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.
optic lobe; neurogenesis; neuroblast; neuroepithelium; Notch; Jak/Stat
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.
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.
Drosophila; blood; hemangioblast; Notch; MAPK; EGF; FGF
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.
Boule; Planaria; Flatworms; Platyhelminthes; Germline; Hermaphrodite
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.
Drosophila; brain; lineages; connectivity; mapping
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.
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.
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.
Drosophila; brain; lineage; pathfinding; connectivity
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.
engrailed; neuropile; compartment; neuromere; arborization; lineage
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.
glia; Drosophila; brain; lineage
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.
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.
A new software package allows for dense electron microscopy reconstructions of neuronal networks in the fruit fly brain, and reveals specific differences in microcircuits between insects and vertebrates.
The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.
Brains contain a vast number of connections between neurons, termed synapses. The precise patterns of these synaptic contacts form the structural underpinning of electrical microcircuits responsible for animal behavior. Due to their small size, synaptic contacts can be conclusively shown using only high-resolution electron microscopy (EM). Therefore, complete series of ultrathin sections are required to reconstruct neuronal microcircuitry. The acquisition and analysis of EM sections (with 15,000 sections per millimeter of tissue) is practical only by computer-assisted means. In this article, we demonstrate the utility of the software package TrakEM2 to model interconnections of nerve fibers from consecutive EM sections and to efficiently reconstruct the neural networks encountered in different parts of a small brain, the early larval brain of the fruit fly Drosophila melanogaster. Neuronal networks are composed of patterns of axons and dendrites (neuronal extensions that transmit and receive signals, respectively), and using TrakEM2, we describe the most common motifs they form. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect brains.
The fly brain is formed by approximately hundred paired lineages of neurons, each lineage derived from one neuroblast. Embryonic neuroblasts undergo a small number of divisions and produce the primary neurons that form the functioning larval brain. In the larva, neuroblasts produce the secondary lineages that make up the bulk of the adult brain. Axons of a given secondary lineage fasciculate with each other and form a discrete bundle, the secondary axon tract (SAT). Secondary axon tracts prefigure the long axon connections of the adult brain, and therefore pathway choices of SATs made in the larva determine adult brain circuitry. Drosophila Shotgun/E-cadherin (DE-cad) and its binding partner Armadillo/β-catenin (β-cat) are expressed in newly born secondary neurons and their axons. The fact that the highly diverse, yet invariant pattern of secondary lineages and SATs has been recently mapped in the wild-type brain enabled us to investigate the role of DE-cad and β-cat with the help of MARCM clones. Clones were validated by their absence of DE-cad immuno-reactivity. The most significant phenotype consists in the defasciculation and an increased amount of branching of SATs at the neuropile-cortex boundary, as well as subtle changes in the trajectory of SATs within the neuropile. In general, only a fraction of mutant clones in a given lineage showed structural abnormalities. Furthermore, although they all globally express DE-cad and β-cat, lineages differ in their requirement for DE-cad function. Some lineages never showed morphological abnormalities in MARCM clones, whereas others reacted with abnormal branching and changes in SAT trajectory at a high frequency. We conclude that DE-cad/β-cat form part of the mechanism that control branching and trajectory of axon tracts in the larval brain.
DE-Cadherin; Drosophila; brain; lineage; pathfinding
The neuroarchitecture of Acoela has been at the center of morphological debates. Some authors, using immunochemical tools, suggest that the nervous system in Acoela is organized as a commissural brain that bears little resemblance to the central, ganglionic type brain of other flatworms, and bilaterians in general. Others, who used histological staining on paraffin sections, conclude that it is a compact structure (an endonal brain; e.g., Raikova 2004; von Graff 1891; Delage Arch Zool Exp Gén 4:109-144, 1886). To address this question with modern tools, we have obtained images from serial transmission electron microscopic sections of the entire hatchling of Symsagittifera roscoffensis. In addition, we obtained data from wholemounts of hatchlings labeled with markers for serotonin and tyrosinated tubulin. Our data show that the central nervous system of a juvenile S. roscoffensis consists of an anterior compact brain, formed by a dense, bilobed mass of neuronal cell bodies surrounding a central neuropile. The neuropile flanks the median statocyst and contains several types of neurites, classified according to their types of synaptic vesicles. The neuropile issues three pairs of nerve cords that run at different dorso-ventral positions along the whole length of the body. Neuronal cell bodies flank the cords, and neuromuscular synapses are abundant. The TEM analysis also reveals different classes of peripheral sensory neurons and provides valuable information about the spatial relationships between neurites and other cell types within the brain and nerve cords. We conclude that the acoel S. roscoffensis has a central brain that is comparable in size and architecture to the brain of other (rhabditophoran) flatworms.
