Drosophila cortex and neuropile glia influence secondary axon tract growth, pathfinding, and fasciculation in the developing larval brain

Glial cells play important roles in the developing brain during axon fasciculation, growth cone guidance, and neuron survival. In the Drosophila brain, three main classes of glia have been identified including surface, cortex, and neuropile glia. While surface glia ensheaths the brain and is involved in the formation of the blood-brain-barrier and the control of neuroblast proliferation, the range of functions for cortex and neuropile glia is less well understood. In this study, we use the nirvana2-GAL4 driver to visualize the association of cortex and neuropile glia with axon tracts formed by different brain lineages and selectively eliminate these glial populations via induced apoptosis. The larval central brain consists of approximately 100 lineages. Each lineage forms a cohesive axon bundle, the secondary axon tract (SAT). While entering and traversing the brain neuropile, SATs interact in a characteristic way with glial cells. Some SATs are completely invested with glial processes; others show no particular association with glia, and most fall somewhere in between these extremes. Our results demonstrate that the elimination of glia results in abnormalities in SAT fasciculation and trajectory. The most prevalent phenotype is truncation or misguidance of axon tracts, or abnormal fasciculation of tracts that normally form separate pathways. Importantly, the degree of glial association with a given lineage is positively correlated with the severity of the phenotype resulting from glial ablation. Previous studies have focused on the embryonic nerve cord or adult-specific compartments to establish the role of glia. Our study provides, for the first time, an analysis of glial function in the brain during axon formation and growth in larval development.     

Tracheal development in the Drosophila brain is constrained by glial cells

The Drosophila brain is tracheated by the cerebral trachea, a branch of the first segmental trachea of the embryo. During larval stages the cerebral trachea splits into several main (primary) branches that grow around the neuropile, forming a perineuropilar tracheal plexus (PNP) at the neuropile surface. Five primary tracheal branches whose spatial relationship to brain compartments is relatively invariant can be distinguished, although the exact trajectories and branching pattern of the brain tracheae are surprisingly variable. Immunohistochemical and electron microscopic studies demonstrate that all brain tracheae grow in direct contact with the glial cell processes that surround the neuropile. To investigate the effect of glia on tracheal development, embryos and larvae lacking glial cells as a result of a genetic mutation or a directed ablation were analyzed. In these animals, the tracheal branching pattern was highly abnormal. In particular, the number of secondary branches entering the central neuropile was increased. Wild-type larvae possess only two central tracheae, typically associated with the mushroom body and the antennocerebral tract. In larvae lacking glial cells, six to ten tracheal branches penetrate the neuropile in a variable pattern. This finding indicates that glia-derived signals constrained tracheal growth in the Drosophila brain and restrict the number of branches entering the neuropile.     

Notch and Numb are required for normal migration of peripheral glia in Drosophila

A prominent feature of glial cells is their ability to migrate along axons to finally wrap and insulate them. In the embryonic Drosophila PNS, most glial cells are born in the CNS and have to migrate to reach their final destinations. To understand how migration of the peripheral glia is regulated, we have conducted a genetic screen looking for mutants that disrupt the normal glial pattern. Here we present an analysis of two of these mutants: Notch and numb. Complete loss of Notch function leads to an increase in the number of glial cells. Embryos hemizygous for the weak Notch(B-8X) allele display an irregular migration phenotype and mutant glial cells show an increased formation of filopodia-like structures. A similar phenotype occurs in embryos carrying the Notch(ts1) allele when shifted to the restrictive temperature during the glial cell migration phase, suggesting that Notch must be activated during glial migration. This is corroborated by the fact that cell-specific reduction of Notch activity in glial cells by directed numb expression also results in similar migration phenotypes. Since the glial migration phenotypes of Notch and numb mutants resemble each other, our data support a model where the precise temporal and quantitative regulation of Numb and Notch activity is not only required during fate decisions but also later during glial differentiation and migration.     

