Biomedical subjects

D H Hall

Publications and source records attributed to D H Hall.

At least 19 recordsLinked to original sources

PubMed2001

The Caenorhabditis elegans autosomal dominant polycystic kidney disease gene homologs lov-1 and pkd-2 act in the same pathway.

Autosomal dominant polycystic kidney disease (ADPKD) strikes 1 in 1000 individuals and often results in end-stage renal failure. Mutations in either PKD1 or PKD2 account for 95% of all cases [1-3]. It has recently been demonstrated that polycystin-1 and polycystin-2 (encoded by PKD1 and PKD2, respectively) assemble to form a cation channel in vitro [4]. Here we determine that the Caenorhabditis elegans PKD1 and PKD2 homologs, lov-1 [5] and pkd-2, act in the same pathway in vivo. Mutations in either lov-1 or pkd-2 result in identical male sensory behavioral defects. Also, pkd-2;lov-1 double mutants are no more severe than either of the single mutants, indicating that lov-1 and pkd-2 act together. LOV-1::GFP and PKD-2::GFP are expressed in the same male-specific sensory neurons and are concentrated in cilia and cell bodies. Cytoplasmic, nonnuclear staining in cell bodies is punctate, suggesting that one pool of PKD-2 is localized to intracellular membranes while another is found in sensory cilia. In contrast to defects in the C. elegans autosomal recessive PKD gene osm-5 [6-8], the cilia of lov-1 and pkd-2 single mutants and of lov-1;pkd-2 double mutants are normal as judged by electron microscopy, demonstrating that lov-1 and pkd-2 are not required for ultrastructural development of male-specific sensory cilia.

Animals↗

PubMed2001

mua-3, a gene required for mechanical tissue integrity in Caenorhabditis elegans, encodes a novel transmembrane protein of epithelial attachment complexes.

Normal locomotion of the nematode Caenorhabditis elegans requires transmission of contractile force through a series of mechanical linkages from the myofibrillar lattice of the body wall muscles, across an intervening extracellular matrix and epithelium (the hypodermis) to the cuticle. Mutations in mua-3 cause a separation of the hypodermis from the cuticle, suggesting this gene is required for maintaining hypodermal-cuticle attachment as the animal grows in size postembryonically. mua-3 encodes a predicted 3,767 amino acid protein with a large extracellular domain, a single transmembrane helix, and a smaller cytoplasmic domain. The extracellular domain contains four distinct protein modules: 5 low density lipoprotein type A, 52 epidermal growth factor, 1 von Willebrand factor A, and 2 sea urchin-enterokinase-agrin modules. MUA-3 localizes to the hypodermal hemidesmosomes and to other sites of mechanically robust transepithelial attachments, including the rectum, vulva, mechanosensory neurons, and excretory duct/pore. In addition, it is shown that MUA-3 colocalizes with cytoplasmic intermediate filaments (IFs) at these sites. Thus, MUA-3 appears to be a protein that links the IF cytoskeleton of nematode epithelia to the cuticle at sites of mechanical stress.

Animals↗

PubMed2001

Regulation of neurotransmitter vesicles by the homeodomain protein UNC-4 and its transcriptional corepressor UNC-37/groucho in Caenorhabditis elegans cholinergic motor neurons.

Motor neuron function depends on neurotransmitter release from synaptic vesicles (SVs). Here we show that the UNC-4 homeoprotein and its transcriptional corepressor protein UNC-37 regulate SV protein levels in specific Caenorhabditis elegans motor neurons. UNC-4 is expressed in four classes (DA, VA, VC, and SAB) of cholinergic motor neurons. Antibody staining reveals that five different vesicular proteins (UNC-17, choline acetyltransferase, Synaptotagmin, Synaptobrevin, and RAB-3) are substantially reduced in unc-4 and unc-37 mutants in these cells; nonvesicular neuronal proteins (Syntaxin, UNC-18, and UNC-11) are not affected, however. Ultrastructural analysis of VA motor neurons in the mutant unc-4(e120) confirms that SV number in the presynaptic zone is reduced ( approximately 40%) whereas axonal diameter and synaptic morphology are not visibly altered. Because the UNC-4-UNC-37 complex has been shown to mediate transcriptional repression, we propose that these effects are performed via an intermediate gene. Our results are consistent with a model in which this unc-4 target gene ("gene-x") functions at a post-transcriptional level as a negative regulator of SV biogenesis or stability. Experiments with a temperature-sensitive unc-4 mutant show that the adult level of SV proteins strictly depends on unc-4 function during a critical period of motor neuron differentiation. unc-4 activity during this sensitive larval stage is also required for the creation of proper synaptic inputs to VA motor neurons. The temporal correlation of these events may mean that a common unc-4-dependent mechanism controls both the specificity of synaptic inputs as well as the strength of synaptic outputs for these motor neurons.

