Signaling cascades triggered by receptor tyrosine kinases (RTKs) participate in diverse developmental processes. The active state of these signaling pathways was monitored by examination of the in situ distribution of the active, dual phosphorylated form of mitogen-activated protein kinase (ERK) with a specific monoclonal antibody. Detection of the active state of the Drosophila epidermal growth factor receptor (DER) pathway allowed the visualization of gradients and boundaries of receptor activation, assessment of the distribution of activating ligands, and analysis of interplay with the inhibitory ligand Argos. This in situ approach can be used to monitor other receptor-triggered pathways in a wide range of organisms.

The CNS is thought to develop from self-renewing stem cells that generate neurons, astrocytes, and oligodendrocytes. Other data, however, have suggested that astrocytes and oligodendrocytes are generated from separate progenitor populations. To reconcile these observations, we have prospectively isolated progenitors that do or do not express Olig2, an oligodendrocyte bHLH determination factor. Both Olig2(-) and Olig2(+) progenitors can behave as tripotential CNS stem cells (CNS-SCs) in vitro. Growth in FGF-2 causes induction of Olig2 in the former population, permitting oligodendrocyte differentiation; extinction of Olig2 in the latter cells permits astrocyte differentiation. The induction of Olig2 by FGF-2 is mediated, in part, via endogenous Sonic Hedgehog. These data indicate that clonogenic competence to generate neurons, astrocytes, and oligodendrocytes reflects a deregulation of dorsoventral patterning during expansion in vitro, raising the question of whether such trifatent cells actually exist in vivo.

In the absence of efficient transcription termination correct 3′‐end processing is an essential step in the synthesis of stable chloroplast mRNAs in higher plants. We show here that 3′‐end processing in vitro involves endonucleolytic cleavage downstream from the mature terminus, followed by exonucleolytic processing to a stem‐loop within the 3′‐untranslated region. These processing steps require a high molecular weight complex that contains both endoribonucleases and an exoribonuclease. In the presence of ancillary RNA binding proteins the complex correctly processes the 3′‐end of precursor RNA. In the absence of these ancillary proteins 3′‐end maturation is prevented and plastid mRNAs are degraded. Based on these results we propose a novel mechanism for the regulation of mRNA 3′‐end processing and stability in chloroplasts.

Spitz (Spi) is a TGF␣ homolog that is a cardinal ligand for the

Receptor tyrosine kinases (RTKs) and the signaling cascades that they trigger play central roles in diverse developmental processes. We describe the capacity to follow the active state of these signaling pathways in situ. This is achieved by monitoring, with a specific monoclonal antibody, the distribution of the active, dual phosphorylated form of MAP kinase (ERK). A dynamic pattern is observed during embryonic and larval phases of Drosophila development, which can be attributed, to a large extent, to the known RTKs. This specific detection has enabled us to determine the time of receptor activation, visualize gradients and boundaries of activation, and postulate the distribution of active ligands. Since the antibody was raised against the phosphorylated form of a conserved ERK peptide containing the TEY motif, this approach is applicable to a wide spectrum of multicellular organisms.