<Emphasis Type="Italic">Wnt</Emphasis> gene loss in flatworms

Wnt genes encode secreted glycoproteins that act in cell–cell signalling to regulate a wide array of developmental processes, ranging from cellular differentiation to axial patterning. Discovery that canonical Wnt/β-catenin signalling is responsible for regulating head/tail specification in planarian regeneration has recently highlighted their importance in flatworm (phylum Platyhelminthes) development, but examination of their roles in the complex development of the diverse parasitic groups has yet to be conducted. Here, we characterise Wnt genes in the model tapeworm Hymenolepis microstoma and mine genomic resources of free-living and parasitic species for the presence of Wnts and downstream signalling components. We identify orthologs through a combination of BLAST and phylogenetic analyses, showing that flatworms have a highly reduced and dispersed complement that includes orthologs of only five subfamilies (Wnt1, Wnt2, Wnt4, Wnt5 and Wnt11) and fewer paralogs in parasitic flatworms (5–6) than in planarians (9). All major signalling components are identified, including antagonists and receptors, and key binding domains are intact, indicating that the canonical (Wnt/β-catenin) and non-canonical (planar cell polarity and Wnt/Ca²⁺) pathways are functional. RNA-Seq data show expression of all Hymenolepis Wnts and most downstream components in adults and larvae with the notable exceptions of wnt1, expressed only in adults, and wnt2 expressed only in larvae. The distribution of Wnt subfamilies in animals corroborates the idea that the last common ancestor of the Cnidaria and Bilateria possessed all contemporary Wnts and highlights the extent of gene loss in flatworms.     

Hox genes and the parasitic flatworms: new opportunities, challenges and lessons from the free-living

Research into the roles played by Hox and related homeotic gene families in the diverse and complex developmental programmes exhibited by parasitic flatworms (Platyhelminthes) can hardly be said to have begun, and thus presents considerable opportunity for new research. Although featured in some of the earliest screens for homeotic genes outside Drosophila and mice, surveys in parasitic flatworms are few in number and almost nothing is yet known of where or when the genes are expressed during ontogeny. This contrasts sharply with a significant body of literature concerning Hox genes in free-living flatworms which have long served as models for the study of regeneration and the maintenance of omnipotent cell lines. Nevertheless, available information suggests that the complement of Hox genes and other classes of homeobox-containing genes in parasitic flatworms is typical of their free-living cousins and of other members of the Lophotrochozoa. Recent work on Schistosoma combined with information on Hox gene expression in planarians indicates that at least some disruption of the clustered genomic arrangement of the genes, as well as of the strict spatial and temporal colinear patterns of expression typical in other groups, may be characteristic of flatworms. However, available data on the genomic arrangement and expression of flatworm Hox genes is so limited at present that such generalities are highly tenuous. Moreover, a basic underlying pattern of colinearity is still observed in their spatial expression patterns making them suitable as cell or region-specific markers. I discuss a number of fundamental developmental questions and some of the challenges to addressing them in relation to each of the major parasitic lineages. In addition, I present newly characterized Hox genes from the model tapeworm Hymenolepis and analyze these by Bayesian inference together with >100 Hox and ParaHox homeodomains of flatworms and select lophotrochozoan taxa, providing a phylogenetic scaffold for their identification.     

The Zn Finger protein Iguana impacts Hedgehog signaling by promoting ciliogenesis

Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian Schmidtea mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates.     

Coordinated modulation of cellular signaling through ligand-gated ion channels in Hydra vulgaris (Cnidaria, Hydrozoa)

Cnidarians lack well developed organs, but they have evolved the molecular and cellular components needed to assemble a nervous system. The apparent 'simplicity' of the cnidarian nervous net does not occur at the cellular level, but rather in the organisation of conducting systems. Cnidarian neurons are in fact electrically excitable, show the typical extended morphology and are connected by chemical synapses or gap junctions. They have been regarded as peptidergic, given the wealth of neuropeptides generally distributed along neurites and in cell bodies, supporting the hypothesis of a modulatory role in neurotransmission. However, the presence of clear-cored, as well as dense-cored synaptic vesicles in cnidarian neurons suggests both fast and slow synaptic transmission mechanisms. In fact, biochemical and functional evidence indicates that classical neurotransmitters and their metabolic partners are present in cnidarian tissues, where they are involved in coordinating motility and behavior. We have identified and characterized in Hydra tissues receptors to the inhibitory and excitatory amino acid neurotransmitters, GABA, glycine and NMDA, that are similar to mammalian ionotropic receptors in terms of their biochemical and pharmacological properties. These receptors appear to regulate pacemaker activities and their physiological correlates; in the live animal, they also affect feeding behavior, namely the duration and termination of the response elicited by reduced glutathione, with opposite actions of GABA and glycine or NMDA, respectively. These results suggest that modulation of cellular signaling through ligand-gated-ion channels is an ancient characteristic in the animal kingdom, and that the pharmacological properties of these receptors have been highly conserved during evolution.     