Animals↗

PubMed2001

Evidence that RME-1, a conserved C. elegans EH-domain protein, functions in endocytic recycling.

In genetic screens for new endocytosis genes in Caenorhabditis elegans we identified RME-1, a member of a conserved class of Eps15-homology (EH)-domain proteins. Here we show that RME-1 is associated with the periphery of endocytic organelles, which is consistent with a direct role in endocytic transport. Endocytic defects in rme-1 mutants indicate that the protein is likely to have a function in endocytic recycling. Evidence from studies of mammalian RME-1 also points to a function for RME-1 in recycling, specifically in the exit of membrane proteins from recycling endosomes. These studies show a conserved function in endocytic recycling for the RME-1 family of EH proteins.

Animals↗

PubMed2001

Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia.

The function of epithelial cell sheets depends on the integrity of specialized cell-cell junctions that connect neighbouring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR-HMP (cadherin-catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, and LET-413 mediates the rapid accumulation of both DLG-1 and AJM-1 in the apical domain. In the absence of both dlg-1 and let-413 function AJM-1 is almost completely lost from apical junctions in embryos, whereas HMP-1 (alpha-catenin) localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical junctional domain in C. elegans.

Animals↗

PubMed2001

A putative GDP-GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans.

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc-5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc-5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc-5 interacts genetically with mig-2, which encodes Rho GTPase. These results suggest that EXC-5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.

Amino Acid Sequence↗

PubMed2001

Immuno-EM localization of GFP-tagged yolk proteins in C. elegans using microwave fixation.

Because of the presence of a low-permeability cuticle covering the animal, fixation of C. elegans tissue for immunoelectron microscopy has proved very difficult. Here we applied a microwave fixation protocol to improve penetration of fixatives before postembedding immunogold labeling. Using this technique, we were able to successfully localize several components of yolk (YP170) trafficking in both wild-type and transgenic strains expressing a vitellogenin::green fluorescent protein fusion (YP170::GFP). Green fluorescent protein (GFP) and its variants are commonly used as markers to localize proteins in transgenic C. elegans using fluorescence microscopy. We have developed a robust method to localize GFP at the EM level. This procedure is applicable to the characterization of transgenic strains in which GFP is used to mark particular proteins or cell types and will undoubtedly be very useful for high-resolution analysis of marked structures.

Animals↗

PubMed2000

Expression of ram-5 in the structural cell is required for sensory ray morphogenesis in Caenorhabditis elegans male tail.

Tissue morphogenesis requires complex cellular interaction and communication. The sensory ray in the Caenorhabditis elegans male tail has a simple cellular make-up and a non-essential function, thus providing an ideal model for studying the mechanisms guiding morphogenesis. We present here the analysis of a novel gene, ram-5, mutations of which are characterized by abnormal lumpy rays in the male tail. Microscopic analysis and behavioral studies revealed that lumpy rays contain operational sensory neurons. However, abnormalities were observed in the hypodermis and structural cells as well as in appositions between these two cell types. Molecular cloning and expression studies revealed that the ram-5 gene encodes a transmembrane protein localized in sensory ray support cells, the structural cells. Expression of ram-5 in these cells is required for normal ray morphogenesis. ram-5-dependent cell-cell communication is implicated in organizing the structural cell and the hypodermis, potentially through adhesion at the structural cell-hypodermal cell border.

Amino Acid Sequence↗

PubMed2000

Gap junctions in the chicken pineal gland.

The chicken pineal gland, which contains a heterogeneous cell population, sustains a circadian rhythm of activity. Synchronization of cellular activity of heterogeneous cells might be facilitated by gap junctional intercellular channels which are permeable to ions and second messengers. To test this possibility, we looked for morphologically identifiable gap junctions between the different pineal cells, used antibodies and cDNA probes to screen for the presence of connexins, and tested for functional intercellular coupling. By transmission electron microscopy and immunocytochemistry, gap junctions and connexins were observed between pinealocyte cell bodies, stromal cells, astrocytes, and astrocyte and pinealocyte processes. Two gap junctional proteins, connexin43 and connexin45, were detected by immunocytochemistry, immunoblotting and RNA blot analysis. Functional intercellular coupling was observed in the gland by transfer of low molecular weight dyes. Dye transferred between homologous and heterologous cells. These data suggest that homologous and heterologous gap junctions may provide a mechanism for coordination of the cellular responses of the elements of the biological clock which are induced by lighting cues to produce the circadian rhythm of pineal activity.

