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1.  Genetic control of intestinal stem cell specification and development: a comparative view 
Stem cell reviews  2012;8(2):597-608.
Stem cells of the adult vertebrate intestine (ISCs) are responsible for the continuous replacement of intestinal cells, but also serve as site of origin of intestinal neoplasms. The interaction between multiple signaling pathways, including Wnt/Wg, Shh/Hh, BMP, and Notch, orchestrate mitosis, motility, and differentiation of ISCs. Many fundamental questions of how these pathways carry out their function remain unanswered. One approach to gain more insight is to look at the development of stem cells, to analyze the “programming” process which these cells undergo as they emerge from the large populations of embryonic progenitors. This review intends to summarize pertinent data on vertebrate intestinal stem cell biology, to then take a closer look at recent studies of intestinal stem cell development in Drosophila. Here, stem cell pools and their niche environment consist of relatively small numbers of cells, and questions concerning the pattern of cell division, niche-stem cell contacts, or differentiation can be addressed at the single cell level. Likewise, it is possible to analyze the emergence of stem cells during development more easily than in vertebrate systems: where in the embryo do stem cells arise, what structures in their environment do they interact with, and what signaling pathways are active sequentially as a result of these interactions. Given the high degree of conservation among genetic mechanisms controlling stem cell behavior in all animals, findings in Drosophila will provide answers that inform research in the vertebrate stem cell field.
PMCID: PMC3950647  PMID: 22529012
2.  A novel tissue in an established model system: the Drosophila pupal midgut 
Development genes and evolution  2011;221(2):69-81.
The Drosophila larval and adult midgut are derived from two populations of endodermal progenitors that separate from each other in the early embryo. As larval midgut cells differentiate into an epithelial layer, adult midgut progenitors (AMPs) remain as small clusters of proliferating, undifferentiated cells attached to the basal surface of the larval gut epithelium. During the first few hours of metamorphosis, AMPs merge into a continuous epithelial tube that overgrows the larval layer and differentiates into the adult midgut; at the same time, the larval midgut degenerates. As shown in this paper, there is a second, transient pupal midgut that develops from the AMPs at the beginning of metamorphosis, and that intercalates between the adult and larval midgut epithelia. Cells of the transient pupal midgut form a multilayered tube that exhibits signs of differentiation, in the form of septate junctions and rudimentary apical microvilli. Some cells of the pupal midgut develop as endocrine cells. The pupal midgut remains closely attached to the degenerating larval midgut cells. Along with these cells, pupal midgut cells are sequestered into the lumen where they form the compact “yellow body”. The formation of a pupal midgut has been reported from several other species, and may represent a general feature of intestinal metamorphosis in insects.
PMCID: PMC3950650  PMID: 21556856
Drosophila; midgut; metamorphosis; pupa; ultrastructure
3.  Drosophila brain development: Closing the gap between a macroarchitectural and microarchitectural approach 
Neurobiologists address neural structure, development and function at the level of “macrocircuits” (how are different brain compartments interconnected, what overall pattern of activity do they produce), and at the level of “microcircuits” (how does connectivity and physiology of individual neurons and their processes within a compartment determine the functional output of this compartment). Work in our lab aims at reconstructing the developing Drosophila brain at both levels. Macrocircuits can be approached conveniently by reconstructing the pattern of brain lineages, which form groups of neurons whose projections form cohesive fascicles interconnecting the compartments of the larval and adult brain. The reconstruction of microcircuits requires serial section electron microscopy, due to the small size of terminal neuronal processes and their synaptic contacts. Because of the amount of labor that traditionally comes with this approach, very little is known about microcircuitry in brains across the animal kingdom. Many of the problems of serial EM reconstruction is now solvable with digital image recording and specialized software for both image acquisition and post-processing. In this paper we introduce our efforts to reconstruct the small Drosophila larval brain, and discuss our results in light of the published data on neuropile ultrastructure in other animal taxa.
