Neurogenin 1 (Neurog1) expression in the ventral neural tube is mediated by a distinct enhancer and preferentially marks ventral interneuron lineages.

The bHLH transcription factor Neurog1 (Ngn1, Neurod3, neurogenin 1) is involved in neuronal differentiation and cell-type specification in distinct regions of the developing nervous system. Here, transgenic mouse models were developed that use a Bacterial Artificial Chromosome (BAC) containing 208kb flanking the Neurog1 gene to efficiently drive expression of GFP and Cre in all Neurog1 domains. Two characteristics of Neurog1 gene regulation were uncovered. First, a 4kb region previously shown to be sufficient for driving expression of a reporter gene to a subset of the Neurog1 pattern in the developing midbrain, hindbrain, and spinal cord is required uniformly for high levels of expression in all Neurog1 domains, even those not originally identified as being regulated by this region. Second, a 0.8 kb enhancer was identified that is sufficient to drive Neurog1-like expression specifically in the ventral neural tube. Furthermore, Neurog1 progenitor cells in the ventral neural tube are largely fated to interneuron lineages rather than to motoneurons. These studies provide new tools for directing tissue specific expression in the developing neural tube, define Neurog1 lineages in the spinal cord, and further define the complex genomic structure required for obtaining the correct levels and spatial restriction of the neuronal differentiation gene Neurog1.

Role of TRAF3 and -6 in the activation of the NF-kappa B and JNK pathways by X-linked ectodermal dysplasia receptor.

X-linked ectodermal dysplasia receptor (XEDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to be highly expressed in ectodermal derivatives during embryonic development and binds to ectodysplasin-A2 (EDA-A2). By using a subclone of 293F cells with stable expression of XEDAR, we report that XEDAR activates the NF-kappaB and JNK pathways in an EDA-A2-dependent fashion. Treatment with EDA-A2 leads to the recruitment of TRAF3 and -6 to the aggregated XEDAR complex, suggesting a central role of these adaptors in the proximal aspect of XEDAR signaling. Whereas TRAF3 and -6, IKK1/IKKalpha, IKK2/IKKbeta, and NEMO/IKKgamma are involved in XEDAR-induced NF-kappaB activation, XEDAR-induced JNK activation seems to be mediated via a pathway dependent on TRAF3, TRAF6, and ASK1. Deletion and point mutagenesis studies delineate two distinct regions in the cytoplasmic domain of XEDAR, which are involved in binding to TRAF3 and -6, respectively, and play a major role in the activation of the NF-kappaB and JNK pathways. Taken together, our results establish a major role of TRAF3 and -6 in XEDAR signaling and in the process of ectodermal differentiation.

Selective exclusion by the polyamine transporter as a mechanism for differential radioprotection of amifostine derivatives.

Amifostine metabolites WR-1065 and the disulfide WR-33278 are thiol-containing polyamine analogues with potent radio- and chemoprotective properties. Some studies suggest that amifostine exerts differential cytoprotection in normal versus neoplastic tissues, but this finding remains controversial. To assess the role of the polyamine transport system in radioprotection by amifostine derivatives, human DU-145 prostate cancer cells were transfected with a cDNA that encodes antizyme (OAZ), a polyamine-inducible protein that suppresses polyamine transport under control of a minimal heat shock promoter. Selected clones expressing OAZ displayed heat shock-dependent suppression of polyamine uptake. When added to culture medium under nonreducing conditions, both WR-1065 and WR-33278 were detected as the disulfide form. Each derivative protected both parental and OAZ-transfected DU-145 cells from X-ray-induced cell killing at 37 degrees C. When cultures were heat shocked at 42 degrees C, both derivatives protected parental, but not OAZ-transfected cells from radiation-induced cell killing. Treatment of DU-145 cells with difluoromethylornithine (DFMO) suppressed intracellular putrescine and spermidine content, but increased the uptake of WR-33278-derived aminothiols. The concentration-dependent radioprotection of DU-145 cells by WR-33278 was enhanced by DFMO. Addition of exogenous putrescine reduced WR-33278-mediated radioprotection in both DFMO-treated and untreated DU-145 cells. These data demonstrate that negative regulation of the polyamine transporter, mediated by polyamines or antizyme, suppresses the uptake and radioprotective activity of amifostine derivatives. Selective exclusion of amifostine derivatives by the polyamine transporter could account for differential radio- or chemoprotection in normal versus neoplastic tissues in specific situations.