TrkC-Positive DRG Neurons Are Rescued in Bax/NT-3 Double Knockout Mice
Mice lacking proapoptotic protein Bax allow for distinguishing survival effects of neurotrophins from other effects. Bax-deficient sensory neurons no longer require neurotrophins for survival (White et al. 1998; Patel et al. 2000), thus they can be used to examine axonal effects. We bred NT-3 heterozygote and Bax knockout (KO) mice to obtain mice with double KO of both NT-3 and Bax genes, and examined proprioceptive axonal projections. All NT-3 and double KOs died within 48 h after birth (Tessarollo et al. 1994). We performed TrkA/TrkC double immunohistochemistry (Huang et al. 1999), enabling detection of both proteins in the same sample. TrkC-positive cells (Figure 1A) and fibers (Figure 1E) were absent in NT-3 KOs at embryonic day (E) 15. Two subsets of DRG cells expressing either TrkA or TrkC were detected in double KOs, similar to wild-type (WT) or Bax KO animals. Surprisingly, at postnatal day (P) 0, a few cells expressed TrkC even in NT-3 KO animals in the Bax +/+ genetic background, and some cells co-expressed TrkA and TrkC, regardless of the genotype (Figure 1B). In order to quantify our results, we analyzed the ratio of TrkA and TrkC immunopositive cells from four different DRGs of animals of different genotypes. At all ages studied, Bax/NT-3 double null DRGs had TrkA/TrkC ratios similar to those of Bax null DRGs, and higher than those of NT-3 null DRGs (Figure 1D). TrkC-positive neurons rescued by Bax deletion, however, failed to differentiate properly, as evidenced by the lack of expression of the proprioceptive molecular marker Parvalbumin (PV) (Figure 1C).
Figure 1  TrkA/TrkC and PV Immunohistochemistry in DRG and Spinal Cord
Red represents TrkA, green represents TrkC (and PV in [C]), and yellow represents co-expression.
(A) TrkA/TrkC immunostaining in E15 DRG. TrkC-positive neurons normally eliminated in NT-3 KOs are rescued in double KOs.
(B) TrkA/TrkC immunostaining at P0 in DRG.
(C) PV immunostaining in P0 DRG. Rescued TrkC-positive cells fail to express PV.
(D) Ratio of TrkA-immunopositive cells to TrkC-immunopositive cells in E15 and P0 DRGs. Data are presented as percentage of cells with standard deviation. Double KOs always had similar ratios to Bax KOs, and NT-3 KOs had the least amount of TrkC-positive cells, if any.
(E) TrkA/TrkC immunostaining in E15 spinal cord. Arrow points to group Ia fibers. Dorsal is up.
Scale bar: 50 μm (A–C), 1 mm (E).