Ultrastructure of glial cells in the nervous system of <Emphasis Type="Italic">Grillotia erinaceus</Emphasis>

Until now, there has been no answer to the question of whether specialized glial cells exist in the nervous system of platyhelminths. The identification of these cells in parasitic flatworms is difficult due to their organization as parenchymal animals. The goal of this study was to reveal and describe structural elements corresponding to the term glia in the CNS of the parasitic flatworm Grillotia erinaceus (Cestoda: Trypanorhyncha). Three types of glial cells are revealed. The first type consists of fibroblast-like cells located in the cerebral ganglia that contain fibrils and excrete onto the surface fibrillar material and possess desmosomes; the presumable function of fibroblast-like glial cells is the isolation and support of ganglionar neurons. Glial cells of the second type form a myelin-like envelope of giant axons and bulbar nerves of the scolex and have laminar cytoplasm; they are numerous and exceed the number of neurons in the composition of nerves. Glial cells of the third type form multilayer envelopes in the main nerve cords and make contacts with the excretory epithelium; however, specialized junctions with neurons were not found. The existence of glia in other free living and parasitic flatworms is discussed.     

Identification and Expression of the Family of Classical Protein-Tyrosine Phosphatases in Zebrafish

Protein-tyrosine phosphatases (PTPs) have an important role in cell survival, differentiation, proliferation, migration and other cellular processes in conjunction with protein-tyrosine kinases. Still relatively little is known about the function of PTPs in vivo. We set out to systematically identify all classical PTPs in the zebrafish genome and characterize their expression patterns during zebrafish development. We identified 48 PTP genes in the zebrafish genome by BLASTing of human PTP sequences. We verified all in silico hits by sequencing and established the spatio-temporal expression patterns of all PTPs by in situ hybridization of zebrafish embryos at six distinct developmental stages. The zebrafish genome encodes 48 PTP genes. 14 human orthologs are duplicated in the zebrafish genome and 3 human orthologs were not identified. Based on sequence conservation, most zebrafish orthologues of human PTP genes were readily assigned. Interestingly, the duplicated form of ptpn23, a catalytically inactive PTP, has lost its PTP domain, indicating that PTP activity is not required for its function, or that ptpn23b has lost its PTP domain in the course of evolution. All 48 PTPs are expressed in zebrafish embryos. Most PTPs are maternally provided and are broadly expressed early on. PTP expression becomes progressively restricted during development. Interestingly, some duplicated genes retained their expression pattern, whereas expression of other duplicated genes was distinct or even mutually exclusive, suggesting that the function of the latter PTPs has diverged. In conclusion, we have identified all members of the family of classical PTPs in the zebrafish genome and established their expression patterns. This is the first time the expression patterns of all members of the large family of PTP genes have been established in a vertebrate. Our results provide the first step towards elucidation of the function of the family of classical PTPs.     

Fine structure of glial cells in the central nervous system of the tapeworm Grillotia erinaceus (Cestoda: Trypanorhyncha)

The problem of glial cells existing in parasitic and free living flatworms is correlated with organization of parenchyma in platyhelmintes. In the contrary to the widespread opinion that myelin-like envelopes and glial cells do not exist in the nervous system of parasitic flatworms, it has been shown by ultrastructural researches that Amphilina foliacea (Cestoda, Amphilinidea) has well developed glial cells and myelin-like envelopes in the ganglia and main cords, which include both glial cells and intercellular components. The aim of our research was to reveal and investigate in details structural components corresponding to the concept of the glial cell in the CNS of Grillotia erinaceus (Cestoda: Trypanorhyncha). Three types of glial cells have been found. The first type is the fibroblast-like glial cells; cells locate in the cerebral ganglion, contain in cytoplasm and extract out fibrillar matrix, form desmosomes and have supporting function. The glial cells of the second type form myeline-like envelope of the giant axons and bulbar nerves in scolex and have laminar cytoplasm. These cells are numerous and exceed in number the neurons bodies into the nerve. The glial cells of the third type form multilayer envelopes in the main nerve cords; extra cellular fibers and gap-junctions take place between the layers. There are contacts between the glial cells of the third type and excretory epithelium but specialized contacts with neurons have been not found. The existing of glial cells in free living and parasitic flatworms is discussed.     

