total ganglionic neuronal population. These data suggest that in vitro as in vivo, some embryonic sympathetic neuroblasts do not require NT-3 for survival; however, the size of the NT-3-independent population may still be affected by environmental factors or cell–cell interactions.
We next determined whether nn cell CM could induce TrkA expression in isolated neuroblasts. CM from B2−
nn cells pretreated with rhGGF2 or PDGF up-regulated trkA
mRNA expression in isolated B2+
neuroblasts (, right; compare GGF, PDGF, with no add). This CM activity was attenuated by a function-blocking anti-NT-3 antibody (, GGF plus NT-3 AB, PDGF plus NT-3 AB). The anti-NT-3 antibody did not affect the TrkA-inducing activity of CM from cells exposed to CNTF (which has TrkA-inducing activity on its own [Verdi and Anderson, 1994
]); this demonstrates that the antibody does not interfere nonspecifically with TrkA induction. As in the survival assay, rhGGF2 had no effect on TrkA expression when added directly to isolated neuroblasts (, left, GGF). These data suggest that rhGGF2 and PDGF promote secretion of sufficient NT-3 from nonneuronal cells to induce trkA expression in neuroblasts. In contrast, CM from nn cells grown without these growth factors had little measurable effect on TrkA expression (, right, compare no add with no add plus NT-3 AB). The enhancement of neuroblast survival (), but not TrkA expression, by untreated nn cell CM most likely reflects the fact that higher levels of NT-3 are required to induce TrkA expression than to promote survival (Verdi and Anderson, 1994
Conditioned Medium from B2− Nonneuronal Cell Cultures Induces TrkA Expression in Cultures of Isolated Sympathetic Neuroblasts, via NT-3
Sympathetic Neuroblasts Contain mRNA for Various Neuregulin Isoforms
The observation that growth factors such as GGF2/neuregulin and PDGF can up-regulate NT-3
mRNA expression and associated bioactivities in ganglionic nn cells raised the question of whether sympathetic neuroblasts could be a source of such NT-3-inducing factors. It has previously been demonstrated that a number of different CNS and PNS neuronal populations, including embryonic sympathetic neurons, make PDGF (Yeh et al., 1991
). Neuregulins have also been demonstrated to be expressed in the developing nervous system (Marchionni et al., 1993
; Meyer and Birchmeier, 1994
; Corfas et al., 1995
), but their expression in sympathetic neuroblasts at these stages has not specifically been examined.
We therefore assayed neuregulin mRNA production in isolated E14.5 B2+ neuroblasts by RT–PCR. Using exon I-specific primers that identify mRNA encoding GGF2 (the form used in the cell culture experiments), strong signals were detected in E14.5 and E15.5 B2+ cells, but were lower in E16.5 and E17.5 cells (). To determine whether other neuregulin isoforms besides GGF2 were expressed in these cells, we performed RT–PCR with several different primer combinations on mRNA isolated from E14.5 neuroblasts. Signals were detected by use of primers from the immunoglobulin and EGF domains, as well as from the EGF and cytoplasmic domains (). These data, although qualitative, indicate that various forms of neuregulins including membrane-bound forms are synthesized by embryonic sympathetic neuroblasts. Importantly, little or no neuregulin mRNA was amplified from E14.5 B2− nn cell cDNA (data not shown).
Relative Levels of GGF2/Neuregulin in Freshly Isolated B2+ Sympathetic Neuroblasts
Expression of Neuregulin Isoforms by Isolated E14.5 B2+ Sympathetic Neuroblasts
Endogenous Neuregulins Enhance Neuroblast Survival in Cocultures with Nonneuronal Cells
The observations that sympathetic neuroblasts are a potential source of neuregulins, and that neuregulins enhance NT-3 production in nonneuronal cells, suggested that neuroblast-derived neuregulins might enhance neuroblast survival in cocultures with nonneuronal cells. To test this idea, we recombined isolated B2+ neuroblasts together with B2− nonneuronal cells in a ratio (1:10) reflecting their relative proportions in freshly sorted cell suspensions, and monitored neuroblast survival. To exclude possible contributions of de novo neurogenesis to overall neuronal number, cohorts of individual neuroblasts were identified several hours after plating and their survival followed with time.
Overall neuroblast survival in the cocultures at 24 hr was 77% ± 4.9% (), much better than the 45%–50% observed when B2+ neuroblasts are cultured in isolation at the same density (see above). Inclusion of anti-NT-3 antibodies in the cocultures inhibited survival of a portion of the neuroblasts (, anti-NT-3). Importantly, function-blocking antibodies to GGF2/neuregulin (see Experimental Procedures) also inhibited neuroblast survival (). However, at 48 hr, the reduction in survival with anti-GGF was not as great as with anti-NT-3. This finding is consistent with the observation that nn cells make some NT-3 even in the absence of sources of GGF/neuregulin (see above).
Survival of Serially Observed Cohorts of B2+ Cells in Coculture with B2− Nonneural Cells under Various Conditions
These data suggested that endogenous sources of neuregulin can enhance neuroblast survival in cocultures containing nonneuronal cells. We therefore next asked whether neuroblasts enhance NT-3 synthesis in nonneuronal cells via neuregulins.