Animals↗

PubMed2000

The LIM homeobox gene ceh-14 confers thermosensory function to the AFD neurons in Caenorhabditis elegans.

In Caenorhabditis elegans three pairs of neurons, AFD, AIY, and AIZ, play a key role in thermosensation. The LIM homeobox gene ceh-14 is expressed in the AFD thermosensory neurons. ceh-14 mutant animals display athermotactic behaviors, although the neurons are still present and differentiated. Two other LIM homeobox genes, ttx-3 and lin-11, function in the two interneurons AIY and AIZ, respectively. Thus, the three key thermosensory neurons are specified by three different LIM homeobox genes. ceh-14 ttx-3 lin-11 triple mutant animals have a basic cryophilic thermotaxis behavior indicative of a second thermotaxis pathway. Misexpression of ceh-14 in chemosensory neurons can restore thermotactic behavior without impairing the chemosensory function. Thus, ceh-14 confers thermosensory function to neurons.

Amino Acid Sequence↗

PubMed1999

Cystic canal mutants in Caenorhabditis elegans are defective in the apical membrane domain of the renal (excretory) cell.

The excretory cell extends a tubular process, or canal, along the basolateral surface of the epidermis to form the nematode renal epithelium. This cell can undergo normal tubulogenesis in isolated cell culture. Mutations in 12 genes cause excretory canal cysts in Caenorhabditis elegans. Genetic interactions, and their similar phenotypes, suggest these genes may encode functionally related proteins. Depending upon genotype and individual canal, defects range from focal cysts, flanked by normal width segments, to regional cysts involving the entire tubule. Oftentimes the enlarged regions are convoluted or partially septated. In mutants with very large cysts, renal function is measurably impaired. Based on histology and ultrastructure, canal cysts likely result from defects of the apical membrane domain. These mutants provide a model of tubulocystic disease without hyperplasia or basement membrane abnormalities. Similar apical mechanisms could regulate tubular morphology of vertebrate nephrons.

Animals↗

PubMed1999

Ultrastructural features of the adult hermaphrodite gonad of Caenorhabditis elegans: relations between the germ line and soma.

Genetic and embryological experiments have established the Caenorhabditis elegans adult hermaphrodite gonad as a paradigm for studying the control of germline development and the role of soma-germline interactions. We describe ultrastructural features relating to essential germline events and the soma-germline interactions upon which they depend, as revealed by electron and fluorescence microscopy. Gap junctions were observed between oocytes and proximal gonadal sheath cells that contract to ovulate the oocyte. These gap junctions must be evanescent since individual oocytes lose contact with sheath cells when they are ovulated. In addition, proximal sheath cells are coupled to each other by gap junctions. Within proximal sheath cells, actin/myosin bundles are anchored to the plasma membrane at plaque-like structures we have termed hemi-adherens junctions, which in turn are closely associated with the gonadal basal lamina. Gap junctions and hemi-adherens junctions are likely to function in the coordinated series of contractions required to ovulate the mature oocyte. Proximal sheath cells are fenestrated with multiple small pores forming conduits from the gonadal basal lamina to the surface of the oocyte, passing through the sheath cell. In most instances where pores occur, extracellular yolk particles penetrate the gonadal basal lamina to directly touch the underlying oocytes. Membrane-bounded yolk granules were generally not found in the sheath cytoplasm by either electron microscopy or fluorescence microscopy. Electron microscopic immunocytochemistry was used to confirm and characterize the appearance of yolk protein in cytoplasmic organelles within the oocyte and in free particles in the pseudocoelom. The primary route of yolk transport apparently proceeds from the intestine into the pseudocoelom, then through sheath pores to the surface of the oocyte, where endocytosis occurs. Scanning electron microscopy was used to directly visualize the distal tip cell which extends tentacle-like processes that directly contact distal germ cells. These distal tip cell processes are likely to play a critical role in promoting germline mitosis. Scanning electron microscopy also revealed thin filopodia extending from the distal sheath cells. Distal sheath filopodia were also visualized using a green fluorescent protein reporter gene fusion and confocal microscopy. Distal sheath filopodia may function to stretch the sheath over the distal arm.

