Norepinephrine transport-mediated gene expression in noradrenergic neurogenesis

Abstract

Background: We have identified a differential gene expression profile in neural crest stem cells that is due to deletion of the norepinephrine transporter (NET) gene. NET is the target of psychotropic substances, such as tricyclic antidepressants and the drug of abuse, cocaine. NET mutations have been implicated in depression, anxiety, orthostatic intolerance and attention deficit hyperactivity disorder (ADHD). NET function in adult noradrenergic neurons of the peripheral and central nervous systems is to internalize norepinephrine from the synaptic cleft. By contrast, during embryogenesis norepinephrine (NE) transport promotes differentiation of neural crest stem cells and locus ceruleus progenitors into noradrenergic neurons, whereas NET inhibitors block noradrenergic differentiation. While the structure of NET und the regulation of NET function are well described, little is known about downstream target genes of norepinephrine (NE) transport.

Results: We have prepared gene expression profiles of in vitro differentiating wild type and norepinephrine transporter-deficient (NETKO) mouse neural crest cells using long serial analysis of gene expression (LongSAGE). Comparison analyses have identified a number of important differentially expressed genes, including genes relevant to neural crest formation, noradrenergic neuron differentiation and the phenotype of NETKO mice. Examples of differentially expressed genes that affect noradrenergic cell differentiation include genes in the bone morphogenetic protein (BMP) signaling pathway, the Phox2b binding partner Tlx2, the ubiquitin ligase Praja2, and the inhibitor of Notch signaling, Numbl. Differentially expressed genes that are likely to contribute to the NETKO phenotype include dopamine-beta-hydroxylase (Dbh), tyrosine hydroxylase (Th), the peptide transmitter 'cocaine and amphetamine regulated transcript' (Cart), and the serotonin receptor subunit Htr3a. Real-time PCR confirmed differential expression of key genes not only in neural crest cells, but also in the adult superior cervical ganglion and locus ceruleus. In addition to known genes we have identified novel differentially expressed genes and thus provide a valuable database for future studies.

Conclusion: Loss of NET function during embryonic development in the mouse deregulates signaling pathways that are critically involved in neural crest formation and noradrenergic cell differentiation. The data further suggest deregulation of signaling pathways in the development and/or function of the NET-deficient peripheral, central and enteric nervous systems.

Figure 1

Time course of expression of NET mRNA and NET function in cultured mouse neural crest cells. (A) Semi-quantitative RT-PCR of NET mRNA on culture days 4 – 8 (upper) and Hprt (lower) at 28, 30 and 32 cycles each. Based on these results NET was subsequently amplified at 30 cycles. (B) Ratio Net/Hprt in triplicate during culture days 4 – 9 at 30 cycles of amplification. Data are expressed as average of three samples; error bars represent standard deviation. (C) Morphology of 3H-NE uptake-positive cells at day 3 of culture resembles immature neural crest cells. (D) At day 7 of culture, many 3H-NE uptake-positive cells have the morphology of mature sympathetic neuroblasts (arrow); NET uptake positive cells with undifferentiated morphology were still present (arrowhead). These observations indicated that at day 7, culture contain NE uptake-positive progenitors as well as neuroblasts with a functional NET. They are multipolar and extend long processes. Bar, (C, D) 100 μm.

Figure 2

Altered morphology and reduced numbers of DBH-immunoreactive cells in day 7 NETKO neural crest cultures. (A) NETKO neural crest cells have no or short processes only (e.g., arrow). (B) By contrast, wild type DBH-positive cells have long processes (e.g., arrow). (C) Quantification of the number of DBH-immunoreactive cells expressed in day 7 wild type and NETKO neural crest cultures. Approximately half the number of DBH-positive cells is expressed in NETKO cultures compared to wild type cultures. Bar, (A, B) 100 μm.

Figure 3

Co-localization of CART and DBH immunoreactivities in wild type and NETKO neural crest cells, superior cervical ganglion and locus ceruleus. (A, A', B, B'), NETKO and wild type neural crest cultures, respectively, at day 7. (C, C', D, D'), superior cervical ganglion of 10 week-old mice; (E, E', F, F'), Locus ceruleus from 10-week old wild type and NETKO mice. Wild type cells express CART at low levels. DBH and CART immunoreactivities co-localize in all three tissues. CART and DBH immunoreactivity appear somewhat brighter in NETKO tissue than in wild type tissues, suggesting increase in expression not only at the RNA but also at the protein level. It needs to be noted that immunofluorescence was not quantified. Bar, (A – F') 100 μm.