PMCID: PMC3950651  PMID: 20028843
Drosophila; brain; lineage; connectivity; elctron microscopy
4.  Structure and Development of Glia in Drosophila 
Glia  2011;59(9):1237-1252.
Insect glia represents a conspicuous and diverse population of cells and plays a role in controlling neuronal progenitor proliferation, axonal growth, neuronal differentiation and maintenance, and neuronal function. Genetic studies in Drosophila have elucidated many aspects of glial structure, function and development. Just as in vertebrates, it appears as if different classes of glial cells are specialized for different functions. Based on topology and cell shape, glial cells of the central nervous system fall into three classes (Fig. 1A–C): (i) surface glia that extend sheath-like processes to wrap around the entire brain; (ii) cortex glia (also called cell body-associated glia) that encapsulate neuronal somata and neuroblasts which form the outer layer (cortex) of the central nervous system; (iii) neuropile glia that are located at the interface between the cortex and the neuropile, the central domain of the nervous system formed by the highly branched neuronal processes and their synaptic contacts. Surface glia is further subdivided into an outer, perineurial layer, and an inner, subperineurial layer. Likewise, neuropile glia comprises a class of cells that remain at the surface of the neuropile (ensheathing glia), and a second class that forms profuse lamellar processes around nerve fibers within the neuropile (astrocyte-like or reticular glia). Glia also surrounds the peripheral nerves and sensory organs; here, one also recognizes perineurial and subperineurial glia, and a third type called “wrapping glia” that most likely corresponds to the ensheathing glia of the central nervous system. Much more experimental work is needed to determine how fundamental these differences between classes of glial cells are, or how and when during development they are specified. To aid in this work the following review will briefly summarize our knowledge of the classes of glial cells encountered in the Drosophila nervous system, and then survey their development from the embryo to adult.
PMCID: PMC3950653  PMID: 21438012
5.  Conserved Genetic Pathways Controlling the Development of the Diffuse Endocrine System in Vertebrates and Drosophila 
The midgut epithelium is formed by absorptive enterocytes, secretory cells and endocrine cells. Each of these lineages is derived from the pluripotent progenitors that constitute the embryonic endoderm; the mature midgut retains pools of self-renewing stem cells that continue to produce all lineages. Recent findings in vertebrates and Drosophila shed light on the genetic mechanism that specifies the fate of the different lineages. A pivotal role is played by the Notch signaling pathway that, in a manner that appears to be very similar to the way in which Notch signaling selects neural progenitors within the neurectoderm, distinguishes the fate of secretory/endocrine cells and enterocytes. Proneural genes encoding bHLH transcription factors are expressed and required in prospective endocrine cells; activation of the Notch pathways restricts the number of these cells and promotes enterocyte development. In this review we compare the development of the intestinal endocrine cells in vertebrates and insects and summarize recent findings dealing with genetic pathways controlling this cell type.
PMCID: PMC3950663  PMID: 20005229
Drosophila; vertebrate; endocrine; gut; development; stem cell; Notch
6.  Neuronal fiber tracts connecting the brain and ventral nerve cord of the early Drosophila larva 
Using a combination of dye injections, clonal labeling, and molecular markers we have reconstructed the axonal connections between brain and ventral nerve cord of the first instar Drosophila larva. Out of the approximately 1400 neurons that form the early larval brain hemisphere, less than 50 cells have axons descending into the ventral nerve cord. Descending neurons fall into four topologically defined clusters located in the antero-medial, antero-lateral, dorsal, and baso-posterior brain, respectively. The antero-lateral cluster represents a lineage derived from a single neuroblast. Terminations of descending neurons are almost exclusively found in the anterior part of the ventral nerve cord, represented by the gnathal and thoracic neuromeres. This region also contains small numbers of neurons with axons ascending into the brain. Terminals of the ascending axons are found in the same basal brain regions that also contain descending neurons. We have mapped ascending and descending axons to the previously described scaffold of longitudinal fiber tracts that interconnect different neuromeres of the ventral nerve cord and the brain. This work provides a structural framework for functional and genetic studies addressing the control of Drosophila larval behavior by brain circuits.
