A reciprocal cell-cell interaction mediated by NT-3 and neuregulins controls the early survival and development of sympathetic neuroblasts - PubMed (original) (raw)

A reciprocal cell-cell interaction mediated by NT-3 and neuregulins controls the early survival and development of sympathetic neuroblasts

J M Verdi et al. Neuron. 1996 Mar.

Abstract

Neurotrophin 3 (NT-3) can support the survival of some embryonic sympathetic neuroblasts before they become nerve growth factor dependent. We show that NT-3 is produced in vivo by nonneuronal cells neighboring embryonic sympathetic ganglia. NT-3 mRNA is produced by these nonneuronal cells in vitro and is up-regulated by platelet-derived growth factor, ciliary neurotrophic factor, and glial growth factor 2 (a neuregulin). Nonneuronal cell-conditioned medium promotes survival and induces TrkA expression in isolated sympathetic neuroblasts, and this activity is blocked by anti-NT-3 antibody. Neuroblasts also enhance NT-3 production by nonneural cells. Neuroblasts synthesize several forms of neuregulin, and antibodies to neuregulin attenuate the effect of the neuroblasts on the nonneuronal cells. These data suggest a reciprocal cell-cell interaction, in which neuroblast-derived neuregulins promote NT-3 production by neighboring nonneuronal cells, which in turn promotes neuroblast survival and further differentiation.

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Figures

Figure 1

Figure 1

Schematics Showing the Switch in Neurotrophin Responsiveness by Embryonic Rat Sympathetic Neuroblasts, and the Regulatory Circuits Underlying the Switch (A) The switch in neurotrophin responsiveness by embryonic rat sympathetic neuroblasts (Birren et al., 1993; DiCicco-Bloom et al., 1993). (B) The regulatory circuits underlying the switch. The induction of TrkA expression in neuroblasts exposed to NT-3 or CNTF appears to be primarily a consequence of mitotic arrest (Verdi and Anderson, 1994). In vitro, exposure of TrkA-expressing neuroblasts to NGF results in both an induction of p75 expression (Wyatt and Davies, 1993; Verdi and Anderson, 1994) and a down-regulation of TrkC expression (Verdi et al., 1994b).

Figure 2

Figure 2

Expression of NT-3 in Sympathetic Ganglionic Nonneuronal Cells In Vivo Transverse sections through the thoracic region of E13.5 mouse embryos containing two copies of the lacZ gene inserted into the NT-3 locus by homologous recombination. In (A), the sections are counterstained with antibody to peripherin; arrows indicate the sympathetic ganglia. In (B), the sections are counterstained with antibody to p75, which reveals nonneuronal cells adjacent to the sympathetic ganglion. Ao, dorsal aorta; sg, sympathetic ganglion.

Figure 3

Figure 3

Expression of NT-3 in Isolated p75+ Ganglionic Nonneuronal Cells Rat E14.5 thoracic sympathetic ganglia were dissociated and sorted by use of anti-p75 monoclonal antibody. The isolated p75+ cells were plated overnight, fixed, and stained with rabbit anti-NT-3 antibody (see Experimental Procedures) at a 1:1000 dilution (A and B) or preimmune serum at the same dilution as a control (C and D). (A) and (C) are phase contrast micrographs of the bright-field images in (B) and (D), respectively.

Figure 4

Figure 4

Schematic Diagram Illustrating Design of Conditioned Medium Experiments Survival or TrkA expression by isolated B2+ neuroblasts is measured after culture in the presence of specific growth factors (A), or in conditioned media (CM) derived from B2– nonneuronal cells cultured either alone (B) or in the presence of neuroblasts (C). The CMs are prepared in the presence or absence of various growth factors, or in the presence or absence of blocking antibodies to GGF/neuregulin (C). The CMs are applied to the isolated neuroblasts in the presence or absence of blocking antibodies to NT-3. The survival or expression of TrkA is measured in the isolated neuroblast preparations.

