The Drosophila adult has a complex tracheal system that forms during the pupal period. We have studied the derivation of part of this system, the air sacs of the dorsal thorax. During the third larval instar, air sac precursor cells bud from a tracheal branch in response to FGF, and then they proliferate and migrate to the adepithelial layer of the wing imaginal disc. In addition, FGF induces these air sac precursors to extend cytoneme-like filopodia to FGF-expressing cells. These findings provide evidence that FGF is a mitogen in Drosophila, correlate growth factor signaling with filopodial contact between signaling and responding cells, and suggest that FGF can act on differentiated tracheal cells to induce a novel behavior and role.

The signaling molecules Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless(Wg) function as morphogens and organize wing patterning in Drosophila. In the screen for mutations that alter the morphogen activity, we identified novel mutants of two Drosophila genes, sister of tout-velu (sotv) and brother of tout-velu(botv), and new alleles of toutvelu (ttv). The encoded proteins of these genes belong to an EXT family of proteins that have or are closely related to glycosyltransferase activities required for biosynthesis of heparan sulfate proteoglycans (HSPGs). Mutation in any of these genes impaired biosynthesis of HSPGs in vivo, indicating that, despite their structural similarity, they are not redundant in the HSPG biosynthesis. Protein levels and signaling activities of Hh, Dpp and Wg were reduced in the cells mutant for any of these EXT genes to a various degree, Wg signaling being the least sensitive. Moreover, all three morphogens were accumulated in the front of EXT mutant cells, suggesting that these morphogens require HSPGs to move efficiently. In contrast to previous reports that ttv is involved exclusively in Hh signaling, we found that ttv mutations also affected Dpp and Wg. These data led us to conclude that each of three EXT genes studied contribute to Hh, Dpp and Wg morphogen signaling. We propose that HSPGs facilitate the spreading of morphogens and therefore, function to generate morphogen concentration gradients.

Neural stem cells called neuroblasts (NBs) generate a variety of neuronal and glial cells in the central nervous system of the Drosophila embryo. These NBs, few in number, are selected from a field of neuroepithelial (NE) cells. In the optic lobe of the third instar larva, all NE cells of the outer optic anlage (OOA) develop into either NBs that generate the medulla neurons or lamina neuron precursors of the adult visual system. The number of lamina and medulla neurons must be precisely regulated because photoreceptor neurons project their axons directly to corresponding lamina or medulla neurons. Here, we show that expression of the proneural protein Lethal of scute [L(1)sc] signals the transition of NE cells to NBs in the OOA. L(1)sc expression is transient, progressing in a synchronized and ordered 'proneural wave' that sweeps toward more lateral NEs. l(1)sc expression is sufficient to induce NBs and is necessary for timely onset of NB differentiation. Thus, proneural wave precedes and induces transition of NE cells to NBs. Unpaired (Upd), the ligand for the JAK/STAT signaling pathway, is expressed in the most lateral NE cells. JAK/STAT signaling negatively regulates proneural wave progression and controls the number of NBs in the optic lobe. Our findings suggest that NBs might be balanced with the number of lamina neurons by JAK/STAT regulation of proneural wave progression, thereby providing the developmental basis for the formation of a precise topographic map in the visual center.

The regulation of floral organ number is closely associated with floral meristem size. Mutations in the gene FLORAL ORGAN NUMBER1(FON1) cause enlargement of the floral meristem in Oryza sativa (rice), resulting in an increase in the number of all floral organs. Ectopic floral organs develop in the whorl of each organ and/or in the additional whorls that form. Inner floral organs are more severely affected than outer floral organs. Many carpel primordia develop indeterminately, and undifferentiated meristematic tissues remain in the center in almost-mature flowers. Consistent with this result, OSH1, a molecular marker of meristematic indeterminate cells in rice, continues to be expressed in this region. Although floral meristems are strongly affected by the fon1-2mutation, vegetative and inflorescence meristems are largely normal, even in this strong allele. We isolated the FON1 gene by positional cloning and found that it encodes a leucine-rich repeat receptor-like kinase most similar to CLAVATA1 (CLV1) in Arabidopsis thaliana. This suggests that a pathway similar to the CLV signaling system that regulates meristem maintenance in Arabidopsis is conserved in the grass family. Unlike CLV1, which is predominantly expressed in the L3 layer of the shoot meristem, FON1 is expressed throughout the whole floral meristem,suggesting that small modifications to the CLV signaling pathway may be required to maintain the floral meristem in rice. In addition, FON1transcripts are detected in all meristems responsible for development of the aerial part of rice, suggesting that genes sharing functional redundancy with FON1 act in the vegetative and inflorescence meristems to mask the effects of the fon1 mutation.