PMCID: PMC3950755  PMID: 19459219
Drosophila; brain; ventral nerve cord; connectivity; ascending axons; descending axons
7.  Hematopoiesis and Hematopoietic Organs in Arthropods 
Development genes and evolution  2013;223(0):10.1007/s00427-012-0428-2.
Hemocytes (blood cells) are motile cells moving throughout the extracellular space and exist in all clades of the animal kingdom. Hemocytes play an important role in shaping the extracellular environment and in the immune response. Developmentally, hemocytes are closely related to the epithelial cells lining the vascular system (endothelia) and body cavity (mesothelia). In vertebrates and insects, common progenitors, called hemangioblasts, give rise to the endothelia and blood cells. In the adult animal, many differentiated hemocytes seem to retain the ability to proliferate; however, in most cases investigated closely, the bulk of hemocyte proliferation takes place in specialized hematopoietic organs. Hematopoietic organs provide an environment where undifferentiated blood stem cells are able to self renew, and at the same time generate offspring that differentiate into different blood cell types. Hematopoiesis in vertebrates, taking place in the bone marrow, has been subject to intensive research by immunologists and stem cell biologists. Much less is known about blood cell formation in invertebrate animals. In this review we will survey structural and functional properties of invertebrate hematopoietic organs, with a main focus on insects and other arthropod taxa. We will then discuss similarities, at the molecular and structural level, that are apparent when comparing the development of blood cells in hematopoietic organs of vertebrates and arthropods. Our comparative review is intended to elucidate aspects of the biology of blood stem cells that are more easily missed when focusing on one or a few model species.
PMCID: PMC3873168  PMID: 23319182
blood; hematopoiesis; stem cell; invertebrate; arthropod
8.  Stem cells and lineages of the intestine: a developmental and evolutionary perspective 
Development genes and evolution  2012;223(0):10.1007/s00427-012-0422-8.
The intestine consists of epithelial cells that secrete digestive enzymes and mucus (gland cells), absorb food particles (enterocytes), and produce hormones (endocrine cells). Intestinal cells are rapidly turned over and need to be replaced. In cnidarians, mitosis of differentiated intestinal cells accounts for much of the replacement; in addition, migratory, multipotent stem cells (interstitial cells) contribute to the production of intestinal cells. In other phyla, intestinal cell replacement is solely the function of stem cells entering the gut from the outside (such as in case of the neoblasts of platyhelmints) or intestinal stem cells located within the midgut epithelium (as in both vertebrates or arthropods). We will attempt in the following to review important aspects of midgut stem cells in different animal groups: where are they located, what types of lineages do they produce, and how do they develop. We will start out with a comparative survey of midgut cell types found across the animal kingdom; then briefly look at the specification of these cells during embryonic development; and finally focus on the stem cells that regenerate midgut cells during adult life. In a number of model systems, including mouse, zebrafish and Drosophila, the molecular pathways controlling ISC proliferation and the specification of intestinal cell types are under intensive investigation. We will highlight findings of the recent literature, focusing on aspects that are shared between the different models and that point at evolutionary ancient mechanisms of intestinal cell formation.
PMCID: PMC3873164  PMID: 23179635
intestine; stem cell; cell fate; development; evolution
9.  Stem cells in the context of evolution and development 
Development genes and evolution  2012;223(0):10.1007/s00427-012-0430-8.
PMCID: PMC3882194  PMID: 23223955
10.  Gene expression patterns in primary neuronal clusters of the Drosophila embryonic brain 
Gene expression patterns : GEP  2007;7(5):584-595.