Animals↗

PubMed1999

Morphogenesis of the Caenorhabditis elegans male tail tip.

Using electron microscopy and immunofluorescent labeling of adherens junctions, we have reconstructed the changes in cell architecture and intercellular associations that occur during morphogenesis of the nematode male tail tip. During late postembryonic development, the Caenorhabditis elegans male tail is reshaped to form a copulatory structure. The most posterior hypodermal cells in the tail define a specialized, sexually dimorphic compartment in which cells fuse and retract in the male, changing their shape from a tapered cone to a blunt dome. Developmental profiles using electron microscopy and immunofluorescent staining suggest that cell fusions are initiated at or adjacent to adherens junctions. Anterior portions of the tail tip cells show the first evidence of retractions and fusions, consistent with our hypothesis that an anterior event triggers these morphogenetic events. Available mutations that interfere with morphogenesis implicate particular regulatory pathways and suggest loci at which evolutionary changes could have produced morphological diversity.

Animals↗

PubMed1998

Active currents regulate sensitivity and dynamic range in C. elegans neurons.

Little is known about the physiology of neurons in Caenorhabditis elegans. Using new techniques for in situ patch-clamp recording in C. elegans, we analyzed the electrical properties of an identified sensory neuron (ASER) across four developmental stages and 42 unidentified neurons at one stage. We find that ASER is nearly isopotential and fails to generate classical Na+ action potentials. Rather, ASER displays a high sensitivity to input currents coupled to a depolarization-dependent reduction in sensitivity that may endow ASER with a wide dynamic range. Voltage clamp revealed depolarization-activated K+ and Ca2+ currents that contribute to high sensitivity near the zero-current potential. The depolarization-dependent reduction in sensitivity can be attributed to activation of K+ current at voltages where it dominates the net membrane current. The voltage dependence of membrane current was similar in all neurons examined, suggesting that C. elegans neurons share a common mechanism of sensitivity and dynamic range.

Animals↗

PubMed1998

The role of the p53 protein in the selective vulnerability of the inner retina to transient ischemia.

PURPOSE: To determine whether the p53 protein plays a role in the selective vulnerability of the inner retina to transient ischemia. METHODS: Transient retinal ischemia was induced using a high intraocular pressure (HIOP) model in the Sprague-Dawley rat for 60 minutes. Histopathologic outcome was determined 7 days after ischemia. In addition, analysis for evidence for apoptosis (TdT-dUTP terminal nick-end label [TUNEL] staining) and p53 protein expression (immunohistochemistry) was performed at several points during the reperfusion period. In a separate set of experiments, wild-type mice and two groups of transgenic mice, one homozygous and the other heterozygous for the p53 null gene, were also subjected to HIOP for 60 minutes, and histopathology was performed 7 days later. RESULTS: At 7 days subsequent to 60 minutes of ischemia in the rat, there was marked thinning of the inner retinal layers. There were scattered TUNEL-positive cells within the inner retina, peaking at 24 to 48 hours and persisting for at least 7 days. p53 immunochemistry demonstrated elevated protein levels within the inner retina; this finding peaked at 24 to 48 hours but was no longer present at 4 days after ischemia. TUNEL staining of the inner retina of the mouse was most prominent 24 hours subsequent to ischemia but persisted at 48 hours. Seven days subsequent to 60 minutes of ischemia in the wild-type and transgenic mice, histopathologic evaluation demonstrated preservation of the retinal histoarchitecture in the heterozygous group compared with the wild-type or homozygous animals. CONCLUSIONS: These data further support the hypothesis that the delayed cell death that occurs after transient retinal ischemia is, in part, apoptotic. In addition, they suggest a role for the p53 protein in the selective vulnerability of the inner retina to transient ischemia. p53 protein may be a target for future therapeutic agents in the treatment of disorders of the retina where ischemia plays a pathogenetic role.

Animals↗

PubMed1997

The POU gene ceh-18 promotes gonadal sheath cell differentiation and function required for meiotic maturation and ovulation in Caenorhabditis elegans.

