Notch and neurotrophins control neuronal shape, but it is not known whether their signaling pathways intersect. Here we report results from hippocampal neuronal cultures that are in support of this possibility. We found that low cell density or blockade of Notch signaling by a soluble Delta-Fc ligand decreased the mRNA levels of the nuclear targets of Notch, the homologues of enhancer-of-split 1 and 5 (Hes1/5). This effect was associated with enhanced sprouting of new dendrites or dendrite branches. In contrast, high cell density or exposure of low-density cultures to NGF increased the Hes1/5 mRNA, reduced the number of primary dendrites and promoted dendrite elongation. The NGF effects on both Hes1/5 expression and dendrite morphology were prevented by p75-antibody (a p75 NTR -blocking antibody) or transfection with enhancer-of-split 6 (Hes6), a condition known to suppress Hes activity. Nuclear translocation of NF-kappaB was identified as a link between p75 NTR and Hes1/5 because it was required for the up-regulation of these two genes. The convergence of the Notch and p75 NTR signaling pathways at the level of Hes1/5 illuminates an unexpected mechanism through which a diffusible factor (NGF) could regulate dendrite growth when cell-cell interaction via Notch is not in action.

We have previously shown that dendrite morphology of cultured hippocampal neurones is controlled by Notch receptor activation or binding of nerve growth factor (NGF) to its low affinity receptor p75 NTR , i.e. processes that up-regulate the expression of the Homologue of enhancer of split 1 and 5. Thus, the increased expression of these genes decreases the number of dendrites, whereas abrogation Neurotrophins have been shown to regulate dendrite morphology in a variety of experimental models (Mcallister et al. 1995(Mcallister et al. , 1997Baker et al. 1998;Jin et al. 2003). As neurotrophins are released in an activity-dependent manner, they may be fundamental in orchestrating the structural modifications that developing and mature neuronal circuits undergo (Whitford et al. 2002). However, the signalling pathways underlying the effects of neurotrophins on dendrite morphology are not fully understood. While most studies in this area have emphasized the importance of the Trk receptors, there is evidence that nerve growth factor (NGF) regulates dendrite morphology by binding to p75 NTR , the common neurotrophin receptor (Salama-Cohen et al. 2005) Address correspondence and reprint requests to Dr A. Rodríguez-Tébar, Instituto Cajal, CSIC, Avenue. Doctor Arce, 37, 28002 Madrid, Spain. E-mail: rodriguez@cajal.csic.esAbbreviations used: DIV, days in vitro; EGFP, enhanced green fluorescent protein; E/I, ratio, ratio of excitatory to inhibitory; GAD, glutamic acid decarboxylase; Hes, homologue of enhancer of split; Mash1: mouse achaete scute homologue 1; MAP, microtubule-associated protein; NGF, nerve growth factor; Ngn3, neurogenin 3; p75-Ab, p75 NTR blocking antibody; ROI, region of interest; Syp, synaptophysin; VgluT, vesicular glutamate transporter; VIAAT, vesicular inhibitory amino acid transporter.

In multicellular organisms morphogenesis highly depends on cell proliferation. Morphogenesis relates to the genetic mechanisms that determine specific sizes and shapes. Morphogenetic analyses need a detailed description of growth in terms of cell lineages. Cell lineage studies reveal spatial and numerical parameters of ordered cell proliferation, an indication of genetic control of cell behavior. The wing disk of Drosophila melanogaster possibly is, in this sense, the best-studied growing anlage. The imaginal wing disk is a monolayer of cells that give rise to the adult epidermis of the dorsal mesothorax, including notum and wing. Cell lineage analyses of the disk have been carried out with mitotic recombination clones labeled with mutant but gratuitous cell markers (1, 2). These clonal analyses have revealed clonal restrictions that separate so-called ''compartments,'' subdividing the early anlage in four major compartments, anterior͞posterior (A͞P) and dorsal͞ventral (D͞V). A subsequent subdivision separates notum and pleura from the wing proper (3, 4). New clonal restrictions, less stringent than in compartment boundaries, later symmetrically subdivide the dorsal and ventral wing compartments into sectors delimited by the veins (5, 6). Cell proliferation within these compartments and wing sectors is more undetermined with clone borders overlapping in the same regions of different wings. The shape of these clones is, however, region characteristic, symmetrical in both dorsal and ventral surfaces and near symmetrical in both anterior and posterior compartments (1, 2); see ref. 7 for review.In the wing disk and the presumptive wing blade in particular, cell proliferation increases the number of cells in an exponential mode, with an average cycle time of 8.5 h (8). The wing disk primordium in the embryo contains about 20 cells and the proliferation period ends with about 50,000, the equivalent to 10-11 rounds of cell division (8). Direct observation of growing imaginal discs has shown that clusters of neighboring cells, not clonally related, enter both the S phase of the cell cycle and mitosis in synchrony (9). Anaphases in a cluster are randomly oriented in the planar axis, but subsequently the two daughter cells allocate along either the A͞P axis (y axis) or the proximodistal axis (x axis) (9). Moreover, the shape of the clones in the growing disk corresponds with that of the clones seen in the adult wing, indicating that there are no major changes in the relative position of neighboring cells during the eversion of the disk at metamorphosis (9). During the larval and pupal periods cell death in the disk affects a very low number of cells in the hinge. There are therefore no major morphogenetic changes associated with cell death in late larval or pupal stages (10).But how do these morphogenetic parameters relate to the final constant wing shape and size? It has been proposed that compartment boundaries work as ''organizers'' of compartment growth and patterning. Along the growing A͞P boundary, the selector gene...