The brain of Drosophila is formed by approximately 100 lineages, each lineage being derived from a stem cell-like neuroblast that segregates from the procephalic neurectoderm of the early embryo. A neuroblast map has been established in great detail for the early embryo, and a suite of molecular markers has been defined for all neuroblasts included in this map (Urbach and Technau, 2003a). However, the expression of these markers was not followed into later embryonic or larval stages, mainly due to the fact that anatomical landmarks to which expression patterns could be related had not been defined. Such markers, in the form of stereotyped clusters of neurons whose axons project along cohesive bundles (“primary axon bundles” or “PABs”) are now available (Younossi-Hartenstein et al., 2006). In the present study we have mapped the expression of molecular markers in relationship to primary neuronal clusters and their PABs. The markers we analyzed include many of the genes involved in patterning of the brain along the anteroposterior axis (cephalic gap genes, segment polarity genes) and dorso-ventral axis (columnar patterning genes), as well as genes expressed in the dorsal protocerebrum and visual system (early eye genes). Our analysis represents an important step along the way to identify neuronal lineages of the mature brain with genes expressed in the early embryo in discrete neuroblasts. Furthermore, the analysis helped us to reconstruct the morphogenetic movements that transform the two-dimensional neuroblast layer of the early embryo into the three-dimensional larval brain and provides the basis for deeper understanding of how the embryonic brain develops.
PMCID: PMC3928073  PMID: 17300994
Drosophila; embryonic brain; brain development; Hox genes; pair rule genes; segment polarity genes; head gap genes; retinal patterning genes; columnar patterning genes
11.  Development of the Drosophila entero-endocrine lineage and its specification by the Notch signaling pathway 
Developmental biology  2011;353(2):10.1016/j.ydbio.2011.01.039.
In this paper we have investigated the developmental-genetic steps that shape the entero-endocrine system of Drosophila melanogaster from the embryo to the adult. The process starts in the endoderm of the early embryo where precursors of endocrine cells and enterocytes of the larval midgut, as well as progenitors of the adult midgut, are specified by a Notch signaling-dependent mechanism. In a second step that occurs during the late larval period, enterocytes and endocrine cells of a transient pupal midgut are selected from within the clusters of adult midgut progenitors. As in the embryo, activation of the Notch pathway triggers enterocyte differentiation, and inhibits cells from further proliferation or choosing the endocrine fate. The third step of entero-endocrine cell development takes place at a mid-pupal stage. Before this time point, the epithelial layer destined to become the adult midgut is devoid of endocrine cells. However, precursors of the intestinal midgut stem cells (pISCs) are already present. After an initial phase of symmetric divisions which causes an increase in their own population size, pISCs start to spin off cells that become postmitotic and express the endocrine fate marker, Prospero. Activation of Notch in pISCs forces these cells into an enterocyte fate. Loss of Notch function causes an increase in the proliferatory activity of pISCs, as well as a higher ratio of Prospero-positive cells.
PMCID: PMC3873147  PMID: 21382366
entero-endocrine cell; midgut; stem cell; Drosophila; Notch
12.  The Drosophila larval visual system: high-resolution analysis of a simple visual neuropil 
Developmental biology  2011;358(1):10.1016/j.ydbio.2011.07.006.
The task of the visual system is to translate light into neuronal encoded information. This translation of photons into neuronal signals is achieved by photoreceptor neurons (PRs), specialized sensory neurons, located in the eye. Upon perception of light the PRs will send a signal to target neurons, which represent a first station of visual processing. Increasing complexity of visual processing stems from the number of distinct PR-subtypes and their various types of target neurons that are contacted. The visual system of the fruit fly larva represents a simple visual system (larval optic neuropil, LON) that consists of 12 PRs falling into two classes: blue-senstive PRs expressing Rhodopsin 5 (Rh5) and green-sensitive PRs expressing Rhodopsin 6 (Rh6). These afferents contact a small number of target neurons, including optic lobe pioneers (OLPs) and lateral clock neurons (LNs). We combine the use of genetic markers to label both PR-subtypes and the distinct, identifiable sets of target neurons with a serial EM reconstruction to generate a high-resolution map of the larval optic neuropil. We find that the larval optic neuropil shows a clear bipartite organization consisting of one domain innervated by PRs and one devoid of PR axons. The topology of PR projections, in particular the relationship between Rh5 and Rh6 afferents, is maintained from the nerve entering the brain to the axon terminals. The target neurons can be subdivided according to neurotransmitter or neuropeptide they use as well as the location within the brain. We further track the larval optic neuropil through development from first larval instar to its location in the adult brain as the accessory medulla.
