Multipotent neural stem cells generate glial cells of the central complex through transit amplifying intermediate progenitors in Drosophila brain development

The neural stem cells that give rise to the neural lineages of the brain can generate their progeny directly or through transit amplifying intermediate neural progenitor cells (INPs). The INP-producing neural stem cells in Drosophila are called type II neuroblasts, and their neural progeny innervate the central complex, a prominent integrative brain center. Here we use genetic lineage tracing and clonal analysis to show that the INPs of these type II neuroblast lineages give rise to glial cells as well as neurons during postembryonic brain development. Our data indicate that two main types of INP lineages are generated, namely mixed neuronal/glial lineages and neuronal lineages. Genetic loss-of-function and gain-of-function experiments show that the gcm gene is necessary and sufficient for gliogenesis in these lineages. The INP-derived glial cells, like the INP-derived neuronal cells, make major contributions to the central complex. In postembryonic development, these INP-derived glial cells surround the entire developing central complex neuropile, and once the major compartments of the central complex are formed, they also delimit each of these compartments. During this process, the number of these glial cells in the central complex is increased markedly through local proliferation based on glial cell mitosis. Taken together, these findings uncover a novel and complex form of neurogliogenesis in Drosophila involving transit amplifying intermediate progenitors. Moreover, they indicate that type II neuroblasts are remarkably multipotent neural stem cells that can generate both the neuronal and the glial progeny that make major contributions to one and the same complex brain structure.     

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.     

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.     

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.     

Altered neuronal lineages in the facial ganglia of Hoxa2 mutant mice

Neurons of cranial sensory ganglia are derived from the neural crest and ectodermal placodes, but the mechanisms that control the relative contributions of each are not understood. Crest cells of the second branchial arch generate few facial ganglion neurons and no vestibuloacoustic ganglion neurons, but crest cells in other branchial arches generate many sensory neurons. Here we report that the facial ganglia of Hoxa2 mutant mice contain a large population of crest-derived neurons, suggesting that Hoxa2 normally represses the neurogenic potential of second arch crest cells. This may represent an anterior transformation of second arch neural crest cells toward a fate resembling that of first arch neural crest cells, which normally do not express Hoxa2 or any other Hox gene. We additionally found that overexpressing Hoxa2 in cultures of P19 embryonal carcinoma cells reduced the frequency of spontaneous neuronal differentiation, but only in the presence of cotransfected Pbx and Meis Hox cofactors. Finally, expression of Hoxa2 and the cofactors in chick neural crest cells populating the trigeminal ganglion also reduced the frequency of neurogenesis in the intact embryo. These data suggest an unanticipated role for Hox genes in controlling the neurogenic potential of at least some cranial neural crest cells.     

Patterns of growth, axonal extension and axonal arborization of neuronal lineages in the developing Drosophila brain

The Drosophila central brain is composed of approximately 100 paired lineages, with most lineages comprising 100-150 neurons. Most lineages have a number of important characteristics in common. Typically, neurons of a lineage stay together as a coherent cluster and project their axons into a coherent bundle visible from late embryo to adult. Neurons born during the embryonic period form the primary axon tracts (PATs) that follow stereotyped pathways in the neuropile. Apoptotic cell death removes an average of 30-40% of primary neurons around the time of hatching. Secondary neurons generated during the larval period form secondary axon tracts (SATs) that typically fasciculate with their corresponding primary axon tract. SATs develop into the long fascicles that interconnect the different compartments of the adult brain. Structurally, we distinguish between three types of lineages: PD lineages, characterized by distinct, spatially separate proximal and distal arborizations; C lineages with arborizations distributed continuously along the entire length of their tract; D lineages that lack proximal arborizations. Arborizations of many lineages, in particular those of the PD type, are restricted to distinct neuropile compartments. We propose that compartments are “scaffolded” by individual lineages, or small groups thereof. Thereby, the relatively small number of primary neurons of each primary lineage set up the compartment map in the late embryo. Compartments grow during the larval period simply by an increase in arbor volume of primary neurons. Arbors of secondary neurons form within or adjacent to the larval compartments, resulting in smaller compartment subdivisions and additional, adult specific compartments.     

