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Harald Hutter

Publications and source records attributed to Harald Hutter.

17 recordsLinked to original sources
PubMed2007

Axon guidance genes identified in a large-scale RNAi screen using the RNAi-hypersensitive Caenorhabditis elegans strain nre-1(hd20) lin-15b(hd126).

The navigation of axons toward their targets is a highly dynamic and precisely regulated process during nervous system development. The molecular basis of this navigation process is only partly understood. In Caenorhabditis elegans, we isolated the RNAi-hypersensitive strain nre-1(hd20) lin-15b(hd126), which allows us to phenocopy axon guidance defects of known genes by feeding RNAi. We used this mutant strain to systematically screen 4,577 genes on chromosomes I and III for axon guidance phenotypes. We identified 93 genes whose down-regulation led to penetrant ventral cord fasciculation defects or motoneuron commissure outgrowth defects. These genes encode various classes of proteins, ranging from secreted or putative cell surface proteins to transcription factors controlling gene expression. A majority of the genes is evolutionary conserved and previously uncharacterized. In addition, we found axon guidance functions for known genes like pry-1, a component of the Wnt-signaling pathway, and ced-1, a receptor required for the engulfment of neurons undergoing apoptosis during development. Our screen provides insights into molecular pathways operating during the generation of neuronal circuits and provides a basis for a more detailed analysis of gene networks regulating axon navigation.

Animals↗
PubMed2006

A differential cytolocalization assay for analysis of macromolecular assemblies in the eukaryotic cytoplasm.

We have developed a differential cytolocalization assay (DCLA) that allows the observation of cytoplasmic protein/protein interactions in vivo. In the DCLA, interactions are visualized as a relocalization of a green fluorescent protein-tagged "prey" by a membrane-bound "bait." This assay was tested and utilized in Caenorhabditis elegans to probe interactions among proteins involved in RNA interference (RNAi) and nonsense-mediated decay (NMD) pathways. Several previously documented interactions were confirmed with DCLA, whereas uniformly negative results were obtained in several controls in which no interaction was expected. Novel interactions were also observed, including the association of SMG-5, a protein required for NMD, to several components of the RNAi pathway. The DCLA can be readily carried out under diverse conditions, allowing a dynamic assessment of protein interactions in vivo. We used this property to test a subset of the RNAi and NMD interactions in animals in which proteins central to each mechanism were mutated; several key associations in each machinery that can occur in vivo in the absence of a functional process were identified.

Amino Acid Sequence↗
PubMed2006

Hemicentin assembly in the extracellular matrix is mediated by distinct structural modules.

Hemicentins are conserved extracellular matrix proteins characterized by a single von Willebrand A (VWA) domain at the amino terminus, a long stretch (>40) of tandem immunoglobulin domains, multiple tandem epidermal growth factors (EGFs), and a single fibulin-like carboxyl-terminal module. In Caenorhabditis elegans, hemicentin is secreted from muscle and gonadal leader cells and assembles at multiple locations into discrete tracks that constrict broad regions of cell contact into adhesive and flexible line-shaped junctions. To determine hemicentin domains critical for function and assembly, we have expressed fragments of hemicentin as GFP tagged fusion proteins in C. elegans. We find that a hemicentin fragment containing the VWA domain can target to multiple assembly sites when expressed under the control of either endogenous hemicentin regulatory sequences or the muscle-specific unc-54 promoter. A hemicentin fragment containing the EGF and fibulin-like carboxyl-terminal modules can co-assemble with existing hemicentin polymers in wild-type animals but has no detectable function in the absence of endogenous hemicentin. The data suggest that the VWA domain is a cell binding domain whose function is to target hemicentin to sites of assembly and the EGF/fibulin-like carboxyl-terminal modules constitute an assembly domain that mediates direct interactions between hemicentin monomers during the hemicentin assembly process.

Animals↗
PubMed2006

Synaptic tetraspan vesicle membrane proteins are conserved but not needed for synaptogenesis and neuronal function in Caenorhabditis elegans.

Tetraspan vesicle membrane proteins (TVPs) comprise a major portion of synaptic vesicle proteins, yet their contribution to the synaptic vesicle cycle is poorly understood. TVPs are grouped in three mammalian gene families: physins, gyrins, and secretory carrier-associated membrane proteins (SCAMPs). In Caenorhabditis elegans, only a single member of each of these families exists. These three nematode TVPs colocalize to the same vesicular compartment when expressed in mammalian cells, suggesting that they could serve overlapping functions. To examine their function, C. elegans null mutants were isolated for each gene, and a triple mutant was generated. Surprisingly, these animals develop normally and exhibit normal neuronal architecture and synaptic contacts. In addition, functions of the motor and sensory systems are normal as determined by pharmacological, chemotaxis, and thermotaxis assays. Finally, direct electrophysiological analysis of the neuromuscular junction revealed no phenotype in the TVP mutants. We therefore conclude that TVPs are not needed for the basic neuronal machinery and instead may contribute to subtle higher order functions.