Neuroblasts Up-regulate NT-3 mRNA Expression in Nonneuronal Cells via Neuregulins
B2+ neuroblasts were recombined with B2− nonneuronal cells in a 1:10 ratio, and the expression of NT-3 mRNA in these cocultures was subsequently measured by RT–PCR. After 48 hr, NT-3 mRNA levels were 13-fold higher in such cocultures than in sister cultures of nn cells grown alone (see , B2+ neuroblasts). To determine whether the NT-3 mRNA produced in these cocultures was derived from the nn cells, the neuroblasts were removed from the nn cells by use of EDTA (see Experimental Procedures), and both cell populations were assayed for NT-3 mRNA by RT–PCR. A strong NT-3 signal was detected in the nn cell cDNA, whereas no signal was detected in similar amounts of neuroblast cDNA amplified for the same number of cycles (data not shown). These data are consistent with our finding that NT-3 mRNA is detectable by RT–PCR in freshly isolated B2− but not B2+ cells from E14.5 ganglia (see above). Thus, these coculture experiments suggest that B2+ neuroblasts, as well as rhGGF2, PDGF, and CNTF, can up-regulate NT-3 mRNA production by B2− nn cells.
To determine whether the effect of B2+ neuroblasts in up-regulating NT-3 mRNA production in nn cells was mediated by endogenous neuregulins, we examined NT-3 mRNA levels in cocultures grown with or without the function-blocking anti-GGF2/neuregulin antibody. In control experiments, the antibody strongly attenuated the up-regulation of NT-3 mRNA by rhGGF2, but had no effect on NT-3 mRNA up-regulation by PDGF (), demonstrating the specificity of the inhibition. Anti-GGF2/neuregulin also attenuated the up-regulation of NT-3 mRNA caused by coculture with E14.5 B2+ neuroblasts (, compare B2+ neurons with B2+ neurons plus GGF AB). This indicated that the neuroblast enhancement of NT-3 mRNA production in nonneuronal cells is, at least in part, mediated by neuregulins. The failure of the antibody to produce a quantitative inhibition of either the neuroblast- or rhGGF2-mediated effect () suggests that the antibody may not have been saturating, or that not all neuregulin isoforms are blocked; alternatively, the neuroblasts may produce other NT-3-inducing signals (e.g., PDGF) not inhibited by the antibody.
Induction of NT-3 mRNA in Nonneuronal Cells by Coculture with Neuroblasts Is Attenuated by Neutralizing Anti-GGF Antibody
Neuroblast-Derived Neuregulins Promote Up-regulation of NT-3-Mediated Bioactivities Secreted by Nonneuronal Cells
We next sought to determine whether the neuroblast enhancement of NT-3 mRNA expression in nn cells was accompanied by an increased secretion of NT-3-dependent bioactivities. Therefore, both TrkA induction and neuroblast survival assays were performed on neuroblasts cultured in CMs derived from untreated B2− nn cells, or from cocultures of B2− nn cells and neuroblasts (design illustrated in ). As shown in , conditioned medium from cocultures up-regulated trkA expression in isolated B2+ neuroblasts, whereas little effect on TrkA expression was observed with CM from nn cells alone (see , compare B2+ neurons with no add). The effect of the coculture CM was attenuated by anti-NT-3 antibody (see , B2+ neurons plus NT-3 AB). This suggests that most or all of the TrkA-inducing activity in CM derived from cocultures of neuroblasts and nonneuronal cells is due to NT-3.
CM from cocultures also enhanced neuroblast survival more effectively than did CM from untreated nn cells (, CM [untreated B2− cells] versus CM [B2− plus B2+ cells, 10:1]; experiments I and III). In one of three independent experiments, coculture and untreated nn cell CM enhanced survival to a similar extent (, experiment II), but the nn cell CM had a higher level of survival-promoting activity to begin with than it did in the other experiments (, row 2). Importantly, all the survival-promoting activity of the coculture CM was inhibited by the anti-NT-3 antibody (, CM [B2− plus B2+] plus anti-NT-3). Taken together, these data indicate that neuroblasts not only enhance the expression of NT-3 mRNA in nn cells, but also promote the secretion of NT-3-dependent bioactivities by these cells.
Finally, we asked whether the anti-rhGGF2/neuregulin antibody eliminated the enhanced secretion of survival-promoting activity by cocultures of neuroblasts and nonneuronal cells. For this experiment, cocultures of B2− and B2+ cells were allowed to condition their medium in the presence or absence of anti-rhGGF2 antibody; the survival activity of CM from these cultures was then compared with that of CM from untreated nn cell cultures (design illustrated in ). In three independent experiments, pretreatment with the anti-GGF/neuregulin antibody attenuated the survival-promoting activity of the coculture-derived CM (, compare CM [B2− plus B2+ plus anti-GGF] with CM [B2− plus B2+ cells, 10:1]). Moreover, in two of three experiments, the anti-GGF antibody reduced the survival-promoting activity of the coculture CM to almost exactly that seen with CM from untreated nn cell cultures (, experiments I and III; compare CM [untreated B2− cells] with CM [B2− plus B2+ plus anti-GGF]). Control experiments indicated that the antibody effect was overcome by addition of excess rhGGF2 (, CM [B2− plus B2+ plus anti-GGF plus GGF]). Additional controls showed that the anti-GGF2/neuregulin antibody had no effect on neuroblasts grown in basal medium, or in medium containing insulin (provided to enhance survival by an NT-3-independent means) (data not shown). Taken together, these data indicated that neuroblast-derived neuregulin not only promotes NT-3 mRNA synthesis in nn cells, but also increases secretion of NT-3-dependent neuroblast survival activity from the nn cells.