In Caenorhabditis elegans, specialized contractile myoepithelial cells of the somatic gonad, the gonadal sheath cells, are closely apposed to oocytes and are required for normal meiotic maturation and ovulation. Previously we found that mutations in the ceh-18 gene, which encodes a POU-class homeoprotein expressed in sheath cells, result in oocyte defects. To determine the basis for these oocyte defects, we have used time-lapse video Nomarski microscopy to observe meiotic maturation, ovulation, and early embryogenesis in ceh-18 mutants. In ceh-18 mutants sheath cell contractions are weaker, less frequent, and uncoordinated throughout the sequence of ovulation events, and ovulation is defective. Defective ovulation can result in the formation of endomitotic oocytes in the gonad, the formation of haploid embryos, and reversals in embryonic polarity. ceh-18 mutant oocytes exhibit defects prior to nuclear envelope breakdown, suggesting that they are physiologically different from the wild type. We observed delays in meiotic maturation, as well as maturation out of the normal spatial and temporal sequence, suggesting that proximal sheath cells directly or indirectly promote and spatially restrict meiotic maturation. Analysis of sheath cell differentiation in ceh-18 mutants using antibodies to proteins of the contractile apparatus reveals that although contractile proteins are expressed, the sheath cells appear disorganized. Transmission electron microscopy reveals that ceh-18 mutant sheath cells are morphologically irregular and only loosely cover oocytes. Taken together, these observations indicate that ceh-18 is a crucial determinant of sheath cell differentiation, a function required for normal meiotic maturation and ovulation.

Animals↗

PubMed1997

Neuropathology of degenerative cell death in Caenorhabditis elegans.

In Caenorhabditis elegans necrosis-like neuronal death is induced by gain-of-function (gf) mutations in two genes, mec-4 and deg-1, that encode proteins similar to subunits of the vertebrate amiloride-sensitive epithelial Na+ channel. We have determined the progress of cellular pathology in dying neurons via light and electron microscopy. The first detectable abnormality is an infolding of the plasma membrane and the production of small electron-dense whorls. Later, cytoplasmic vacuoles and larger membranous whorls form, and the cell swells. More slowly, chromatin aggregates and the nucleus invaginates. Mitochondria and Golgi are not dramatically affected until the final stages of cell death when organelles, and sometimes the cells themselves, lyse. Certain cells, including some muscle cells in deg-1 animals, express the abnormal gene products and display a few membrane abnormalities but do not die. These cells either express the mutant genes at lower levels, lack other proteins needed to form inappropriately functioning channels, or are better able to compensate for the toxic effects of the channels. Overall, the ultrastructural changes in these deaths suggest that enhanced membrane cycling precedes vacuolation and cell swelling. The pathology of mec-4(gf) and deg-1(gf) cells shares features with that of genetic disorders with alterations in channel subunits, such as hypokalemic periodic paralysis in humans and the weaver mouse, and with degenerative conditions, e.g., acute excitotoxic death. The initial pathology in all of these conditions may reflect attempts by affected cells to compensate for abnormal membrane proteins or functions.

Animals↗

PubMed1997

Functional and sequence analysis of splicing defective nrdB mutants of bacteriophage T4 reveal new bases and a new sub-domain required for group I intron self-splicing.

The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 nuclelotide group 1 self splicing intron. In order to study the functional domains for self-splicing of this intron, 23 nrdB splicing defective intron mutants were analyzed for both sequence and functional changes. These mutants cluster towards the ends in regions of conserved structural elements of the intron. These 23 mutants have single base changes at 14 different sites. Interestingly two of these sites that seemed to map within the intron are actually located on the flanking exon sequences on both sides of the intron. A high frequency (4/12) of the mutation sites are in bases not thought to be base-paired in the standard model of group I intron structure. The mutation sites in pairing regions P3, P7, P8, P9 and between P6[3'] and P7[5'] are identical to changes found in the well studied td (encoding dTMP synthase) intron. However, five new mutation sites (S61, SL1, S29, SL11, SL196 and SL126) are unique to the nrdB intron and disrupt self-splicing. A mutation (S61) in the P7.1 pairing region is especially significant because no mutations have been found in this pairing, thus defining a new sub-domain essential for RNA splicing. Like the td intron, the mutation site in P9 of the nrdB intron is a hot spot for mutations, but unlike td, the nrdB intron does not show a mutational hot spot in the P6[5'] region. Our molecular dissection of the nrdB intron also supports the P9.0 and P10 pairings that have been postulated to help form a complex tertiary structure required to give the RNA sequence its catalytic activity: particularly 3' splice site selection, cleavage and exon ligation.

Bacteriophage T4↗