Dissecting Drosophila embryonic brain development using photoactivated gene expression

The Drosophila brain is generated by a complex series of morphogenetic movements. To better understand brain development and to provide a guide for experimental manipulation of brain progenitors, we created a fate map using photoactivated gene expression to mark cells originating within specific mitotic domains and time-lapse microscopy to dynamically monitor their progeny. We show that mitotic domains 1, 5, and 9 give rise to discrete cell populations within specific regions of the brain. Two novel observations were that the antennal sensory system, composed of four disparate cell clusters, arose from mitotic domain 5 and that mitotic domain B produced glial cells, while neurons were produced from mitotic domains 1, 5, and 9. Time-lapse analysis of marked cells showed complex mitotic and migratory patterns for cells derived from these mitotic domains. Photoactivated gene expression was also used either to kill, to induce ectopic divisions, or to alter cell fate. This revealed that deficits were not repopulated, while ectopic cells were removed and extra glia were tolerated.     

Genetic dissection of trophic interactions in the larval optic neuropil of Drosophila melanogaster

The larval visual system of Drosophila melanogaster consists of two bilateral clusters of 12 photoreceptors, which express Rhodopsin 5 and 6 (Rh5 and Rh6) in a non-overlapping manner. These neurons send their axons in a fascicle, the larval optic nerve (LON), which terminates in the larval optic neuropil. The LON is required for the development of a serotonergic arborization originating in the central brain and for the development of the dendritic tree of the circadian pacemakers, the small ventral lateral neurons (LNv) [Malpel, S., Klarsfeld, A., Rouyer, F., 2002. Larval optic nerve and adult extra-retinal photoreceptors sequentially associate with clock neurons during Drosophila brain development. Development 129, 1443-1453; Mukhopadhyay, M., Campos, A.R., 1995. The larval optic nerve is required for the development of an identified serotonergic arborization in Drosophila melanogaster. Dev. Biol., 169, 629-643]. Here, we show that both Rh5- and Rh6-expressing fibers overlap equally with the 5-HT arborization and that it, in turn, also contacts the dendritic tree of the LNv. The experiments described here aimed at determining whether Rh5- or Rh6-expressing fibers, as well as the LNv, influence the development of this serotonergic arborization. We conclude that Rh6-expressing fibers play a unique role in providing a signal required for the outgrowth and branching of the serotonergic arborization. Moreover, the innervation of the larval optic neuropil by the 5-HT arborization depends on intact Rac function. A possible role for these serotonergic processes in modulating the larval circadian rhythmicity and photoreceptor function is discussed.     

Fondue and transglutaminase in the Drosophila larval clot

Hemolymph coagulation is vital for larval hemostasis and important in immunity, yet the molecular basis of coagulation is not well understood in insects. Of the larval clotting factors identified in Drosophila, Fondue is not conserved in other insects, but is notable for its effects on the clot's physical properties, a possible function in the cuticle, and for being a substrate of transglutaminase. Transglutaminase is the only mammalian clotting factor found in Drosophila, and as it acts in coagulation in other invertebrates, it is also likely to be important in clotting in Drosophila. Here we describe a Fondue-GFP fusion construct that labels the cuticle and clot, and show that chemical inhibition and RNAi knockdown of the Drosophila transglutaminase gene affect clot properties and composition in ways similar to knockdown of the fon gene. Thus, Fondue appears to be incorporated into the cuticle and is a key transglutaminase substrate in the clot. This is also the first direct functional confirmation that transglutaminase acts in coagulation in Drosophila.     

Drosophila brain tumor metastases express both neuronal and glial cell type markers

Loss of either lgl or brat gene activity in Drosophila larvae causes neoplastic brain tumors. Fragments of tumorous brains from either mutant transplanted into adult hosts over-proliferate, and kill their hosts within 2 weeks. We developed an in vivo assay for the metastatic potential of tumor cells by quantifying micrometastasis formation within the ovarioles of adult hosts after transplantation and determined that specific metastatic properties of lgl and brat tumor cells are different. We detected micrometastases in 15.8% of ovarioles from wild type host females 12 days after transplanting lgl tumor cells into their abdominal cavities. This frequency increased significantly with increased proliferation time. We detected micrometastases in 15% of ovarioles from wild type host females 10 days after transplanting brat tumor cells into their abdominal cavities. By contrast, this frequency did not change significantly with increased proliferation time. We found that nearly all lgl micrometastases co-express the neuronal cell marker, ELAV, and the glial cell marker, REPO. These markers are not co-expressed in normal brain cells nor in tumorous brain cells. This indicates deregulated gene expression in these metastatic cells. By contrast, most of the brat micrometastases expressed neither marker. While mutations in both lgl and brat cause neoplastic brain tumors, our results reveal that metastatic cells arising from these tumors have quite different properties. These data may have important implications for the treatment of tumor metastasis.     

