Figure 8.

BMP-2 and BMP-4 promote the expression of TrkC and the acquisition of dependence on NT-3 for survival. A, TrkC-immunoreactive neurons expressed as a percentage of total cells per culture exposed only to vehicle (121.9 ± 34.7 × 10³; 3 cultures). Crest-derived cells were immunoselected from the bowel at E12 and maintained invitrofor 4-5 d. TrkC expression was determined immunocytochemically. BMP-2 and BMP-4 (20 ng/ml) increase the proportion of TrkC-expressing neurons in the cultures, but NT-3 (40 ng/ml) does not. B, Crest-derived cells were immunoselected from the bowel as in A, but at E14, and maintained similarly. Data were normalized to the total number of cells per culture exposed only to vehicle (287.9 ± 32.5 × 10³; 13 cultures). NT-3 increases the proportion of TrkC-expressing neurons; this increase is significantly greater when NT-3 is combined with BMP-2 or BMP-4. Note that the BMP-2- or BMP-4-associated increases in TrkC expression are greater at E12 (A) than at E14. C, Withdrawal of NT-3. Crest-derived cells were obtained at E14 (as in B) and cultured for an initial period of 4 d. They were then maintained for an additional 18 hr with or without NT-3. Data were normalized to the total number of TrkC-immunoreactive neurons in the cultures in each of the indicated conditions. (The total of TrkC-immunoreactive neurons per culture in these experiments ranged from 5.9 × 10³ to 28.0 × 10³.) When NT-3, rather than vehicle, was present throughout incubation, TrkC-expressing neurons were significantly protected from apoptosis (†); moreover, the proportion of TrkC-expressing neurons exhibiting apoptosis was significantly greater when NT-3 was withdrawn (-) during the follow-up period (^*). The inclusion of NT-3 with BMP-2 or BMP-4 decreased the proportion of TrkC-expressing neurons exhibiting apoptosis but enhanced the magnitude of apoptosis that accompanied the 18 hr withdrawal of NT-3 (^*). D, Rescue from apoptosis by NT-3. Crest-derived cells were obtained at E14 (as in C) and cultured for an initial period of 2 d. They were then maintained for an additional 2 d with or without NT-3. Data were normalized to the total number of TrkC-immunoreactive neurons in the cultures in each of the indicated conditions. The total of TrkC-immunoreactive neurons per culture ranged from 2.9 × 10³ to 14.5 ± 1.2 × 10³. The presence of BMP-2 or BMP-4 during the initial period of incubation increased the proportion of TrkC-expressing neurons undergoing apoptos is during a subsequent period of incubation in the absence of NT-3. This increase was prevented when NT-3 was present during the follow-up period of incubation (^*). ns, Not significant.