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.     

Morphogenesis and proliferation of the larval brain glia in Drosophila

Glial cells subserve a number of essential functions during development and function of the Drosophila brain, including the control of neuroblast proliferation, neuronal positioning and axonal pathfinding. Three major classes of glial cells have been identified. Surface glia surround the brain externally. Neuropile glia ensheath the neuropile and form septa within the neuropile that define distinct neuropile compartments. Cortex glia form a scaffold around neuronal cell bodies in the cortex. In this paper we have used global glial markers and GFP-labeled clones to describe the morphology, development and proliferation pattern of the three types of glial cells in the larval brain. We show that both surface glia and cortex glia contribute to the glial layer surrounding the brain. Cortex glia also form a significant part of the glial layer surrounding the neuropile. Glial cell numbers increase slowly during the first half of larval development but show a rapid incline in the third larval instar. This increase results from mitosis of differentiated glia, but, more significantly, from the proliferation of neuroblasts.     

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.     

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.     

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.     

Drosophila homologue of the Rothmund-Thomson syndrome gene: essential function in DNA replication during development

Members of the RecQ family play critical roles in maintaining genome integrity. Mutations in human RecQL4 cause a rare genetic disorder, Rothmund-Thomson syndrome. Transgenic mice experiments showed that the RecQ4 null mutant causes embryonic lethality. Although biochemical evidence suggests that the Xenopus RecQ4 is required for the initiation of DNA replication in the oocyte extract, its biological functions during development remain to be elucidated. We present here our results in establishing the use of Drosophila as a model system to probe RecQ4 functions. Immunofluorescence experiments monitoring the cellular distribution of RecQ4 demonstrated that RecQ4 expression peaks during S phase, and RecQ4 is expressed only in tissues active in DNA replication, but not in quiescent cells. We have isolated Drosophila RecQ4 hypomorphic mutants, recq^EP and recq4²³, which specifically reduce chorion gene amplification of follicle cells by 4-5 fold, resulting in thin and fragile eggshells, and female sterility. Quantitative analysis on amplification defects over a 14-kb domain in chorion gene cluster suggests that RecQ4 may have a specific function at or near the origin of replication. A null allele recq4¹⁹ causes a failure in cell proliferation, decrease in DNA replication, chromosomal fragmentation, and lethality at the stage of first instar larvae. The mosaic analysis indicates that cell clones with homozygous recq4¹⁹ fail to proliferate. These results indicate that RecQ4 is essential for viability and fertility, and is required for most aspects of DNA replication during development.     

Drosophila mars is required for organizing kinetochore microtubules during mitosis

Spindle assembly is essential for the equal distribution of genetic material to the daughter cells during mitosis. The process of spindle assembly is complicated and involves multiple levels of molecular regulation. It is generally accepted that mitotic spindles are emanated from the centrosomes and are assembled in the vicinity of chromosomes. However, the molecular mechanism involved in the spindle assembly during mitosis remains unclear. In this study, we have provided several lines of evidence to show that Drosophila Mars is required for the assembly and stabilization of kinetochore microtubules. In an immunocytochemical study, we show that Mars is mainly localized on the kinetochore microtubules during mitosis. Using RNA interference to deplete the Mars expression in Drosophila S2 cells resulted in the malformation of mitotic spindle that mainly lacked the kinetochore microtubules. The spindle defect resulted in mitotic delays by increasing the percentage of uncongressed chromosomes both in vitro and in vivo. In summary, this study has extended our previous study of Mars in cell cycle regulation and provided further evidence showing that Mars is required for the assembly of kinetochore microtubules.     

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.     

The pleiohomeotic gene is required for maintaining expression of genes functioning in ventral appendage formation in Drosophila melanogaster

Polycomb group (PcG) proteins are negative regulators that maintain the expression of homeotic genes and affect cell proliferation. Pleiohomeotic (Pho) is a unique PcG member with a DNA-binding zinc finger motif and was proposed to recruit other PcG proteins to form a complex. The pho null mutants exhibited several mutant phenotypes such as the transformation of antennae to mesothoracic legs. We examined the effects of pho on the identification of ventral appendages and proximo-distal axis formation during postembryogenesis. In the antennal disc of the pho mutant, Antennapedia (Antp), which is a selector gene in determining leg identity, was ectopically expressed. The homothorax (hth), dachshund (dac) and Distal-less (Dll) genes involved in proximo-distal axis formation were also abnormally expressed in both the antennal and leg discs of the pho mutant. The engrailed (en) gene, which affects the formation of the anterior-posterior axis, was also misexpressed in the anterior compartment of antennal and leg discs. These mutant phenotypes were enhanced in the mutant background of Posterior sex combs (Psc) and pleiohomeotic-like (phol), which are another PcG genes. These results suggest that pho functions in maintaining expression of genes involved in the formation of ventral appendages and the proximo-distal axis.     

The egghead gene is required for compartmentalization in Drosophila optic lobe development

The correct targeting of photoreceptor neurons (R-cells) in the developing Drosophila visual system requires multiple guidance systems in the eye-brain complex as well as the precise organization of the target area. Here, we report that the egghead (egh) gene, encoding a glycosyltransferase, is required for a compartment boundary between lamina glia and lobula cortex, which is necessary for appropriate R1-R6 innervation of the lamina. In the absence of egh, R1-R6 axons form a disorganized lamina plexus and some R1-R6 axons project abnormally to the medulla instead of the lamina. Mosaic analysis demonstrates that this is not due to a loss of egh function in the eye or in the neurons and glia of the lamina. Rather, as indicated by clonal analysis and cell-specific genetic rescue experiments, egh is required in cells of the lobula complex primordium which transiently abuts the lamina and medulla in the developing larval brain. In the absence of egh, perturbation of sheath-like glial processes occurs at the boundary region delimiting lamina glia and lobula cortex, and inappropriate invasion of lobula cortex cells across this boundary region disrupts the pattern of lamina glia resulting in inappropriate R1-R6 innervation. This finding underscores the importance of the lamina/lobula compartment boundary in R1-R6 axon targeting.