Expression of TrkB receptor isoforms in the developing avian visual system.

The expression of novel TrkB receptor transcripts has been characterized to understand the potentially diverse roles of brain-derived neurotrophic factor (BDNF) in the developing avian visual system. In situ localization with an extracellular domain probe common to all TrkB transcripts labeled a sub-population of large retinal ganglion cells as well as many associated visual nuclei, including the neuronal layers within the tectum that receive retinal innervation. Because of the potential for structurally and functionally distinct receptors derived from the TrkB gene locus, cDNA cloning and reverse transcription-PCR analysis were used to further analyze receptor isoform expression in the retina and tectum. Receptor isoforms were sequenced that contained a deletion of the N terminus, a deletion in the putative ligand-binding domain, or a deletion in the cytoplasmic juxtamembrane (JM) domain. Two novel JM insertion sequences also were identified, one of which exhibits weak homology to beta-actin and was found in both kinase-containing (TK+) and kinase deletion (KD) receptor isoforms. In the developing retina, TK+ receptor mRNA is upregulated during the period of retinal ganglion cell (RGC) death, consistent with the proposed role of BDNF as a tectal-derived survival factor for RGCs. However, the expression of TK+ transcripts in the tectum indicates that this structure also contains cells responsive to BDNF throughout development. Because BDNF is expressed in both the retina and tectum, it is conceivable that TrkB also mediates autocrine/paracrine signaling within these structures or anterograde retinotectal trophic support.

trkC-mediated NT-3 signaling is required for the early development of a subpopulation of neurogenic neural crest cells.

Soon after they segregate from the neural tube, trunk neural crest cells disperse on two spatially and temporally distinct pathways. Only crest cells that migrate early and ventromedially give rise to neurons of the peripheral nervous system. It is also known that neural crest cell-derived populations require appropriate environmental cues early in development in order to generate neurons, and for the subsequent survival of differentiated neurons. We examined whether neurotrophin-3 (NT-3), a survival factor for subsets of peripheral neurons, is also involved in the regulation of neurogenesis by neural crest cells. First, we found that premigratory and migrating neural crest cells on the medial migration pathway of Embryonic Day 2.5 (E 2.5) embryos express mRNAs encoding multiple isoforms of the NT-3 receptor, trkC, as do cells in the neural tube and epithelial dermamyotome. On E4, a subpopulation of neurons in nascent sensory ganglia express trkC message. Second, we demonstrate that trkC mRNA is only expressed in neural crest cell populations that possess neurogenic potential. Third, we show that the presence of NT-3, during the initial development of cultured neural crest cells, is required for neurogenesis by a subpopulation of neurogenic neural crest-derived cells. These results suggest that a subpopulation of neurogenic neural crest cells expresses functional trkC receptors and requires the timely availability of NT-3 for their development before reaching their final embryonic locations. We suggest that developmental heterogeneity exists in the identity and requirements of neural crest cell subsets that harbor neurogenic potential. We also suggest that the "paradoxical" expression of trkC receptors by the somitic dermamyotome may, in fact, play a role in the exclusive development of crest-derived neurogenic precursors on the medial pathway by limiting the availability of NT-3 on the lateral pathway.

Muscle sensory neurons require neurotrophin-3 from peripheral tissues during the period of normal cell death.

To determine if muscle sensory neurons require neurotrophin-3 (NT3) during the period of normal cell death, we used an NT3-specific antiserum to deplete NT3 from peripheral tissues during this period in chick embryos. DiI staining of dorsal roots indicated that limb injections of anti-NT3 reduced the spinal projection of muscle spindle afferents. In contrast, injection of the antiserum into the spinal cord had no demonstrable effect, indicating that the reduced projection following limb injection was due to peripheral blockade of NT3 signaling. Counts of neurons retrogradely labeled from muscle and cutaneous nerves showed that peripheral blockade of NT3 selectively reduced the survival of muscle sensory neurons without affecting the survival of cutaneous sensory neurons or motoneurons. In situ hybridization with trkC probes indicated that, during the period of cell death, most large diameter muscle sensory neurons express trkC transcripts, whereas few cutaneous neurons express this receptor for NT3. We conclude that large diameter muscle afferents, including spindle afferents, require NT3 from peripheral tissues to survive the normal period of sensory neuron death in vivo.

Isoforms of the avian TrkC receptor: a novel kinase insertion dissociates transformation and process outgrowth from survival.

TrkC receptor isoforms have been identified by cDNA cloning and RT-PCR analysis of embryonic chick brain RNA. An N-terminal truncation motif is missing from the signal sequence and first cysteine cluster of the extracellular domain. Within the cytoplasmic dimain, a kinase truncation motif retains part of the kinase domain, but appeared to lack activity. Finally, a kinase insert (KI) motif introduces a 25 amino acid sequence distinct from the known mammalian inserts. KI receptors, like full-length receptors, were tyrosine phosphorylated in response to NT-3 and mediated the transformation of chick embryo fibroblasts and process outgrowth from rat PC12 cells. However, KI receptors supported little, if any, survival of serum-deprived PC12 cells. These results indicate that alternative splicing of trkC transcripts is an important mechanism for regulating cellular responses to NT-3.