Animals↗
PubMed2006

Global cell sorting in the C. elegans embryo defines a new mechanism for pattern formation.

4D microscopic observations of Caenorhabditis elegans development show that the nematode uses an unprecedented strategy for development. The embryo achieves pattern formation by sorting cells, through far-ranging movements, into coherent regions before morphogenesis is initiated. This sorting of cells is coupled to their particular fate. If cell identity is altered by experiment, cells are rerouted to positions appropriate to their new fates even across the whole embryo. This cell behavior defines a new mechanism of pattern formation, a mechanism that is also found in other animals. We call this new mechanism "cell focusing". When the fate of cells is changed, they move to new positions which also affect the shape of the body. Thus, this process is also important for morphogenesis.

Animals↗
PubMed2006

AST-1, a novel ETS-box transcription factor, controls axon guidance and pharynx development in C. elegans.

Neurons send out axons and dendrites over large distances into target areas where they eventually form synapses with selected target cells. Axonal navigation is controlled by a variety of extracellular signals and neurons express receptors only for that subset of signals they need to navigate to their own target area. How the expression of axon guidance receptors is regulated is not understood. In genetic screens for mutants with axon guidance defects, we identified an ETS-domain transcription factor, AST-1, specifically required for axon navigation in certain classes of interneurons. In addition, ast-1 has a role in the differentiation of the ventral cord pioneer neuron AVG. Outside the nervous system, ast-1 is essential for morphogenesis of the pharynx. Ast-1 is transiently expressed in several classes of neurons (including AVG) during neuronal differentiation with a peak expression during late stages of neuronal differentiation and axon outgrowth. Ast-1 genetically interacts with other transcription factors controlling neuronal differentiation like lin-11 and zag-1 as well as components of the netrin pathway suggesting that ast-1 might control the expression of components of the netrin signal transduction machinery.

Amino Acid Sequence↗
PubMed2006

Fluorescent reporter methods.

The identification and cloning of the green fluorescent protein (GFP) from jellyfish marks the beginning of a new era of fluorescent reporters. In Caenorhabditis elegans, genetically encoded markers like the fluorescent proteins of the GFP family became the reporter of choice for gene expression studies and protein localization. The small size and transparency of the worm allows the visualization of in vivo dynamics, which increases the number of potential applications for fluorescent reporters tremendously. In combination with subcellular tags, GFP can be used to label subcellular structures like synapses allowing novel approaches to study developmental processes like synapse formation. Other fluorescent labels like small organic dyes, which are in widespread use in cell culture systems, are rarely used in C. elegans owing to difficulties in applying these labels through the impenetrable cuticle or eggshell of the animal. A notable exception is the use of lipophilic dyes, which are taken up by certain sensory neurons in the intact animal and can be introduced into the embryo after puncturing of the egg shell. This chapter covers the use of fluorescent dyes and fluorescent proteins in C. elegans. Emphasis is placed on microscopic techniques including wide field and confocal microscopy as well as time-lapse recordings. The use of fluorescent proteins as transgenic markers and image processing of fluorescence images are briefly discussed.

Animals↗
PubMed2005

Fibulin-1C and Fibulin-1D splice variants have distinct functions and assemble in a hemicentin-dependent manner.

Fibulins are a family of extracellular glycoproteins associated with basement membranes and elastic fibers in vertebrates. Conservation of the fibulin-1 gene throughout metazoan evolution includes fibulin-1C and fibulin-1D alternate splice variants, although little is known about variant specific functions that would justify this striking structural conservation. We have therefore investigated the structure, localization and loss-of-function phenotype specific to both fibulin-1 variants in C. elegans. We find that fibulin-1C has specific roles during pharynx, intestine, gonad and muscle morphogenesis, being required to regulate cell shape and adhesion, whereas fibulin-1D assembles in flexible polymers that connect the pharynx and body-wall-muscle basement membranes. The assembly of fibulin-1C and fibulin-1D in multiple locations is dependent upon the presence of hemicentin, a recently described extracellular member of the immunoglobulin superfamily. We suggest that the distinct developmental roles and hemicentin-dependent assembly for fibulin-1 splice variants demonstrated here may be relevant to fibulin-1 and possibly other fibulin family members in non-nematode species.

Abdominal Muscles↗
PubMed2005

Novel genes controlling ventral cord asymmetry and navigation of pioneer axons in C. elegans.

