Expression of Trk receptors in the developing mouse trigeminal ganglion: in vivo evidence for NT-3 activation of TrkA and TrkB in addition to TrkC

Abstract

Animals lacking neurotrophin-3 (NT-3) are born with deficits in almost all sensory ganglia. Among these, the trigeminal ganglion is missing 70% of the normal number of neurons, a deficit which develops during the major period of neurogenesis between embryonic stages (E) 10.5 and E13.5. In order to identify the mechanisms for this deficit, we used antisera specific for TrkA, TrkB, and TrkC to characterize and compare the expression patterns of each Trk receptor in trigeminal ganglia of wild type and NT-3 mutants between E10.5 and E15.5. Strikingly, TrkA, TrkB, and TrkC proteins appear to be exclusively associated with neurons, not precursors. While some neurons show limited co-expression of Trk receptors at E11.5, by E13. 5 each neuron expresses only one Trk receptor. Neuronal birth dating and cell counts show that in wild-type animals all TrkB- and TrkC-expressing neurons are generated before E11.5, while the majority of TrkA-expressing neurons are generated between E11.5 and E13.5. In mice lacking NT-3, the initial formation of the ganglion, as assessed at E10.5, is similar to that in wild-type animals. At E11.5, however, the number of TrkC-expressing neurons is dramatically reduced and the number of TrkC-immunopositive apoptotic profiles is markedly elevated. By E13.5, TrkC-expressing neurons are virtually eliminated. At E11.5, compared to wild type, the number of TrkB-expressing neurons is also reduced and the number of TrkB immunoreactive apoptotic profiles is increased. TrkA neurons are also reduced in the NT-3 mutants, but the major deficit develops between E12.5 and E13.5 when elevated numbers of TrkA-immunoreactive apoptotic profiles are detected. Normal numbers of TrkA- and TrkB-expressing neurons are seen in a TrkC-deficient mutant. Therefore, our data provide evidence that NT-3 supports the survival of TrkA-, TrkB- and TrkC-expressing neurons in the trigeminal ganglion by activating directly each of these receptors in vivo.

Fig. 1

Specificity of Trk receptor antisera. (A) Western blot analysis of lysates of COS cells transiently transfected with full length rat TrkA, TrkB or TrkC, or of untransfected COS cells. Lysates were subjected to western blot analysis with RTA IgG (1 μg/ml; left panel), affinity purified RTB (1 μg/ml; center panel), or affinity purified RTC (1 μg/ml; right panel). Each antiserum recognizes protein bands of specific molecular weights in lysates of COS cells transfected with corresponding Trk receptor cDNA. (B–D) Immunohistochemical staining of E13.5 IX-X ganglion complex. The intracranial superior jugular ganglion is at the top in these images and the extracranial nodose-petrosal complex is toward the bottom. RTA (B) identifies neurons in the superior jugular ganglion, but not the nodose-petrosal complex. RTB (C) recognizes neurons in the nodose-petrosal complex but not in the superior jugular ganglion and RTC (D) recognizes a small number of neurons in the nodose-petrosal complex. Scale bars (B–D) 200 μm.

Fig. 2

Trk receptor proteins are expressed exclusively in neurons, not precursors. (A,C) Confocal images showing (A) RTC (red) and neurofilament (green) and (C) RTB (red) and neurofilament (green) colocalization in E11.5 trigeminal ganglion. Cells that are immunoreactive for RTC or RTB, but not neurofilament (arrowheads), in some confocal plane almost always show colocalization with neurofilament in another confocal planes (inset) (B,D). Confocal images showing RTC or RTB (both in red) and BrdU (green) immunoreactivity in E11.5 trigeminal ganglion. The nuclei of (B) RTC- and (D) RTB-immunopositive cells do not incorporate BrdU. (E) Merged confocal image showing RTA and NF- 160 colocalization (yellow) in E11.5 trigeminal ganglion. (FG) Confocal images of E13.5 trigeminal ganglion stained with neurofilament (green) and RTC (F) or RTB (G) (red for both). RTB and RTC immunoreactivity is confined to neurons. Scale bars (A,C) 50 μm; (E) 50 μm; (B,D,F) and (G) is 100 μm.