PMCID: PMC3873161  PMID: 21781960
Drosophila; larval visual system; serial EM analysis; photoreceptors; sensory systems
13.  Fiji - an Open Source platform for biological image analysis 
Nature methods  2012;9(7):10.1038/nmeth.2019.
Fiji is a distribution of the popular Open Source software ImageJ focused on biological image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image processing algorithms. Fiji facilitates the transformation of novel algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
PMCID: PMC3855844  PMID: 22743772
14.  X11/Mint Genes Control Polarized Localization of Axonal Membrane Proteins in Vivo 
The Journal of Neuroscience  2013;33(19):8575-8586.
Mislocalization of axonal proteins can result in misassembly and/or miswiring of neural circuits, causing disease. To date, only a handful of genes that control polarized localization of axonal membrane proteins have been identified. Here we report that Drosophila X11/Mint proteins are required for targeting several proteins, including human amyloid precursor protein (APP) and Drosophila APP-like protein (APPL), to axonal membranes and for their exclusion from dendrites of the mushroom body in Drosophila, a brain structure involved in learning and memory. Axonal localization of APP is mediated by an endocytic motif, and loss of X11/Mint results in a dramatic increase in cell-surface levels of APPL, especially on dendrites. Mutations in genes required for endocytosis show similar mislocalization of these proteins to dendrites, and strongly enhance defects seen in X11/Mint mutants. These results suggest that X11/Mint-dependent endocytosis in dendrites may serve to promote the axonal localization of membrane proteins. Since X11/Mint binds to APP, and abnormal trafficking of APP contributes to Alzheimer's disease, deregulation of X11/Mint may be important for Alzheimer's disease pathogenesis.
PMCID: PMC3865514  PMID: 23658195
15.  Concomitant requirement for Notch and Jak/Stat signaling during neuro-epithelial differentiation in the Drosophila optic lobe 
Developmental biology  2010;346(2):284-295.
The optic lobe forms a prominent compartment of the Drosophila adult brain that processes visual input from the compound eye. Neurons of the optic lobe are produced during the larval period from two neuroepithelial layers called the outer and inner optic anlage (OOA, IOA). In the early larva, the optic anlagen grow as epithelia by symmetric cell division. Subsequently, neuroepithelial cells (NE) convert into neuroblasts (NB) in a tightly regulated spatio-temporal progression that starts at the edges of the epithelia and gradually move towards its centers. Neuroblasts divide at a much faster pace in an asymmetric mode, producing lineages of neurons that populate the different parts of the optic lobe. In this paper we have reconstructed the complex morphogenesis of the optic lobe during the larval period, and established a role for the Notch and Jak/Stat signaling pathways during the NE-NB conversion. After an early phase of complete overlap in the OOA, signaling activities sort out such that Jak/Stat is active in the lateral OOA which gives rise to the lamina, and Notch remains in the medial cells that form the medulla. During the third instar, a wave front of enhanced Notch activity progressing over the OOA from medial to lateral controls the gradual NE-NB conversion. Neuroepithelial cells at the medial edge of the OOA, shortly prior to becoming neuroblasts, express high levels of Delta, which activates the Notch pathway and thereby maintains the OOA in an epithelial state. Loss of Notch signaling, as well as Jak/Stat signaling, results in a premature NE-NB conversion of the OOA, which in turn has severe effects on optic lobe patterning. Our findings present the Drosophila optic lobe as a useful model to analyze the key signaling mechanisms controlling transitions of progenitor cells from symmetric (growth) to asymmetric (differentiative) divisions.
PMCID: PMC3551593  PMID: 20692248
optic lobe; neurogenesis; neuroblast; neuroepithelium; Notch; Jak/Stat
16.  TrakEM2 Software for Neural Circuit Reconstruction 
PLoS ONE  2012;7(6):e38011.
