An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. Here we show that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. We show that Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors.

Hedgehog (Hh) proteins specify tissue pattern in metazoan embryos by forming gradients that emanate from discrete sites of expression and elicit concentration-dependent cellular differentiation or proliferation responses1,2. Cellular responses to Hh and the movement of Hh through tissues are both precisely regulated, and abnormal Hh signaling has been implicated in human birth defects and cancer3-7. Hh signaling is mediated by its N-terminal domain (HhN), which is dually lipidated and secreted as part of a multivalent lipoprotein particle8-10. Reception of the HhN signal is modulated by several cell-surface proteins on responding cells, including Patched (Ptc), Smoothened (Smo), Ihog/CDO and the vertebrate-specific proteins Hip and Gas111. Drosophila Ihog and its vertebrate homologs CDO and BOC contain multiple immunoglobulin (Ig) and fibronectin type III (FNIII) repeats, and the first FNIII repeat of Ihog binds Drosophila HhN in a heparin-dependent manner12,13. Surprisingly, pull-down experiments suggest that mammalian Sonic hedgehog (ShhN) binds a nonorthologous FNIII repeat of CDO12,14. We report here biochemical, biophysical, and X-ray structural studies of a complex between ShhN and the third FNIII repeat of CDO. We show that the ShhN-CDO interaction is completely unlike the HhN-Ihog interaction and requires calcium, which binds at a previously undetected site on ShhN. This site is conserved in nearly all Hh proteins and is a hot spot for mediating interactions between ShhN and CDO, Ptc, Hip, and Gas1. Mutations in vertebrate Hh proteins causing holoprosencephaly and brachydactyly type A1 map to this calcium-binding site and disrupt interactions with these partners.

Although the transporter-like protein Patched (Ptc) is genetically implicated in reception of the extracellular Hedgehog (Hh) protein signal, a clear definition of the Hh receptor is complicated by the existence of additional Hh-binding proteins and, in Drosophila, by the lack of physical evidence for direct binding of Hh to Ptc. Here we show that activity of Ihog (Interference hedgehog), or of its close relative Boi (Brother of Ihog), is absolutely required for Hh biological response and for sequestration of the Hh protein to limit long-range signaling. We demonstrate that Ihog interacts directly with Ptc, is required for presentation of Ptc on the cell surface, and that Ihog and Ptc are both required for high-affinity Hh binding. On the basis of their joint roles in ligand binding, signal transduction, and receptor trafficking, we conclude that Ihog and Ptc together constitute the Drosophila Hh receptor.[Keywords: Brother of Ihog; Hedgehog receptor; Hedgehog signaling; Ihog; Patched] Supplemental material is available at http://www.genesdev.org.

Besides 19,008 possible ectodomains, Drosophila Dscam contains two alternative transmembrane/juxtamembrane segments, respectively, derived from exon 17.1 and exon 17.2. We wondered whether specific Dscam isoforms mediate formation and segregation of axonal branches in the Drosophila mushroom bodies (MBs). Removal of various subsets of the 12 exon 4s does not affect MB neuronal morphogenesis, while expression of a Dscam transgene only partially rescues Dscam mutant phenotypes. Interestingly, differential rescuing effects are observed between two Dscam transgenes that each possesses one of the two possible exon 17s. Axon bifurcation/segregation abnormalities are better rescued by the exon 17.2-containing transgene, but coexpression of both transgenes is required for rescuing mutant viability. Meanwhile, exon 17.1 targets ectopically expressed Dscam-GFP to dendrites while Dscam[exon 17.2]-GFP is enriched in axons; only Dscam[exon 17.2] affects MB axons. These results suggest that exon 17.1 is minimally involved in axonal morphogenesis and that morphogenesis of MB axons probably involves multiple distinct exon 17.2-containing Dscam isoforms.

H edgehog (Hh) is a secreted signaling molecule that mediates key tissue-patterning events during both vertebrate and invertebrate development (1-3). Hh also plays an important role in the maintenance and regulation of stem cells in adult organisms (4, 5). Abnormal activation of the Hh signaling pathway has been implicated in the initiation and growth of many human tumors (6), and drugs targeting the Hh pathway are under development (7).Hh is secreted but undergoes two lipid modifications that restrict its free diffusion and facilitate transport to appropriate target sites (8,9). Hh binding to Ptc, a 12-pass integral membrane protein with homology to bacterial resistance-nodulationdivision (RND) transporters (10) is a central event in Hh signaling and blocks the ability of Ptc to inhibit the seven-pass integral membrane protein Smoothened (Smo), a positive regulator of Hh responses (10). Recent genetic and RNAi experiments implicate the membrane-associated proteins Ihog and dally-like protein (Dlp) in Hh responsiveness (11)(12)(13)(14). Dlp, a member of the glypican family of heparan sulfate proteoglycans (HSPGs) (15), has dual roles in mediating Hh responsiveness and in transport of the Hh signal to distant cells (12)(13)(14). Heparan sulfate glycosaminoglycan chains, in particular, have also been implicated in Hh movement by the observation that tout velu, which encodes a heparan sulfate copolymerase (16, 17) that acts on Dlp (12), is required for normal Hh transport.Ihog, a Drosophila protein previously known as CG9211, is a type I transmembrane protein with four Ig domains followed by two FNIII domains, a membrane-spanning region, and a cytoplasmic region of no known function (11). Ihog is homologous to another Drosophila protein, CG32796 or brother of Ihog (BOI), and two mammalian proteins, CDO and BOC (18,19), that are also components of the Hh signaling pathway (11,20,21). Several observations indicate that Ihog may function as a coreceptor for Hh: (i) reduction of Ihog expression results in diminished Hh binding and responsiveness, (ii) epistasis experiments place Ihog function upstream or at the level of Ptc, (iii) the Ihog extracellular region is able to pull down HhN from conditioned medium, and (iv) coexpression of Ptc and Ihog results in a synergistic increase in Hh binding to the cell surface (11).The first FNIII domain of Ihog (IhogFn1) is necessary and sufficient to pull down HhN, but both FNIII domains are needed to synergize with Ptc and reconstitute Ihog function in cell-based signaling assays (11). Complicating interpretation of Ihog function is the observation that purified HhN and Ihog extracellular regions do not appreciably interact (see Fig. 2 A), indicating that a binary interaction between Ihog and Hh cannot explain Ihog function. To investigate the role of Ihog in Hh signaling, we initiated structural and biophysical studies of functional fragments of Drosophila melanogaster Ihog and Hh. Results and DiscussionStructure of Ihog FNIII Domains. The crystal structure of IhogFn1 was determ...