The components required for amino acid neurotransmitter signaling are present in adipose tissues

The adipocyte does not only serve as fuel storage but produces and secretes compounds with modulating effects on food intake and energy homeostasis. Although there is firm evidence for a centrally mediated regulation of adipocyte function via the autonomous nervous system, little is known about signaling between adipocytes. Amino acid neurotransmitters are candidates for such paracrine signaling. Here, we applied immunohistochemistry to detect components required for amino acid transmitter signaling in rat fat depots. In interscapular brown adipose tissue as well as in interscapular, mesenteric, perirenal, and epididymal white adipose tissues, we demonstrate robust immunosignals for the excitatory neurotransmitter glutamate, the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), and the GABA-synthesizing enzyme glutamate decarboxylase (GAD) isoforms GAD65 and GAD67. Moreover, all adipose tissues stained for the vesicular glutamate transporter VGLUT1 and the vesicular GABA transporter VGAT in addition to the vesicle marker synaptophysin. Electron microscopic immunocytochemistry showed that VGLUT1 and VGAT, but not VGLUT2 or VGLUT3, are localized in vesicular organelles in adipocytes. The receptors for glutamate (subunits GluR2/3 and NR1 but not mGluR2) and for GABA (GABA(A)Ralpha2) were present in the adipocytes. The presence of glutamate, GABA, their vesicular transporters, and their receptors indicates a paracrine signaling role for amino acids in adipose tissues.     

Identification of motifs that are conserved in 12 Drosophila species and regulate midline glia vs. neuron expression

Functional complexity of the central nervous system (CNS) is reflected by the large number and diversity of genes expressed in its many different cell types. Understanding the control of gene expression within cells of the CNS will help reveal how various neurons and glia develop and function. Midline cells of Drosophila differentiate into glial cells and several types of neurons and also serve as a signaling center for surrounding tissues. Here, we examine regulation of the midline gene, wrapper, required for both neuron–glia interactions and viability of midline glia. We identify a region upstream of wrapper required for midline expression that is highly conserved (87%) between 12 Drosophila species. Site-directed mutagenesis identifies four motifs necessary for midline glial expression: (1) a Single-minded/Tango binding site, (2) a motif resembling a pointed binding site, (3) a motif resembling a Sox binding site, and (4) a novel motif. An additional highly conserved 27 bp are required to restrict expression to midline glia and exclude it from midline neurons. These results suggest short, highly conserved genomic sequences flanking Drosophila midline genes are indicative of functional regulatory regions and that small changes within these sequences can alter the expression pattern of a gene.     

Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence

Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2) from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements.     

Human Intellectual Disability Genes Form Conserved Functional Modules in Drosophila

Intellectual Disability (ID) disorders, defined by an IQ below 70, are genetically and phenotypically highly heterogeneous. Identification of common molecular pathways underlying these disorders is crucial for understanding the molecular basis of cognition and for the development of therapeutic intervention strategies. To systematically establish their functional connectivity, we used transgenic RNAi to target 270 ID gene orthologs in the Drosophila eye. Assessment of neuronal function in behavioral and electrophysiological assays and multiparametric morphological analysis identified phenotypes associated with knockdown of 180 ID gene orthologs. Most of these genotype-phenotype associations were novel. For example, we uncovered 16 genes that are required for basal neurotransmission and have not previously been implicated in this process in any system or organism. ID gene orthologs with morphological eye phenotypes, in contrast to genes without phenotypes, are relatively highly expressed in the human nervous system and are enriched for neuronal functions, suggesting that eye phenotyping can distinguish different classes of ID genes. Indeed, grouping genes by Drosophila phenotype uncovered 26 connected functional modules. Novel links between ID genes successfully predicted that MYCN, PIGV and UPF3B regulate synapse development. Drosophila phenotype groups show, in addition to ID, significant phenotypic similarity also in humans, indicating that functional modules are conserved. The combined data indicate that ID disorders, despite their extreme genetic diversity, are caused by disruption of a limited number of highly connected functional modules.     

Identification and Expression Analysis of Zebrafish Glypicans during Embryonic Development

Heparan sulfate Proteoglycans (HSPG) are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG’s, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.