A key challenge in neuroscience is the expeditious reconstruction of neuronal circuits. For model systems such as Drosophila and C. elegans, the limiting step is no longer the acquisition of imagery but the extraction of the circuit from images. For this purpose, we designed a software application, TrakEM2, that addresses the systematic reconstruction of neuronal circuits from large electron microscopical and optical image volumes. We address the challenges of image volume composition from individual, deformed images; of the reconstruction of neuronal arbors and annotation of synapses with fast manual and semi-automatic methods; and the management of large collections of both images and annotations. The output is a neural circuit of 3d arbors and synapses, encoded in NeuroML and other formats, ready for analysis.
PMCID: PMC3378562  PMID: 22723842
17.  The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the Drosophila mesoderm 
Developmental Biology  2011;353(1):105-118.
Blood progenitors arise from a pool of pluripotential cells (“hemangioblasts”) within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos, is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. We show in this paper that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters (“cardiogenic clusters”) that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the Mitogen Activated Protein Kinase (MAPK) pathway by Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor (FGFR) is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts.
PMCID: PMC3312814  PMID: 21382367
Drosophila; blood; hemangioblast; Notch; MAPK; EGF; FGF
18.  Boule-like genes regulate male and female gametogenesis in the flatworm Macrostomum lignano 
Developmental Biology  2011;357(1):117-132.
Members of the DAZ (Deleted in AZoospermia) gene family are important players in the process of gametogenesis and their dysregulation accounts for 10% of human male infertility. Boule, the ancestor of the family, is mainly involved in male meiosis in most organisms. With the exception of Drosophila and C. elegans, nothing is known on the function of boule in non-vertebrate animals. In the present study, we report on three boule orthologues in the flatworm Macrostomum lignano. We demonstrate that macbol1 and macbol2 are expressed in testes whilst macbol3 is expressed in ovaries and developing eggs. Macbol1 RNAi blocked spermatocyte differentiation whereas macbol2 showed no effect upon RNAi treatment. Macbol3 RNAi resulted in aberrant egg maturation and led to female sterility. We further demonstrated the evolutionary functional conservation of macbol1 by introducing this gene into Drosophila bol1 mutants. Macbol1 was able to rescue the progression of fly meiotic divisions. In summary, our findings provide evidence for an involvement of boule genes in male and female gamete development in one organism. Furthermore, boule gene function is shown here for the first time in a lophotrochozoan. Our results point to a more diverse functional assignment of boule genes. Therefore, a better understanding of boule function in flatworms can help to elucidate the molecular mechanisms of and concomitant infertility in higher organisms including humans.
► The function of boule genes is shown for the first time in a lophotrochozoan. ► There are three distinct macbol orthologues in the flatworm Macrostomum lignano. ► Macbol1 and macbol2 are testis-specific, macbol3 is essential for oogenesis. ► RNAi arrests male meiosis (macbol1) and impedes egg maturation (macbol3). ► Macbol1 is able to rescue meiotic divisions in boule-mutant Drosophila melanogaster.
PMCID: PMC3158854  PMID: 21740899
Boule; Planaria; Flatworms; Platyhelminthes; Germline; Hermaphrodite
19.  A development-based compartmentalization of the Drosophila central brain 
The Journal of comparative neurology  2010;518(15):2996-3023.
The neuropile of the Drosophila brain is subdivided into anatomically discrete compartments. Compartments are rich in terminal neurite branching and synapses; they are the neuropile domains in which signal processing takes place. Compartment boundaries are defined by more or less dense layers of glial cells, as well as long neurite fascicles. These fascicles are formed during the larval period when the approximately 100 neuronal lineages that constitute the Drosophila central brain differentiate. Each lineage forms an axon tract with a characteristic trajectory in the neuropile; groups of spatially related tracts congregate into the brain fascicles that can be followed from the larva throughout metamorphosis into the adult stage. In this paper we provide a map of the adult brain compartments and the relevant fascicles defining compartmental boundaries. We have identified the neuronal lineages contributing to each fascicle, which allowed us to directly compare compartments of the larval and adult brain. Most adult compartments can be recognized already in the early larval brain where they form a “protomap” of the later adult compartments. Our analysis highlights the morphogenetic changes shaping the Drosophila brain; the data will be important for studies that link early acting genetic mechanisms to the adult neuronal structures and circuits controlled by these mechanisms.
