the-c-elegans-intestine-as-model-for-intercellular-lumen

The <em>C. elegans</em> Intestine As a Model for Intercellular Lumen Morphogenesis and <em>In Vivo </em>Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Harvard Medical School

Teneurins are transmembrane proteins that assist morphogenetic processes in many organisms. ten-1 is the C. elegans teneurin homolog with two transcripts, ten-1a and ten-1b, that respectively encode a long (TEN-1L) and short (TEN-1S) form of the protein. We previously isolated a C. elegans mutant where one pharyngeal neuron was frequently misplaced, and now show that it corresponds to a novel allele of ten-1.

C. elegans ten-1 is synthetic lethal with mutations in cytoskeleton regulators, and enhances many axon guidance defective mutants.

The organs of animal embryos are typically covered with an extracellular matrix (ECM) that must be carefully remodeled as these organs enlarge during post-embryonic growth; otherwise, their shape and functions may be compromised. We previously described the twisting of the Caenorhabditis elegans pharynx (here called the Twp phenotype) as a quantitative mutant phenotype that worsens as that organ enlarges during growth. Mutations previously known to cause pharyngeal twist affect membrane proteins with large extracellular domains (DIG-1 and SAX-7), as well as a C. elegans septin (UNC-61). Here we show that two novel alleles of the C. elegans papilin gene, mig-6(et4) and mig-6(sa580), can also cause the Twp phenotype. We also show that overexpression of the ADAMTS protease gene mig-17 can suppress the pharyngeal twist in mig-6 mutants and identify several alleles of other ECM-related genes that can cause or influence the Twp phenotype, including alleles of fibulin (fbl-1), perlecan (unc-52), collagens (cle-1, dpy-7), laminins (lam-1, lam-3), one ADAM protease (sup-17), and one ADAMTS protease (adt-1). The Twp phenotype in C. elegans is easily monitored using light microscopy, is quantitative via measurements of the torsion angle, and reveals that ECM components, metalloproteinases, and ECM attachment molecules are important for this organ to retain its correct shape during post-embryonic growth. The Twp phenotype is therefore a promising experimental system to study ECM remodeling and diseases.

Genetics of extracellular matrix remodeling during organ growth using the Caenorhabditis elegans pharynx model.

The T-box transcription factor mab-9 has been shown to be required for the correct fate of the male-specific blast cells B and F, normal posterior hypodermal morphogenesis, and for the correct axon migration of motor neurons that project circumferential commissures to dorsal muscles. In this study, an RNAi screen designed to identify upstream transcriptional regulators of mab-9 showed that silencing of unc-4 (encoding a paired-class homeodomain protein) increases mab-9::gfp expression in the nervous system, specifically in posterior DA motor neurons. Over-expression of unc-4 from a heat-shock promoter has the opposite effect, causing repression of mab-9 in various cells. We find that mab-9 expression in unc-37 mutants is also elevated in DA motor neurons, consistent with known roles for UNC-37 as a co-repressor with UNC-4. These results identify mab-9 as a novel target of the UNC-4/UNC-37 repressor complex in motor neurons, and suggest that mis-expression of mab-9 may contribute to the neuronal wiring defects in unc-4 and unc-37 mutants.

The UNC-4 homeobox protein represses mab-9 expression in DA motor neurons in Caenorhabditis elegans.

Serotonin [5-hydroxytryptamine (5-HT)]-absorbing neurons use serotonin reuptake transporter (SERT) to uptake 5-HT from extracellular space but do not synthesize it. While 5-HT-absorbing neurons have been identified in diverse organisms from Caenorhabditis elegans to humans, their function has not been elucidated. Here, we show that SERT in 5-HT-absorbing neurons controls behavioral response to food deprivation in C. elegans. The AIM and RIH interneurons uptake 5-HT released from chemosensory neurons and secretory neurons. Genetic analyses suggest that 5-HT secreted by both synaptic vesicles and dense core vesicles diffuse readily to the extrasynaptic space adjacent to the AIM and RIH neurons. Loss of mod-5/SERT function blocks the 5-HT absorption. mod-5/SERT mutants have been shown to exhibit exaggerated locomotor response to food deprivation. We found that transgenic expression of MOD-5/SERT in the 5-HT-absorbing neurons fully corrected the exaggerated behavior. Experiments of cell-specific inhibition of synaptic transmission suggest that the synaptic release of 5-HT from the 5-HT-absorbing neurons is not required for this behavioral modulation. Our data point to the role of 5-HT-absorbing neurons as temporal-spatial regulators of extrasynaptic 5-HT. Regulation of extrasynaptic 5-HT levels by 5-HT-absorbing neurons may represent a fundamental mechanism of 5-HT homeostasis, integrating the activity of 5-HT-producing neurons with distant targets in the neural circuits, and could be relevant to some actions of selective serotonin reuptake inhibitors in humans.