The ventral cord in C. elegans is the major longitudinal axon tract containing essential components of the motor circuit. In genetic screens using transgenic animals expressing neuron specific GFP reporters, we identified twelve genes required for the correct outgrowth of interneuron axons of the motor circuit. In mutant animals, axons fail to navigate correctly towards the ventral cord or fail to fasciculate correctly within the ventral cord. Several of those mutants define previously uncharacterized genes. Two of the genes, ast-4 and ast-7, are involved in the generation of left-right asymmetry of the two ventral cord axon tracts. Three other genes specifically affect pioneer-follower relationships between early and late outgrowing axons, controlling either differentiation of a pioneer neuron (lin-11) or the ability of axons to follow a pioneer (ast-2, unc-130). Navigation of the ventral cord pioneer neuron AVG itself is defective in ast-4, ast-6 and unc-130 mutants. Correlation of these defects with navigation defects in different classes of follower axons revealed a true pioneer role for AVG in the guidance of interneurons in the ventral cord. Taken together, these genes provide a basis to address different aspects of axon navigation within the ventral cord of C. elegans.

Animals↗
PubMed2004

A novel nuclear receptor/coregulator complex controls C. elegans lipid metabolism, larval development, and aging.

Environmental cues transduced by an endocrine network converge on Caenorhabditis elegans nuclear receptor DAF-12 to mediate arrest at dauer diapause or continuous larval development. In adults, DAF-12 selects long-lived or short-lived modes. How these organismal choices are molecularly specified is unknown. Here we show that coregulator DIN-1 and DAF-12 physically and genetically interact to instruct organismal fates. Homologous to human corepressor SHARP, DIN-1 comes in long (L) and short (S) isoforms, which are nuclear localized but have distinct functions. DIN-1L has embryonic and larval developmental roles. DIN-1S, along with DAF-12, regulates lipid metabolism, larval stage-specific programs, diapause, and longevity. Epistasis experiments reveal that din-1S acts in the dauer pathways downstream of lipophilic hormone, insulin/IGF, and TGFbeta signaling, the same point as daf-12. We propose that the DIN-1S/DAF-12 complex serves as a molecular switch that implements slow life history alternatives in response to diminished hormonal signals.

Aging↗
PubMed2004

UNC-39, the C. elegans homolog of the human myotonic dystrophy-associated homeodomain protein Six5, regulates cell motility and differentiation.

Mutations in the unc-39 gene of C. elegans lead to migration and differentiation defects in a subset of mesodermal and ectodermal cells, including muscles and neurons. Defects include mesodermal specification and differentiation as well a neuronal migration and axon pathfinding defects. Molecular analysis revealed that unc-39 corresponds to the previously named gene ceh-35 and that the UNC-39 protein belongs to the Six4/5 family of homeodomain transcription factors and is similar to human Six5, a protein implicated in the pathogenesis of type I myotonic dystrophy (DM1). We show that human Six5 and UNC-39 are functional homologs, suggesting that further characterization of the C. elegans unc-39 gene might provide insight into the etiology of DM1.

Amino Acid Sequence↗
PubMed2003

Extracellular cues and pioneers act together to guide axons in the ventral cord of C. elegans.

The ventral cord is the major longitudinal axon tract in C. elegans containing essential components of the motor circuit. Previous studies have shown that axons grow out sequentially and that there is a single pioneer for the right axon tract which is important for the correct outgrowth of follower axons. Here, the dependencies between early and late outgrowing axons in the ventral cord were studied systematically with laser ablation experiments and a detailed analysis of mutants using multi-color GFP markers. Different classes of axon were affected to a different extent when the AVG pioneer neuron was eliminated. In the majority of the animals, axons were able to grow out normally even in the absence of the pioneer, suggesting that its presence is not absolutely essential for the correct outgrowth of follower axons. The transcription factor LIN-11 was found to be essential for the differentiation and pioneering function of the AVG neuron. UNC-30 appears to play a similar role for the PVP pioneer neurons. Later outgrowing axons typically do not simply follow earlier outgrowing ones, but subtle dependencies between certain groups of early and late outgrowing axons do exist. Different groups of axons growing in the same axon bundle apparently use different combinations of guidance cues for their navigation and can navigate largely independently.

