Lateral inhibition: In proneural clusters (green), proneural bHLH proteins are expressed, e.g. Achaete (middle panel). The interplay between proneural proteins and the Notch signalling pathway (Dl and Ser shown in red and blue, respectively) results in specifying one or two sensory organ precursor cells per proneural cluster (right panel).

TRANSCRIPTIONAL REGULATION OF NEURAL DEVELOPMENT
basic-helix-loop-helix (bHLH) proteins constitute a large family of transcriptional regulators that play master regulatory roles in the development of many cell types, inasmuch as they can trigger entire differentiation programs when ectopically expressed. In both invertebrates and vertebrates, a subclass of these proteins, the proneural proteins, are responsible for imparting ectodermal cells with neural potential. Neural precursors cells are specified at places and times when these proteins are active. The Drosophila proneural proteins, Achaete, Scute, Lethal of Scute, Atonal and Amos are responsible for different subdomains of the central and peripheral nervous system. Each of these heterodimerizes via its amphipathic HLH domain with a ubiquitously expressed bHLH "E-protein" Daughterless (Da) generating a complex that binds a target site called the E A -box, and activates transcription.

The activity of proneural proteins is antagonized by another group of bHLH proteins, the E(spl) proteins in Drosophila and their homologous HES proteins in vertebrates. These heterodimerize amongst themselves, using their HLH domains, and bind another consensus, the E B/C box. E(spl)/ HES proteins belong to a class of bHLH proteins which is structurally distinct from the proneurals and Da, by virtue of possessing an Orange domain; they are therefore dubbed bHLH-O. The Orange domain is a helical domain that enhances homodimerization, as well as interaction with other proteins. bHLH-O proteins act as repressors, recruiting a variety of corepressors, such as Groucho.

The seven E(spl) genes are expressed in response to Notch signalling during lateral inhibition, a process that restricts the number of proneural gene expressing cells that actually go on to adopt the neural precursor fate. We have shown that E(spl) proteins interact with proneural proteins and Da . More specifically, E(spl)m ? and E(spl)m7 are recruited to Sc target promoters via protein-protein interactions with Da and Sc, rather than, or in addition to, direct DNA binding. E(spl) proteins do not use their HLH domain to interact with Da/proneurals, therefore they do not disrupt the Da/proneural bHLH complexes. Instead they interact with transactivation domains (TADs) of the latter. We have characterized the C-terminus of the proneural protein Sc as its TAD. This domain directly interacts with the N-terminus of E(spl)m7. A similar characterization of Da is currently under way. We are also studying post-translational modifications of these factors and how they impact on their stability and activity.

We are studying proneural-E(spl) interactions by a combination of yeast two hybrid, in vitro binding and in vivo reporter assay approaches.

Besides the seven E(spl) proteins, we are studying another bHLH-O protein called Hey. Hey is also a transcriptional target of Notch signalling, but in contrast to E(spl) , it is not expressed in the neuroectoderm, rather it is restricted to differentiating neurons and specifically in a subset of newly born neurons, which receive Notch signalling during their birth. Our results, for the first time, implicate a bHLH-O protein in the process of GMC asymmetric division during both neurogenic phases of the animal, early embryogenesis and larval. Although in the majority of cases Hey is a target of Notch, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed us to perform an study of Notch signalling during the genesis of secondary neurons in the larval CNS. The major conclusion is that newly born neurons receive Notch signalling from within their lineage, namely from their sibs or from earlier born "cousins".

Brain hemisphere showing neurobasts (red -Dpn) and GMCs/ newly born neurons (green- Pros). A subset of the latter express Hey (blue).

NOTCH LIGANDS NEED UBIQUITIN LIGASES
Notch signalling constitutes an evolutionary conserved mechanism that mediates many developmental processes.The main actors in this pathway in Drosophila are the Notch (N) transmembrane receptor and its two alternative transmembrane ligands, Delta (Dl) and Serrate (Ser), belonging to the class of DSL proteins (“Delta-Serrate-Lag2”). Notch is activated by an intracellular juxtamembrane proteolytic cleavage mediated by Presenilin. The released N intracellular fragment translocates to the nucleus and acts as a transcriptional co-activator of target genes [such as the E(spl) ]. DSL binding triggers N intracellular proteolysis and activation by first inducing another extracellular proteolytic event, followed by N ectodomain shedding. Our work has revolved around the mechanisms used by Dl and Ser to activate Notch.

We have characterized Neuralized (Neur) and Mindbomb1 (Mib1), two RING domain E3 ubiquitin ligases, both of which interact with Dl and Ser. They stimulate their signalling at the same time as they increase their endocytosis and turnover at the lysosome. Either Neur or Mib1 can activate the DSL proteins; in fact the two E3 ligases have distinct patterns of expression, thus regulating different Notch-mediated processes. As simultaneous disruption of neur and mib1 completely abolishes Notch signalling, it appears that some ubiquitylation step is a prerequisite for DSL signalling. We are working on characterizing the molecular details of DSL protein activation by the Neur and Mib1 E3 ligases.

Mosaic nota showing different degrees of defects in lateral inhibition. Sens (red) is a marker for sensory organ precursor cells. The rightmost notum has only mild defects.