PMCID: PMC2905803  PMID: 20533357
Drosophila; brain; lineages; connectivity; mapping
20.  Bazooka mediates secondary axon morphology in Drosophila brain lineages 
Neural Development  2011;6:16.
In the Drosophila brain, neural lineages project bundled axon tracts into a central neuropile. Each lineage exhibits a stereotypical branching pattern and trajectory, which distinguish it from other lineages. In this study, we used a multilineage approach to explore the neural function of the Par-complex member Par3/Bazooka in vivo. Drosophila bazooka is expressed in post-mitotic neurons of the larval brain and localizes within neurons in a lineage-dependent manner. The fact that multiple GAL4 drivers have been mapped to several lineages of the Drosophila brain enables investigation of the role of Bazooka from larval to adult stages Bazooka loss-of-function (LOF) clones had abnormal morphologies, including aberrant pathway choice of ventral projection neurons in the BAla1 lineage, ectopic branching in the DALv2 and BAmv1 lineages, and excess BLD5 lineage axon projections in the optic medulla. Exogenous expression of Bazooka protein in BAla1 neurons rescued defective guidance, supporting an intrinsic requirement for Bazooka in the post-mitotic neuron. Elimination of the Par-complex member Par6 recapitulated Bazooka phenotypes in some but not all lineages, suggesting that the Par complex functions in a lineage-dependent manner, and that Bazooka may act independently in some lineages. Importantly, this study highlights the potential of using a multilineage approach when studying gene function during neural development in Drosophila.
PMCID: PMC3107162  PMID: 21524279
21.  Identifying neuronal lineages of Drosophila by sequence analysis of axon tracts 
The Drosophila brain is formed by an invariant set of lineages, each of which is derived from a unique neural stem cell (neuroblast) and forms a genetic and structural unit of the brain. The task of reconstructing brain circuitry at the level of individual neurons can be made significantly easier by assigning neurons to their respective lineages. In this paper we address the automatization of neuron and lineage identification. We focused on the Drosophila brain lineages at the larval stage when they form easily recognizable secondary axon tracts (SATs) that were previously partially characterized. We now generated an annotated digital database containing all lineage tracts reconstructed from five registered wild-type brains, at higher resolution and including some that were previously not characterized. We developed a method for SAT structural comparisons based on a dynamic programming approach akin to nucleotide sequence alignment, and a machine learning classifier trained on the annotated database of reference SATs. We quantified the stereotypy of SATs by measuring the residual variability of aligned wild-type SATs. Next, we employed our method for the identification of SATs within wild-type larval brains, and found it highly accurate (93–99 %). The method proved highly robust for the identification of lineages in mutant brains, and in brains that differed in developmental time or labeling. We describe for the first time an algorithm that quantifies neuronal projection stereotypy in the Drosophila brain, and use the algorithm for automatic neuron and lineage recognition.
PMCID: PMC2905806  PMID: 20519528
22.  Patterns of growth, axonal extension and axonal arborization of neuronal lineages in the developing Drosophila brain 
Developmental biology  2009;335(2):289-304.
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.