Animals↗
PubMed2003

The astacin protein family in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, 40 genes code for astacin-like proteins (nematode astacins, NAS). The astacins are metalloproteases present in bacteria, invertebrates and vertebrates and serve a variety of physiological functions like digestion, hatching, peptide processing, morphogenesis and pattern formation. With the exception of one distorted pseudogene, all the other C. elegans astacins are expressed and are evidently functional. For 13 genes we found splicing patterns differing from the Genefinder predictions in WormBase, sometimes markedly. The GFP expression pattern for NAS-4 shows a specific localization in anterior pharynx cells and in the whole digestive tract (as the secreted form). In contrast, NAS-7 is found in the head of adult hermaphrodites, but not in pharynx cells or in the lumen of the digestive tract. In embryos, NAS-7 fluorescence becomes detectable just before hatching. In C. elegans astacins, three basic structural and functional moieties can be discerned: a prepro portion, the central catalytic chain and long C-terminal extensions with presumably regulatory functions. Within the regulatory moiety, EFG-like, CUB, SXC, and TSP-1 domains can be distinguished. Based on structural differences of the regulatory unit we established six NAS subgroups, which seemingly represented different functional and evolutionary clusters. This pattern deduced exclusively from the domain arrangement in the regulatory moiety is perfectly reflected in an evolutionary tree constructed solely from amino acid sequence information of the catalytic chain. Related catalytic chains tend to have related regulatory extensions. The notable gene, NAS-39 shows a striking resemblance to human BMP-1 and the tolloids.

Animals↗
PubMed2003

Laminin alpha subunits and their role in C. elegans development.

Laminins are heterotrimeric (alpha/beta/gamma) glycoproteins that form a major polymer within basement membranes. Different alpha, beta and gamma subunits can assemble into various laminin isoforms that have different, but often overlapping, distributions and functions. In this study, we examine the contributions of the laminin alpha subunits to the development of C. elegans. There are two alpha, one beta and one gamma laminin subunit, suggesting two laminin isoforms that differ by their alpha subunit assemble in C. elegans. We find that near the end of gastrulation and before other basement membrane components are detected, the alpha subunits are secreted between primary tissue layers and become distributed in different patterns to the surfaces of cells. Mutations in either alpha subunit gene cause missing or disrupted extracellular matrix where the protein normally localizes. Cell-cell adhesions are abnormal: in some cases essential cell-cell adhesions are lacking, while in other cases, cells inappropriately adhere to and invade neighboring tissues. Using electron microscopy, we observe adhesion complexes at improper cell surfaces and disoriented cytoskeletal filaments. Cells throughout the animal show defective differentiation, proliferation or migration, suggesting a general disruption of cell-cell signaling. The results suggest a receptor-mediated process localizes each secreted laminin to exposed cell surfaces and that laminin is crucial for organizing extracellular matrix, receptor and intracellular proteins at those surfaces. We propose this supramolecular architecture regulates adhesions and signaling between adjacent tissues.

Animals↗
PubMed2003

zag-1, a Zn-finger homeodomain transcription factor controlling neuronal differentiation and axon outgrowth in C. elegans.

The nervous system consists of diverse subtypes of neurons, whose identities must be specified during development. One important aspect of the differentiation program of neurons is the expression of the appropriate set of genes controlling axon pathway selection. We have identified a novel Znfinger/homeodomain containing transcription factor, zag-1, required for particular aspects of axonal pathfinding. In zag-1 mutants, motorneuron commissures either branch prematurely or fail to branch at the correct point. Ventral cord interneurons show defects in the guidance towards the ventral cord and also in the ventral cord. Several neurons misexpress differentiation markers, including glutamate receptor subunits and chemosensory receptors. zag-1 is expressed transiently in embryonic and postembryonic neurons during differentiation as well as in some mesodermal tissues. Null mutants of zag-1 are unable to swallow food and die as L1 larvae with a starved appearance, indicating that zag-1 has an additional role in pharynx development. The vertebrate homolog, deltaEF1, is highly conserved and known to act as transcriptional repressor in various tissues. Our data indicate that zag-1 also acts as transcriptional repressor controlling important aspects of terminal differentiation of neurons.

Amino Acid Sequence↗
PubMed2002

Tetraspan vesicle membrane proteins: synthesis, subcellular localization, and functional properties.

Tetraspan vesicle membrane proteins (TVPs) are characterized by four transmembrane regions and cytoplasmically located end domains. They are ubiquitous and abundant components of vesicles in most, if not all, cells of multicellular organisms. TVP-containing vesicles shuttle between various membranous compartments and are localized in biosynthetic and endocytotic pathways. Based on gene organization and amino acid sequence similarities TVPs can be grouped into three distinct families that are referred to as physins, gyrins, and secretory carrier-associated membrane proteins (SCAMPs). In mammals synaptophysin, synaptoporin, pantophysin, and mitsugumin29 constitute the physins, synaptogyrin 1-4 the gyrins, and SCAMP1-5 the SCAMPs. Members of each family are cell-type-specifically synthesized resulting in unique patterns of TVP coexpression and subcellular colocalization. TVP orthologs have been identified in most multicellular organisms, including diverse animal and plant species, but have not been detected in unicellular organisms. They are subject to protein modification, most notably to phosphorylation, and are part of multimeric complexes. Experimental evidence is reviewed showing that TVPs contribute to vesicle trafficking and membrane morphogenesis.

Amino Acid Sequence↗