Figure 5

Figure 5

Conditioned Medium from B2− Nonneuronal Cell Cultures Induces TrkA Expression in Cultures of Isolated Sympathetic Neuroblasts, via NT-3 The left of the graph (factor added directly) shows a series of controls in which factors or antibodies were added directly to isolated B2+ neuroblasts. Note that NT-3 induces trkA mRNA, and that the neutralizing anti-NT-3 antibody blocks this effect. In the right of the graph, expression of TrkA was monitored in cultures of isolated B2+ neuroblasts grown for 24 hr in various conditioned media from nonneuronal cultures. No add means that the conditioned medium was prepared from nn cultures grown with no added factors. Note that GGF strongly enhances the TrkA-inducing activity of nn cell CM, although by itself, GGF has no direct effect on TrkA expression in isolated neuroblasts (left side of the graph). The TrkA-inducing activity of the GGF-treated CM is strongly attenuated by anti-NT-3 antibody. A similar result is obtained with PDGF. The effect of CNTF is, as expected, not blocked by anti-NT-3, because CNTF itself has strong TrkA-inducing activity (Verdi and Anderson, 1994). B2+ neurons indicates that conditioned medium was prepared from a coculture of B2− nonneuronal cells and B2+ neurons recombined in a 10:1 ratio (see Figure 4C). Note that this CM has much higher TrkA-inducing activity than control CM (no add) and that this activity is again attenuated by the blocking anti-NT-3 antibody. The relative levels of trkA mRNA were determined by RT–PCR and are presented as the percentage of trkA mRNA detected in PC12 cell mRNA samples run in parallel; the numbers are the mean ± SEM of replicate samples. Similar results were obtained in two different experiments from separate isolations.

Figure 6

Figure 6

Relative Levels of GGF2/Neuregulin in Freshly Isolated B2+ Sympathetic Neuroblasts cDNA prepared from cells isolated at the indicated ages was amplified with exon 1–specific primers that detect the kringle domain of the GGF2 isoform (see Table 3). The values represent the mean ± SEM of two independent experiments, relative to the level detected at E17.5 after normalization to actin. Levels of GGF2 mRNA at E19.5 were below the detection limit of the assay. Note that GGF2 mRNA was not detectable in samples of B2− nonneuronal cell cDNA run in parallel. Qualitative results obtained with other neuregulin primer sets are presented in Table 3.

Figure 7

Figure 7

Induction of NT-3 mRNA in Nonneuronal Cells by Coculture with Neuroblasts Is Attenuated by Neutralizing Anti-GGF Antibody Relative NT-3 mRNA levels in cultures of B2− nonneuronal cells grown under different conditions for 24 hr were determined by RT–PCR (see Table 1) and normalized to actin mRNA levels. rhGGF2 strongly up-regulates NT-3 (see also Table 1), and this effect is attenuated by anti-GGF antibody. By contrast, the up-regulation of NT-3 obtained with PDGF is unaffected by the anti-GGF antibody. NT-3 mRNA levels are also up-regulated by coculture with B2+ neuroblasts, and this effect is also attenuated by the anti-GGF antibody. The data represent the average ± SEM of two independent experiments.

Figure 8

Figure 8

Summary of Experimental Observations and Model for Reciprocal Cell–Cell Interactions Controlling Early Stages in Sympathetic Gangliogenesis (A, top) Illustration of the data indicating first of all that NT-3 promotes neuroblast survival and mitotic arrest/TrkA expression (see also Verdi and Anderson, 1994); second, that B2− ganglionic nonneuronal cells also promote neuroblast survival and TrkA expression (and express NT-3); and third, that blocking anti-NT-3 antibodies inhibit the survival-promoting and TrkA-inducing activities in conditioned medium from nonneuronal cells. (A, bottom) Illustration of the data indicating first of all that GGF and PDGF (as well as CNTF) up-regulate NT-3 expression in nonneuronal cells; second, that neuroblasts also up-regulate NT-3 expression in non-neuronal cells (and themselves express forms of GGF/neuregulin as well as PDGF [Yeh et al., 1991]); and third, that blocking anti-GGF antibodies inhibit the ability of neuroblasts to up-regulate NT-3 expression in nonneuronal cells. (B) Reciprocal cell–cell interactions that occur in vitro between neuroblasts and nonneuronal cells as suggested by the data.

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