Cellular Localization of Brain‐derived Neurotrophic Factor and Neurotrophin‐3 mRNA Expression in the Early Chicken Embryo

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Abstract
Degenerate primers from conserved regions in nerve growth factor, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were used in the polymerase chain reaction to isolate DNA fragments from the chicken BDNF and NT-3 genes. A genomic clone coding for chicken NT-3 was isolated and the structure of the chicken NT-3 mature protein was subsequently deduced from nucleotide sequence analysis of the isolated chicken NT-3 gene. Comparison of the chicken BDNF and NT-3 with the corresponding rat molecules showed that the avian molecules are very similar to their mammalian homologues. Northern blot analyses of messenger RNA (mRNA) from chicken embryos from embryonic day 3.5 (E3.5), E4.5, E8, E12 and E18 showed that expression of both BDNF and NT-3 mRNA peaked at E4.5 and decreased at later stages of development. Both probes revealed two transcripts; larger mRNAs of 4.5 kilobases (kb) for BDNF and 4.0 kb for NT-3 predominated over the smaller transcripts of 1.4 and 1.3 kb, respectively. The cellular localization of BDNF and NT-3 mRNA in the E4 and E6 embryos was studied by in situ hybridization. In the E4 embryo, labelling for BDNF was seen over cells in restricted parts of the epithelium of the otic vesicle. Analysis of adjacent sections for the low-affinity nerve growth factor receptor mRNA showed that regions in the otic vesicle epithelium which labelled for BDNF mRNA also labelled for low-affinity nerve growth factor receptor mRNA. No labelling for NT-3 was detected in the otic vesicle. Labelling for BDNF mRNA was also found over mesenchyme dorsal to the wing bud, in the wing bud and in the splanchnopleural lining of the stomach. Labelling for NT-3 mRNA was found at E4 over the epidermis on the ventral side in the region of the branchial arches. The labelling extended up the maxillary processes to Rathke's pouch. The closely located infundibulum was weakly labelled for NT-3 mRNA. NT-3 mRNA was also detected in the mesenchyme surrounding the oesophagus and lung buds. The regional expression pattern is in agreement with the established role for BDNF and NT-3 as target-derived neurotrophic factors, but the results also suggest that BDNF may be an intrinsic factor important for the development of the inner ear. The results support the emerging view that neurotrophic factors can play a role in early differentiation of both neuronal and non-neuronal tissues.