Wnt signaling regulates many aspects of metazoan development, including stem cells. In C. elegans, Wnt/MAPK signaling controls asymmetric divisions. A recent model proposed that the POP-1/TCF DNA binding protein works together with SYS-1/beta-catenin to activate transcription of target genes in response to Wnt/MAPK signaling. The somatic gonadal precursor (SGP) divides asymmetrically to generate distal and proximal daughters of distinct fates: only its distal daughter generates a distal tip cell (DTC), which is required for stem cell maintenance. No DTCs are produced in the absence of POP-1/TCF or SYS-1/beta-catenin, and extra DTCs are made upon overexpression of SYS-1/beta-catenin. Here we report that POP-1/TCF and SYS-1/beta-catenin directly activate transcription of ceh-22/nkx2.5 isoforms in SGP distal daughters, a finding that confirms the proposed model of Wnt/MAPK signaling. In addition, we demonstrate that the CEH-22/Nkx2.5 homeodomain transcription factor is a key regulator of DTC specification. We speculate that these conserved molecular regulators of the DTC niche in nematodes may provide insight into specification of stem cell niches more broadly.

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LMP1 is an Epstein-Barr virus (EBV)-Latent membrane protein 1 (LMP1), an Epstein-Barr virus (EBV)-encoded oncoprotein, mimics an active cellular receptor, CD40, but in a ligand-independent manner (15, 36). LMP1 stimulates multiple signaling pathways, including NF-B-, JNK-, and STAT-mediated transcription (10,14,29). As an oncoprotein, LMP1 is essential for maintaining the proliferation of EBV-infected lymphoblasts (9, 26), it transforms rodent fibroblasts to proliferate anchorage independently (1, 38), and it also induces B-cell lymphomas in transgenic mice late in their lives (28). LMP1 is an integral membrane protein consisting of a short amino cytoplasmic domain, a six-membranespanning domain, and a 200-amino-acid carboxy cytoplasmic domain. The carboxy cytoplasmic domain binds TRAFs, TRADD, and JAK3 and is essential for LMP1's ability to maintain proliferation of EBV-positive lymphoblasts and to activate multiple signaling pathways (9,10,14,20,29,30,33).In contrast to its ability to stimulate signaling and cell proliferation positively, LMP1 also inhibits RNA accumulation and general protein synthesis and induces cytostasis when it is expressed at levels as little as twofold higher than the average for EBV-infected cells (12,24,34). This inhibition requires the first two membrane-spanning domains of LMP1, but the mechanism of inhibition is unknown (5). Because all of the studies of this inhibitory activity have been carried out in transfected cells, it was unclear whether the inhibition occurs in EBVinfected cells and thus whether it could affect the EBV life cycle.We have sought to understand this inhibitory function of LMP1 by first determining if it occurs in EBV-infected cells. We have found that in three different clones of EBV-infected lymphoblasts, the levels of expression of LMP1 in individual cells in each clone range over 100-fold. This variation in LMP1 protein levels correlated with LMP1 RNA levels. Cell proliferation was diminished in individual cells expressing high levels of LMP1, which is consistent with the notion that high natural levels of LMP1 inhibit cell proliferation and indicates that the inhibitory effect of LMP1 occurs under physiological conditions. We next investigated the mechanism by which LMP1 inhibits gene expression. We found that higher levels of LMP1 result in the phosphorylation of eukaryotic translation initiation factor 2 (eIF2␣) that is dependent upon LMP1's transmembrane domains but not its carboxy-terminal signaling domain. Importantly, this induction of phosphorylation of eIF2␣ occurs normally in EBV-infected cells. In EBV-infected clones, cells expressing high physiological levels of LMP1 had high levels of phosphorylation of eIF2␣. Our results indicate that LMP1 inhibits gene expression and cell proliferation when expressed at high physiological levels. This inhibition occurs as one extreme of the normal spectrum of activities mediated by LMP1. MATERIALS AND METHODSCell culture and plasmids. 721 is an EBV-positive lymphoblastoid cell line (23). 11/17-2 and 11/17-3 ...

Latent membrane protein 1 (LMP1) is an Epstein±Barr virus (EBV)-encoded, ligand-independent receptor that mimics CD40. We report here that LMP1 signals principally from intracellular compartments. LMP1 associates simultaneously with lipid rafts and with its signaling molecules, tumor necrosis factor-receptor (TNF-R)-associated factors (TRAFs) and TNF-R1-associated death domain protein (TRADD) intracellularly, although it can be detected at low levels at the plasma membrane, indicating that most of LMP1's signaling complex resides in intracellular compartments. LMP1's signaling is independent of its accumulation at the plasma membrane in different cells, and as demonstrated by a mutant of LMP1 which has signi®cantly reduced localization at the plasma membrane yet signals as ef®ciently as does wild-type LMP1. The fusion of the transmembrane domain of LMP1 to signaling domains of CD40, TNF-R1 and Fas activates their signaling; we demonstrate that a fusion of LMP1 with CD40 recruits TRAF2 intracellularly. Our results imply that members of the TNF-R family can signal from intracellular compartments containing lipid rafts and may do so when they act in autocrine loops.

The C. elegans distal tip cell (DTC) provides a niche for germline stem cells in both hermaphrodites and males. The hermaphrodite distal tip cell (hDTC) also provides “leader” function to control gonadal elongation and shape, while in males, leader function is allocated to the linker cell (LC). Therefore, the male distal tip cell (mDTC) serves as a niche but not as a leader. The C. elegans homolog of E/Daughterless, HLH-2, was previously implicated in hDTC specification. Here we report that HLH-2 is also critical for hDTC maintenance, hDTC niche function and hDTC expression of a lag-2/DSL ligand reporter. We also find that HLH-2 functions in males to direct linker cell specification and to promote both mDTC maintenance and the mDTC niche function. We conclude that HLH-2 functions in both sexes to promote leader cell specification and DTC niche function.