Specification and development of the pars intercerebralis and pars lateralis, neuroendocrine command centers in the Drosophila brain

The central neuroendocrine system in the Drosophila brain includes two centers, the pars intercerebralis (PI) and pars lateralis (PL). The PI and PL contain neurosecretory cells (NSCs) which project their axons to the ring gland, a complex of peripheral endocrine glands flanking the aorta. We present here a developmental and genetic study of the PI and PL. The PI and PL are derived from adjacent neurectodermal placodes in the dorso-medial head. The placodes invaginate during late embryogenesis and become attached to the brain primordium. The PI placode and its derivatives express the homeobox gene Dchx1 and can be followed until the late pupal stage. NSCs labeled by the expression of Drosophila insulin-like peptide (Dilp), FMRF, and myomodulin form part of the Dchx1 expressing PI domain. NSCs of the PL can be followed throughout development by their expression of the adhesion molecule FasII. Decapentaplegic (Dpp), secreted along the dorsal midline of the early embryo, inhibits the formation of the PI and PL placodes; loss of the signal results in an unpaired, enlarged placodeal ectoderm. The other early activated signaling pathway, EGFR, is positively required for the maintenance of the PI placode. Of the dorso-medially expressed head gap genes, only tailless (tll) is required for the specification of the PI. Absence of the corpora cardiaca, the endocrine gland innervated by neurosecretory cells of the PI and PL, does not affect the formation of the PI/PL, indicating that inductive stimuli from their target tissue are not essential for early PI/PL development.     

Abnormalities in cell proliferation and apico-basal cell polarity are separable in Drosophila lgl mutant clones in the developing eye

In homozygous mutants of Drosophila lethal-2-giant larvae (lgl), tissues lose apico-basal cell polarity and exhibit ectopic proliferation. Here, we use clonal analysis in the developing eye to investigate the effect of lgl null mutations in the context of surrounding wild-type tissue. lgl⁻ clones in the larval eye disc exhibit ectopic expression of the G1-S regulator, Cyclin E, and ectopic proliferation, but do not lose apico-basal cell polarity. Decreasing the perdurance of Lgl protein in larval eye disc clones, by forcing extra proliferation of lgl⁻ tissue (using a Minute background), leads to a loss in cell polarity and to more extreme ectopic cell proliferation. Later in development at the pupal stage, lgl mutant photoreceptor cells show aberrant apico-basal cell polarity, but this is not associated with ectopic proliferation, presumably because cells are differentiated. Thus in a clonal context, the ectopic proliferation and cell polarity defects of lgl⁻ mutants are separable. Furthermore, lgl⁻ mosaic eye discs have alterations in the normal patterns of apoptosis: in larval discs some lgl⁻ and wild-type cells at the clonal boundary undergo apoptosis and are excluded from the epithelia, but apoptosis is decreased elsewhere in the disc, and in pupal retinas lgl⁻ tissue shows less apoptosis.     

TGFbeta receptor saxophone non-autonomously regulates germline proliferation in a Smox/dSmad2-dependent manner in Drosophila testis

Elucidating the regulatory mechanism of cell proliferation is central to the understanding of cancer development or organ size control. Drosophila spermatogenesis provides an excellent model to study cell proliferation since the germline cells mitotically amplify in a precise manner. However, the underlying molecular mechanism remains elusive. Germ cells derived from each gonialblast develop synchronously as one unit encapsulated by two somatic support cells (called cyst cells). Components of TGFbeta pathway have previously been found to restrict germ cell proliferation via their functions in cyst cells. Here we report that saxophone (sax), a TGFbeta type I receptor, is required in somatic cells to prevent the mitotically dividing spermatogonia from over-amplifying. Using various approaches, we demonstrate that Mad (Mothers against Dpp), a receptor-Smad usually associated with Sax-mediated TGFbeta/BMP signaling, is dispensable in this process. Instead, Smox (Smad on X, Drosophila Smad2), the other receptor-Smad formerly characterized in TGFbeta/activin signaling, is necessary for the precise mitotic divisions of spermatogonia. Furthermore, over-expressing Smox in cyst cells can partially rescue the proliferation phenotype induced by sax mutation. We propose that Smox acts downstream of Sax to prevent spermatogonial over-proliferation in Drosophila.     