Acoel; Central nervous system; Nerve cords; Nerve net; Sensory receptors; Transmission electronic microscopy; 3D modeling software
Motivation: Tiled serial section Transmission Electron Microscopy (ssTEM) is increasingly used to describe high-resolution anatomy of large biological specimens. In particular in neurobiology, TEM is indispensable for analysis of synaptic connectivity in the brain. Registration of ssTEM image mosaics has to recover the 3D continuity and geometrical properties of the specimen in presence of various distortions that are applied to the tissue during sectioning, staining and imaging. These include staining artifacts, mechanical deformation, missing sections and the fact that structures may appear dissimilar in consecutive sections.
Results: We developed a fully automatic, non-rigid but as-rigid-as-possible registration method for large tiled serial section microscopy stacks. We use the Scale Invariant Feature Transform (SIFT) to identify corresponding landmarks within and across sections and globally optimize the pose of all tiles in terms of least square displacement of these landmark correspondences. We evaluate the precision of the approach using an artificially generated dataset designed to mimic the properties of TEM data. We demonstrate the performance of our method by registering an ssTEM dataset of the first instar larval brain of Drosophila melanogaster consisting of 6885 images.
Availability: This method is implemented as part of the open source software TrakEM2 (http://www.ini.uzh.ch/∼acardona/trakem2.html) and distributed through the Fiji project (http://pacific.mpi-cbg.de).
In Drosophila, neurons of the central nervous system are grouped into units called lineages. Each lineage contains cells derived from a single neuroblast. Due to its clonal nature, the Drosophila brain is a valuable model system to study neuron development and circuit formation. To better understand the mechanisms underlying brain development, genetic manipulation tools can be utilized within lineages to visualize, knock down, or over-express proteins. Here, we will introduce the formation and development of lineages, discuss how one can utilize this model system, offer a comprehensive list of known lineages and their respective markers, and then briefly review studies that have utilized Drosophila neural lineages with a look at how this model system can benefit future endeavors.
Drosophila; Brain; Lineage; Neuroblast; Axon
We combine Gal4/UAS, FLP/FRT and fluorescent reporters to generate cell clones that provide spatial, temporal, and genetic information about the origins of individual cells in Drosophila. We name this combination the Gal4 Technique for Real-time and Clonal Expression (G-TRACE). The approach should allow for screening and the identification of real-time and lineage-traced expression patterns on a genomic scale.
The Drosophila melanogaster lymph gland is a haematopoietic organ1–3 in which pluripotent blood cell progenitors proliferate and mature into differentiated haemocytes. Previous work4 has defined three domains, the medullary zone, the cortical zone and the posterior signalling centre (PSC), within the developing third-instar lymph gland. The medullary zone is populated by a core of undifferentiated, slowly cycling progenitor cells, whereas mature haemocytes comprising plasmatocytes, crystal cells and lamellocytes are peripherally located in the cortical zone. The PSC comprises a third region that was first defined as a small group of cells expressing the Notch ligand Serrate5. Here we show that the PSC is specified early in the embryo by the homeotic gene Antennapedia (Antp) and expresses the signalling molecule Hedgehog. In the absence of the PSC or the Hedgehog signal, the precursor population of the medullary zone is lost because cells differentiate prematurely. We conclude that the PSC functions as a haematopoietic niche that is essential for the maintenance of blood cell precursors in Drosophila. Identification of this system allows the opportunity for genetic manipulation and direct in vivo imaging of a haematopoietic niche interacting with blood precursors.
We examined the development of the nervous system in the rhopalium, a medusa-specific sensory structure, in Aurelia sp.1 (Cnidaria, Scyphozoa) using confocal microscopy. The rhopalial nervous system appears primarily ectodermal and contains neurons immunoreactive to antibodies against tyrosinated tubulin, taurine, GLWamide, and FMRFamide. The rhopalial nervous system develops in an ordered manner: the presumptive gravity-sensing organ, consisting of the lithocyst and the touch plate, differentiates first; the “marginal center,” which controls swimming activity, second; and finally, the ocelli, the presumptive photoreceptors. At least seven bilaterally arranged neuronal clusters consisting of sensory and ganglion cells and their neuronal processes became evident in the rhopalium during metamorphosis to the medusa stage. Our analysis provides an anatomical framework for future gene expression and experimental studies of development and functions of scyphozoan rhopalia.
Electronic supplementary material
The online version of this article (doi:10.1007/s00427-009-0291-y) contains supplementary material, which is available to authorized users.
Cnidaria; Scyphozoa; Rhopalia; Nervous system development; Medusa
Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa - the Catenulida, the Acoelomorpha and the Rhabditophora - have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives.
Summary: High-resolution, three-dimensional (3D) imaging of large biological specimens generates massive image datasets that are difficult to navigate, annotate and share effectively. Inspired by online mapping applications like GoogleMaps™, we developed a decentralized web interface that allows seamless navigation of arbitrarily large image stacks. Our interface provides means for online, collaborative annotation of the biological image data and seamless sharing of regions of interest by bookmarking. The CATMAID interface enables synchronized navigation through multiple registered datasets even at vastly different scales such as in comparisons between optical and electron microscopy.
