The long-term goal of the proposed work is to elucidate the mechanisms that underlie cornmitment of the pluripotent neural crest stem cell to the sympathetic neuron cell lineage. Whereas stem cells express the nonselective neurotrophin receptor, p75NTR and the neurotrophin-3 (NT-3)-selective receptor, TrkC, sympathetic neurons express the norepinephrine transporter (NET), tyrosine hydroxylase (TH), dopamine-~-hydroxylase (DBH), the nerve growth factor-selective receptor, TrkA, and p75NTR. The overall hypothesis states that neurotrophin-3 (NT-3) action and norepinephrine transport are critically involved in these developmental changes. The in vitro colony assay that has been developed in this laboratory will be used to assess and quantify the influences of NT-3, norepinephrine (NE) and NE uptake inhibitors on sympathetic neurogenesis. There are 3 specific aims.
In Aim 1 it will be determined by multiplex RT-PCR, fluorescent in situ hybridization, confocal microscopy, immuno-electron microscopy, nuclear runoff assay and a TrkC knock-down strategy whether the basicfibroblast/NT-3 growth factor combination induces transcription of the NET by activating TrkC.
In Aim 2 it will be determined by ratio imaging whether norepinephrine transport causes calcium influx by activating voltage-gated calcium channels.
Aim 3 will start to identify, by use of antibodies against activated signaling mediators and of dominant negative and constitutively active forms of signaling mediators, the signaling pathways by which NE-mediated calcium transients regulate expression of TrkA, TH and DBH. The proposed work will fill in some of the gaps of knowledge of the causal relationship between extracellular signaling and gene expression. A better understanding of the molecular mechanisms that control the early phases of sympathetic neurogenesis is also pertinent to function and disorders of the autonomic nervous system. Moreover, characterization of the properties of the neural crest stem cell may become a useful aid in nervous system regeneration and reconstitution of function. Finally, the fact that the tricyclic antidepressant, desipramine, and the drug of abuse, cocaine, block noradrenergic differentiation in vitro and in the embryo has important implications for mental health. Both types of drug easily cross the placenta and can cause autonomic disturbances in infants born to mothers who have been treated with the antidepressant, or who have used cocaine during pregnancy.
| Hu, Yao Fei; Caron, Marc G; Sieber-Blum, Maya (2009) Norepinephrine transport-mediated gene expression in noradrenergic neurogenesis. BMC Genomics 10:151 |
| Sieber-Blum, Maya (2004) Cardiac neural crest stem cells. Anat Rec A Discov Mol Cell Evol Biol 276:34-42 |
| Ren, Z G; Porzgen, P P; Youn, Y-H et al. (2003) Ubiquitous embryonic expression of the norepinephrine transporter. Dev Neurosci 25:1-13 |
| Ren, Z G; Porzgen, P; Zhang, J M et al. (2001) Autocrine regulation of norepinephrine transporter expression. Mol Cell Neurosci 17:539-50 |
| Sieber-Blum, M; Ren, Z (2000) Norepinephrine transporter expression and function in noradrenergic cell differentiation. Mol Cell Biochem 212:61-70 |