Notch signaling controls proliferation through cell-autonomous and non-autonomous mechanisms in the Drosophila eye

During Drosophila eye development, localized Notch signaling at the dorsal ventral (DV)-midline promotes growth of the entire eye field. This long-range action of Notch signaling may be mediated through the diffusible ligand of the Jak/STAT pathway, Unpaired (Upd), which was recently identified as a downstream target of Notch. However, Notch activity has not been shown to be cell-autonomously required for Upd expression and therefore yet another diffusible signal may be required for Notch activation of Upd. Our results clarify the Notch requirement, demonstrating that Notch activity at the DV-midline leads to cell-autonomous expression of Upd as monitored in loss and gain-of-function Notch clones. In addition, mutations in the Jak/STAT pathway interact genetically with the Notch pathway by suppressing Notch mediated overgrowth. N(act) clones show non-autonomous effects on the cell cycle anterior to the furrow, indicating function of the Jak/STAT pathway. However, cell-autonomous effects of Notch within and posterior to the furrow are independent of Upd. Here, Notch autonomously maintains cells in a proliferative state and blocks photoreceptor differentiation.     

A conserved nuclear receptor, Tailless, is required for efficient proliferation and prolonged maintenance of mushroom body progenitors in the Drosophila brain

The intrinsic neurons of mushroom bodies (MBs), centers of olfactory learning in the Drosophila brain, are generated by a specific set of neuroblasts (Nbs) that are born in the embryonic stage and exhibit uninterrupted proliferation till the end of the pupal stage. Whereas MB provides a unique model to study proliferation of neural progenitors, the underlying mechanism that controls persistent activity of MB-Nbs is poorly understood. Here we show that Tailless (TLL), a conserved orphan nuclear receptor, is required for optimum proliferation activity and prolonged maintenance of MB-Nbs and ganglion mother cells (GMCs). Mutations of tll progressively impair cell cycle in MB-Nbs and cause premature loss of MB-Nbs in the early pupal stage. TLL is also expressed in MB-GMCs to prevent apoptosis and promote cell cycling. In addition, we show that ectopic expression of tll leads to brain tumors, in which Prospero, a key regulator of progenitor proliferation and differentiation, is suppressed whereas localization of molecular components involved in asymmetric Nb division is unaffected. These results as a whole uncover a distinct regulatory mechanism of self-renewal and differentiation of the MB progenitors that is different from the mechanisms found in other progenitors.     

Drosophila melanogaster larval hemolymph protein mapping

With the completion of the genome sequence of Drosophila melanogaster the importance of constructing a proteome map is to be considered. Therefore, with the application of recent advances in proteomic analysis approaches, a protein map of D. melanogaster larvae hemolymph proteins was obtained using 2-DE in the range of pH 3-10. After Coomassie colloidal detection of 289 spots, a total of 105 were excised from the gel and digested with trypsin. Identification was done based on a combination of MALDI-TOF/TOF MS and MS/MS spectra. The 99 proteins identified using this approach include a large number of metabolic enzymes, translational apparatus components, and structural proteins. Among these we emphasize the identification of proteins with molecular chaperone properties (heat shock proteins and PPIases) and protein spots involved in defense responses such as antioxidant and immunological defense mechanisms (thioredoxin, prophenoloxidase, and serine proteases), as well as in signal transduction pathways.     

Metastatic ability of Drosophila tumors depends on MMP activity

We analyzed how cells from tumors caused by mutations in either lgl or brat use matrix metalloproteinases (MMPs) to facilitate metastasis in Drosophila. MMP1 accumulation is dramatically increased in lgl larval imaginal discs compared to both wild type and brat mutants. Removal of Mmp1 gene activity in lgl brain tumor cells reduced their frequency of ovarian micro-metastases after transplantation; whereas, removal of Mmp1 gene activity in brat tumor cells had no such effect. Host ovaries showed increased Mmp1 gene expression in response to transplantation of brat tumors but not of lgl tumors. Reduction of MMP activity in host ovaries by ectopic expression of TIMP significantly reduced both lgl and brat metastases in that organ. These results highlight the mechanisms that lgl and brat tumor cells use to metastasize. Our interpretation of these data is that secretion of MMP1 from lgl tumor cells facilitates their metastasis, while secretion of MMP1 from host ovaries facilitates brat tumor metastasis. This study is the first demonstration that Drosophila tumors utilize MMP activity to metastasize.