Embryonic expression patterns for 6,003 (44%) of the 13,659 protein-coding genes identified in the Drosophila melanogaster genome were documented, of which 40% show tissue-restricted expression.
Cell and tissue specific gene expression is a defining feature of embryonic development in multi-cellular organisms. However, the range of gene expression patterns, the extent of the correlation of expression with function, and the classes of genes whose spatial expression are tightly regulated have been unclear due to the lack of an unbiased, genome-wide survey of gene expression patterns.
We determined and documented embryonic expression patterns for 6,003 (44%) of the 13,659 protein-coding genes identified in the Drosophila melanogaster genome with over 70,000 images and controlled vocabulary annotations. Individual expression patterns are extraordinarily diverse, but by supplementing qualitative in situ hybridization data with quantitative microarray time-course data using a hybrid clustering strategy, we identify groups of genes with similar expression. Of 4,496 genes with detectable expression in the embryo, 2,549 (57%) fall into 10 clusters representing broad expression patterns. The remaining 1,947 (43%) genes fall into 29 clusters representing restricted expression, 20% patterned as early as blastoderm, with the majority restricted to differentiated cell types, such as epithelia, nervous system, or muscle. We investigate the relationship between expression clusters and known molecular and cellular-physiological functions.
Nearly 60% of the genes with detectable expression exhibit broad patterns reflecting quantitative rather than qualitative differences between tissues. The other 40% show tissue-restricted expression; the expression patterns of over 1,500 of these genes are documented here for the first time. Within each of these categories, we identified clusters of genes associated with particular cellular and developmental functions.
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 is surprisingly variable. Immuno-histochemical and electron microscopic 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 antenno-cerebral 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.
Trachea; glia; brain; Drosophila; larva; pattern
Flatworms are characterized by an outstanding stem cell system. These stem cells (neoblasts) can give rise to all cell types including germ cells and power the exceptional regenerative capacity of many flatworm species. Macrostomum lignano is an emerging model system to study stem cell biology of flatworms. It is complementary to the well-studied planarians because of its small size, transparency, simple culture maintenance, the basal taxonomic position and its less derived embryogenesis that is more closely related to spiralians. The development of cell-, tissue- and organ specific markers is necessary to further characterize the differentiation potential of flatworm stem cells. Large scale in situ hybridization is a suitable tool to identify possible markers. Distinguished genes identified in a large scale screen in combination with manipulation of neoblasts by hydroxyurea or irradiation will advance our understanding of differentiation and regulation of the flatworm stem cell system.
We have set up a protocol for high throughput large scale whole mount in situ hybridization for the flatworm Macrostomum lignano. In the pilot screen, a number of cell-, tissue- or organ specific expression patterns were identified. We have selected two stem cell- and germ cell related genes – macvasa and macpiwi – and studied effects of hydroxyurea (HU) treatment or irradiation on gene expression. In addition, we have followed cell proliferation using a mitosis marker and bromodeoxyuridine labeling of S-phase cells after various periods of HU exposure or different irradiation levels. HU mediated depletion of cell proliferation and HU induced reduction of gene expression was used to generate a cDNA library by suppressive subtractive hybridization. 147 differentially expressed genes were sequenced and assigned to different categories.
We show that Macrostomum lignano is a suitable organism to perform high throughput large scale whole mount in situ hybridization. Genes identified in such screens – together with BrdU/H3 labeling – can be used to obtain information on flatworm neoblasts.
As a first step to creating a comprehensive atlas of gene-expression patterns during Drosophila embryogenesis, 2,179 genes have been examinded by in situ hybridization to fixed Drosophila embryos. Of the genes assayed, 63.7% displayed dynamic expression patterns that were documented with 25,690 digital photomicrographs of individual embryos.
Cell-fate specification and tissue differentiation during development are largely achieved by the regulation of gene transcription.
As a first step to creating a comprehensive atlas of gene-expression patterns during Drosophila embryogenesis, we examined 2,179 genes by in situ hybridization to fixed Drosophila embryos. Of the genes assayed, 63.7% displayed dynamic expression patterns that were documented with 25,690 digital photomicrographs of individual embryos. The photomicrographs were annotated using controlled vocabularies for anatomical structures that are organized into a developmental hierarchy. We also generated a detailed time course of gene expression during embryogenesis using microarrays to provide an independent corroboration of the in situ hybridization results. All image, annotation and microarray data are stored in publicly available database. We found that the RNA transcripts of about 1% of genes show clear subcellular localization. Nearly all the annotated expression patterns are distinct. We present an approach for organizing the data by hierarchical clustering of annotation terms that allows us to group tissues that express similar sets of genes as well as genes displaying similar expression patterns.
Analyzing gene-expression patterns by in situ hybridization to whole-mount embryos provides an extremely rich dataset that can be used to identify genes involved in developmental processes that have been missed by traditional genetic analysis. Systematic analysis of rigorously annotated patterns of gene expression will complement and extend the types of analyses carried out using expression microarrays.