Molecular cloning and cellular localization of trk C in the chicken embryo

Abstract

Degenerate primers directed against conserved regions of the trk and trk B amino acid sequences were used in the polymerase chain reaction to isolate a 455 by fragment from embryonic day 3 chicken cDNA encoding the trk C. This fragment was subsequently used to synthesize an anti-sense trk C cRNA probe which was used in a RNase protection assay of total RNA from chicken embryos. trk C mRNA was found in the E2 embryo with increasing levels later in development. In the E9 embryo highest levels were found in brain and spinal cord with intermediate levels in eye, heart, gut and muscle. Low levels were found in kidney, liver, skin and yolk sac. Using the 455 bp trk C fragment as a probe in RNA blot analyses of poly A⁺ RNA, a major transcript of 6.3 kb and two minor transcripts of 3 kb and 10 kb were found. In situ hybridization was performed on embryos taken at three stages of development (embryonic day 3, 9 and 19), using a 48-mer antisense oligonucleotide probe for chicken trk C. Within the sensory nervous system trk C mRNA expression at all ages was confined to the ventrolateral neurons of the spinal sensory and trigeminal ganglia as well as distal ganglia associated with the VIIth, IXth and Xth cranial nerves. Labelling for trk C mRNA was also observed within the developing CNS at E3 and the ganglion of Remak at E19. A barely detectable level of expression was observed in the sympathetic chain and no labelling was evident in the proximal ganglia of the cranial nerves. These results suggest that neurons have a very early capacity to respond to neurotrophin-3 which continues throughout embryonic development. The early expression of trk C mRNA was also support the growing evidence suggesting a role for neurotrophins in neuronal differentiation.