Fig. 3

Dynamic changes in the expression patterns of TrkB- and TrkC-expressing neurons between E11.5-E13.5. (A) TrkB-expressing neurons in E11.5 trigeminal ganglion appear to cluster toward the anterior of the ganglion (to the right in this image). (B) TrkC-expressing neurons in E11.5 trigeminal ganglion are distributed throughout the entire ganglion. (C) In E13.5 trigeminal ganglion, RTB recognizes a smaller proportion of neurons, vascular structures, and the surrounding mesenchyme. (D) Similar to RTB, RTC recognizes only a small fraction of neurons at E13.5. (E–H) High magnification of TrkB at E11.5 (E) and E13.5 (F); TrkC at E11.5 (G) and E13.5 (H). Scale bars: (A–D) 200 μm; (E–H) 50 μm.

Fig. 4

Quantitation of neurons and of Trk-immunoreactive cells in E10.5-E15.5 trigeminal ganglia of wild type and NT-3 mutants. (A) Total numbers of neurons (black squares) and of neurons expressing TrkA (white squares), TrkB (white diamonds), and TrkC (white circles) in wild-type trigeminal ganglion. (B) Numbers of TrkA-expressing neurons in trigeminal ganglion of wild type (black squares) and NT-3 mutants (white squares). (C) Numbers of TrkB-expressing neurons in trigeminal ganglion of wild type (black squares) and NT-3 mutants (white squares). (D) Numbers of TrkC-expressing neurons in trigeminal ganglion of wild type (black squares) and NT-3 mutants (white squares). Counts from at least three animals are shown as mean ± s.e.m. for each time point.

Fig. 5

Limited coexpression of Trk receptor proteins in E11.5, but not in E13.5, trigeminal neurons. (A–C) Confocal images showing coexpression of (A) TrkA (green) and TrkC (red), (B) TrkB (green) and TrkC (red), and (C) TrkA (red) and TrkB (green) in E11.5 trigeminal ganglion. Cells coexpressing both antigens are present as yellow and indicated by arrows. (D–I) At E12.5 (D–F) and E13.5 (G–I), each Trk receptor antiserum labels separate cell populations. Scale bar, 100 μm.

Fig. 6

Neurons expressing different Trk receptors are generated at distinct time intervals during embryogenesis. (A) A schematic diagram indicating the experimental paradigm for birth-dating Trk-expressing neurons. The arrows designate the embryonic days when BrdU was injected intraperitoneally into the pregnant dams and the days when the embryos were collected for immunohistochemistry. (B–D) When pulse labeled with BrdU at E11.5, a significant number of TrkA-expressing neurons (red cytoplasmic staining) at E12.5 and E13.5 were immunoreactive for BrdU (brown nuclear staining) (arrows in B,C). However, when embryos received BrdU injections at E12.5 and were collected at either E13.5 or E14.5, there was no colabeling of BrdU and TrkA in the trigeminal neurons (D) (notice the complete separation of nuclear brown staining and red cytoplasmic staining). (E–G) Embryos labeled with BrdU at E10.5 showed colabeling of BrdU (black nuclear staining) and TrkC (red cytoplasmic staining) at E11.5 or E12.5 in the trigeminal neurons (arrows in E,F). However, in embryos injected with BrdU at E11.5 or E12.5, there was no colabeling of BrdU and TrkC in the trigeminal neurons (G). Scale bar, 20 μm.

Fig. 7

Stage-dependent apoptotic cell death of TrkA-, TrkB- and TrkC-expressing neurons in NT-3 mutant embryos. (A,D) RTA immunoreactivity in wild-type (A) and mutant (D) embryos at E13.5. While most TrkA-expressing neurons in NT-3 mutants show normal morphology, there is increase in apoptotic figures associated with TrkA immunoreactivity (arrows). (B,E) RTB immunoreactivity in wild-type (B) and mutant (E) embryos. In E11.5 NT-3 mutant trigeminal ganglion, the number of TrkB-expressing neurons is reduced and the number of apoptosis associated with TrkB immunoreactivity (arrows) is increased. (C,F) RTC immunoreactivity in wild-type (C) and mutant (F) embryos. Numerous TrkC-immunoreactive apoptoses are identified in NT-3 mutant ganglion at E11.5. Scale bar, 20 μm. (G) Quantitation of Trk receptor immunoreactive apoptosis at different embryonic stages in wild type and NT-3 mutants. Note that the numbers for TrkA-, TrkB- and TrkC-immunoreactive apoptosis peaks at E13.5, E11.5 and E11.5, respectively. Counts are derived from at least two animals and are presented as mean ± s.e.m.