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.     

gcm2 promotes glial cell differentiation and is required with glial cells missing for macrophage development in Drosophila

glial cells missing (gcm) is the primary regulator of glial cell fate in Drosophila. In addition, gcm has a role in the differentiation of the plasmatocyte/macrophage lineage of hemocytes. Since mutation of gcm causes only a decrease in plasmatocyte numbers without changing their ability to convert into macrophages, gcm cannot be the sole determinant of plasmatocyte/macrophage differentiation. We have characterized a gcm homolog, gcm2. gcm2 is expressed at low levels in glial cells and hemocyte precursors. We show that gcm2 has redundant functions with gcm and has a minor role promoting glial cell differentiation. More significant, like gcm, mutation of gcm2 leads to reduced plasmatocyte numbers. A deletion removing both genes has allowed us to clarify the role of these redundant genes in plasmatocyte development. Animals deficient for both gcm and gcm2 fail to express the macrophage receptor Croquemort. Plasmatocytes are reduced in number, but still express the early marker Peroxidasin. These Peroxidasin-expressing hemocytes fail to migrate to their normal locations and do not complete their conversion into macrophages. Our results suggest that both gcm and gcm2 are required together for the proliferation of plasmatocyte precursors, the expression of Croquemort protein, and the ability of plasmatocytes to convert into macrophages.     

Drosophila APC2 and APC1 have overlapping roles in the larval brain despite their distinct intracellular localizations

The tumor suppressor APC and its homologs, first identified for a role in colon cancer, negatively regulate Wnt signaling in both oncogenesis and normal development, and play Wnt-independent roles in cytoskeletal regulation. Both Drosophila and mammals have two APC family members. We further explored the functions of the Drosophila APCs using the larval brain as a model. We found that both proteins are expressed in the brain. APC2 has a highly dynamic, asymmetric localization through the larval neuroblast cell cycle relative to known mediators of embryonic neuroblast asymmetric divisions. Adherens junction proteins also are asymmetrically localized in neuroblasts. In addition they accumulate with APC2 and APC1 in nerves formed by axons of the progeny of each neuroblast-ganglion mother cell cluster. APC2 and APC1 localize to very different places when expressed in the larval brain: APC2 localizes to the cell cortex and APC1 to centrosomes and microtubules. Despite this, they play redundant roles in the brain; while each single mutant is normal, the zygotic double mutant has severely reduced numbers of larval neuroblasts. Our experiments suggest that this does not result from misregulation of Wg signaling, and thus may involve the cytoskeletal or adhesive roles of APC proteins.     

Patterns of growth and tract formation during the early development of secondary lineages in the Drosophila larval brain

The Drosophila brain consists of a relatively small number of invariant, genetically determined lineages which provide a model to study the relationship between gene function and neuronal architecture. In following this long‐term goal, we reconstruct the morphology (projection pattern and connectivity) and gene expression patterns of brain lineages throughout development. In this article, we focus on the secondary phase of lineage morphogenesis, from the reactivation of neuroblast proliferation in the first larval instar to the time when proliferation ends and secondary axon tracts have fully extended in the late third larval instar. We have reconstructed the location and projection of secondary lineages at close (4 h) intervals and produced a detailed map in the form of confocal z‐projections and digital three‐dimensional models of all lineages at successive larval stages. Based on these reconstructions, we could compare the spatio‐temporal pattern of axon formation and morphogenetic movements of different lineages in normal brain development. In addition to wild type, we reconstructed lineage morphology in two mutant conditions. (1) Expressing the construct UAS‐p35 which rescues programmed cell death we could systematically determine which lineages normally lose hemilineages to apoptosis. (2) so‐Gal4‐driven expression of dominant‐negative EGFR ablated the optic lobe, which allowed us to conclude that the global centrifugal movement normally affecting the cell bodies of lateral lineages in the late larva is causally related to the expansion of the optic lobe, and that the central pattern of axonal projections of these lineages is independent of the presence or absence of the optic lobe. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 434–451, 2016