PMCID: PMC2785225  PMID: 19538956
Drosophila; brain; lineage; pathfinding; connectivity
23.  The arborization pattern of engrailed-positive neural lineages reveal neuromere boundaries in the Drosophila brain neuropile 
The Drosophila brain is a highly complex structure composed of thousands of neurons that are interconnected in numerous exquisitely organized neuropile structures such as the mushroom bodies, central complex, antennal lobes, and other specialized neuropiles. While the neurons of the insect brain are known to derive in a lineage-specific fashion from a stereotyped set of segmentally organized neuroblasts, the developmental origin and neuromeric organization of the neuropile formed by these neurons is still unclear. In this report, we use genetic labeling techniques to characterize the neuropile innervation pattern of engrailed-expressing brain lineages of known neuromeric origin. We show that the neurons of these lineages project to and form most arborizations, in particular all of their proximal branches, in the same brain neuropile compartments in embryonic, larval and adult stages. Moreover, we show that engrailed-positive neurons of differing neuromeric origin respect boundaries between neuromere-specific compartments in the brain. This is confirmed by an analysis of the arborization pattern of empty spiracles-expressing lineages. These findings indicate that arborizations of lineages deriving from different brain neuromeres innervate a non-overlapping set of neuropile compartments. This supports a model for neuromere-specific brain neuropile, in which a given lineage forms its proximal arborizations predominantly in the compartments that correspond to its neuromere of origin.
PMCID: PMC2879895  PMID: 19711412
engrailed; neuropile; compartment; neuromere; arborization; lineage
24.  Drosophila cortex and neuropile glia influence secondary axon tract growth, pathfinding, and fasciculation in the developing larval brain 
Developmental biology  2009;334(2):355-368.
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.
PMCID: PMC2776086  PMID: 19646433
glia; Drosophila; brain; lineage
25.  Genome-Wide Analyses Reveal a Role for Peptide Hormones in Planarian Germline Development 
PLoS Biology  2010;8(10):e1000509.
Genomic/peptidomic analyses of the planarian Schmidtea mediterranea identifies >200 neuropeptides and uncovers a conserved neuropeptide required for proper maturation and maintenance of the reproductive system.
Bioactive peptides (i.e., neuropeptides or peptide hormones) represent the largest class of cell-cell signaling molecules in metazoans and are potent regulators of neural and physiological function. In vertebrates, peptide hormones play an integral role in endocrine signaling between the brain and the gonads that controls reproductive development, yet few of these molecules have been shown to influence reproductive development in invertebrates. Here, we define a role for peptide hormones in controlling reproductive physiology of the model flatworm, the planarian Schmidtea mediterranea. Based on our observation that defective neuropeptide processing results in defects in reproductive system development, we employed peptidomic and functional genomic approaches to characterize the planarian peptide hormone complement, identifying 51 prohormone genes and validating 142 peptides biochemically. Comprehensive in situ hybridization analyses of prohormone gene expression revealed the unanticipated complexity of the flatworm nervous system and identified a prohormone specifically expressed in the nervous system of sexually reproducing planarians. We show that this member of the neuropeptide Y superfamily is required for the maintenance of mature reproductive organs and differentiated germ cells in the testes. Additionally, comparative analyses of our biochemically validated prohormones with the genomes of the parasitic flatworms Schistosoma mansoni and Schistosoma japonicum identified new schistosome prohormones and validated half of all predicted peptide-encoding genes in these parasites. These studies describe the peptide hormone complement of a flatworm on a genome-wide scale and reveal a previously uncharacterized role for peptide hormones in flatworm reproduction. Furthermore, they suggest new opportunities for using planarians as free-living models for understanding the reproductive biology of flatworm parasites.
Author Summary
Flatworms cause diseases affecting hundreds of millions of people, so understanding what influences their reproductive activity is of fundamental importance. Neurally derived signals have been suggested to coordinate sexual reproduction in free-living flatworms, yet the neuroendocrine signaling repertoire has not been characterized comprehensively for any flatworm. Neuropeptides are a large diverse group of cell-cell signaling molecules and play many roles in vertebrate reproductive development; however, little is known about their function in reproductive development among invertebrates. Here we use biochemical and bioinformatic techniques to identify bioactive peptides in the genome of the planarian flatworm Schmidtea mediterranea and identify 51 genes encoding >200 peptides. Analysis of these genes in both sexual and asexual strains of S. mediterranea identified a neuropeptide Y superfamily member as important for the normal development and maintenance of the planarian reproductive system. We suggest that understanding peptide hormone function in planarian reproduction could have practical implications in the treatment of parasitic flatworms.
PMCID: PMC2953531  PMID: 20967238

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