Super-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we report an optimized optical clearing agent for high-resolution fluorescence imaging (SeeDB2). SeeDB2 matches the refractive indices of fixed tissues to that of immersion oil (1.518), thus minimizing both light scattering and spherical aberrations. During the clearing process, fine morphology and fluorescent proteins were highly preserved. SeeDB2 enabled super-resolution microscopy of various tissue samples up to a depth of >100 μm, an order of magnitude deeper than previously possible under standard mounting conditions. Using this approach, we demonstrate accumulation of inhibitory synapses on spine heads in NMDA-receptor-deficient neurons. In the fly medulla, we found unexpected heterogeneity in axon bouton orientations among Mi1 neurons, a part of the motion detection circuitry. Thus, volumetric super-resolution microscopy of cleared tissues is a powerful strategy in connectomic studies at synaptic levels.

The Drosophila optic lobe comprises a wide variety of neurons, which form laminar neuropiles with columnar units and topographic projections from the retina. The Drosophila optic lobe shares many structural characteristics with mammalian visual systems. However, little is known about the developmental mechanisms that produce neuronal diversity and organize the circuits in the primary region of the optic lobe, the medulla. Here, we describe the key features of the developing medulla and report novel phenomena that could accelerate our understanding of the Drosophila visual system. The identities of medulla neurons are pre-determined in the larval medulla primordium, which is subdivided into concentric zones characterized by the expression of four transcription factors: Drifter, Runt, Homothorax and Brain-specific homeobox (Bsh). The expression pattern of these factors correlates with the order of neuron production. Once the concentric zones are specified, the distribution of medulla neurons changes rapidly. Each type of medulla neuron exhibits an extensive but defined pattern of migration during pupal development. The results of clonal analysis suggest homothorax is required to specify the neuronal type by regulating various targets including Bsh and cell-adhesion molecules such as N-cadherin, while drifter regulates a subset of morphological features of Drifter-positive neurons. Thus, genes that show the concentric zones may form a genetic hierarchy to establish neuronal circuits in the medulla.

Matrix metalloproteases regulate both physiological and pathological events by processing matrix proteins and growth factors. ADAMTS1 in particular is required for normal ovulation and renal function and has been shown to modulate angiogenesis. Here we report that TSP1 and 2 are substrates of ADAMTS1. Using a combination of mass spectrometry and Edman degradation, we mapped the cleavage sites and characterized the biological relevance of these processing events. ADAMTS1 cleavage mediates the release of polypeptides from the trimeric structure of both TSP1 and 2 generating a pool of antiangiogenic fragments from matrix-bound thrombospondin. Using neo-epitope antibodies we confirmed that processing occurs during wound healing of wild-type mice. However, TSP1 proteolysis is decreased or absent in ADAMTS1 null mice; this is associated with delayed wound closure and increased angiogenic response. Finally, TSP1À/À endothelial cells revealed that the antiangiogenic response mediated by ADAMTS1 is greatly dependent on TSP1. These findings have unraveled a mechanistic explanation for the angiostatic functions attributed to ADAMTS1 and demonstrated in vivo processing of TSP1 under situations of tissue repair.

The brain consists of various types of neurons that are generated from neural stem cells; however, the mechanisms underlying neuronal diversity remain uncertain. A recent study demonstrated that the medulla, the largest component of the Drosophila optic lobe, is a suitable model system for brain development because it shares structural features with the mammalian brain and consists of a moderate number and various types of neurons. The concentric zones in the medulla primordium that are characterized by the expression of four transcription factors, including Homothorax (Hth), Brain-specific homeobox (Bsh), Runt (Run) and Drifter (Drf), correspond to types of medulla neurons. Here, we examine the mechanisms that temporally determine the neuronal types in the medulla primordium. For this purpose, we searched for transcription factors that are transiently expressed in a subset of medulla neuroblasts (NBs, neuronal stem cell-like neural precursor cells) and identified five candidates (Hth, Klumpfuss (Klu), Eyeless (Ey), Sloppy paired (Slp) and Dichaete (D)). The results of genetic experiments at least explain the temporal transition of the transcription factor expression in NBs in the order of Ey, Slp and D. Our results also suggest that expression of Hth, Klu and Ey in NBs trigger the production of Hth/Bsh-, Run- and Drf-positive neurons, respectively. These results suggest that medulla neuron types are specified in a birth order-dependent manner by the action of temporal transcription factors that are sequentially expressed in NBs.