Regulation of extrasynaptic 5-HT by serotonin reuptake transporter function in 5-HT-absorbing neurons underscores adaptation behavior in Caenorhabditis elegans.

Sudden sensorineural hearing loss (SSNHL) is a debilitating condition with an incidence of nearly 20 per 100,000 in populations. Metronidazole-induced ototoxicity is an extremely rare etiology of SSNHL. In this report, we describe a young female with bilateral SSNHL due to oral use of metronidazole. A 23 years old female presented to the emergency department with acute bilateral hearing loss. We found out that her hearing loss had started 4 days after initiation of metronidazole which was administered for treatment of diarrhea. This case report shows that physicians should be aware of the uncommon side effects while prescribing metronidazole to patients in order to manage the possible adverse events on time.

Sudden hearing loss subsequent to diarrhea: what is the missing link?

Mitochondria play an important role in numerous diseases as well as normative aging. Severe reduction in mitochondrial function contributes to childhood disorders such as Leigh Syndrome, whereas mild disruption can extend the lifespan of model organisms. The Caenorhabditis elegans isp-1 gene encodes the Rieske iron-sulfur protein subunit of cytochrome c oxidoreductase (complex III of the electron transport chain). The partial loss of function allele, isp-1(qm150), leads to several pleiotropic phenotypes. To better understand the molecular mechanisms of ISP-1 function, we sought to identify genetic suppressors of the delayed development of isp-1(qm150) animals. Here we report a series of intragenic suppressors, all located within a highly conserved six amino acid tether region of ISP-1. These intragenic mutations suppress all of the evaluated isp-1(qm150) phenotypes, including developmental rate, pharyngeal pumping rate, brood size, body movement, activation of the mitochondrial unfolded protein response reporter, CO2 production, mitochondrial oxidative phosphorylation, and lifespan extension. Furthermore, analogous mutations show a similar effect when engineered into the budding yeast Rieske iron-sulfur protein Rip1, revealing remarkable conservation of the structure-function relationship of these residues across highly divergent species. The focus on a single subunit as causal both in generation and in suppression of diverse pleiotropic phenotypes points to a common underlying molecular mechanism, for which we propose a "spring-loaded" model. These observations provide insights into how gating and control processes influence the function of ISP-1 in mediating pleiotropic phenotypes including developmental rate, movement, sensitivity to stress, and longevity.

Tether mutations that restore function and suppress pleiotropic phenotypes of the C. elegans isp-1(qm150) Rieske iron-sulfur protein.

Several intragenic mutations suppress the C. elegans isp-1(qm150) allele of the mitochondrial Rieske iron-sulfur protein (ISP), a catalytic subunit of Complex III of the respiratory chain. These mutations were located in a helical region of the "tether" span of ISP-1, distant from the primary mutation in the extrinsic head, and suppressed all pleiotropic phenotypes associated with the qm150 allele. Analysis of these suppressors revealed control of electron transfer into Complex III through a "spring-loaded" mechanism involving a binding force for formation of enzyme-substrate complex, counter balanced by forces (a chemical "spring") favoring helix formation in the tether. The primary P→S mutation results in inhibition of electron flow into the Q-cycle by decreasing the binding force, and the tether mutations relieve this inhibition by weakening the "spring." In this commentary we discuss additional control features, and relate the primary inhibition to outcomes at the organismal level. In particular, the sensitivity to hyperoxia and the elevated reactive oxygen species (ROS) seen in isp-1(qm150), likely reflect over-reduction of the quinone pool, which is upstream of the inhibited site; at high O2, this would lead to increased ROS production through complex I. We speculate that alternative NADH:ubiquinone oxidoreductase activity in C. elegans from the worm apoptosis inducing factor (AIF) homolog (WAH-1) might also be involved, and that WAH-1 might have a "canary" function in detection of this adverse state (high O2/reduced pool), and a role in protection of the organism by transformation to AIF-like products, and apoptotic recycling of defective cells.

New functional and biophysical insights into the mitochondrial Rieske iron-sulfur protein from genetic suppressor analysis in C. elegans.

Improving healthspan, defined as the period where organisms live without frailty and/or disease, is a major goal of biomedical research. While healthspan measures in people are relatively easy to identify, developing robust markers of healthspan in model organisms has proven challenging. Studies using the nematode Caenorhabditis elegans have provided vital information on the basic mechanisms of aging; however, worm health is difficult to define, and the impact of interventions that increase lifespan on worm healthspan has been controversial. Here, we describe a marker of population healthspan in C. elegans that we term age-associated vulval integrity defects, or Avid, frequently described elsewhere as rupture or exploding. We connect the presence of this phenotype with temperature, reproduction, diet, and longevity. Our results show that Avid occurs in post-reproductive worms under common laboratory conditions at a frequency that correlates negatively with temperature; Avid is rare in worms kept at 25 °C and more frequent in worms kept at 15 °C. We describe the kinetics of Avid, link the phenotype to oocyte production, and describe how Avid involves the ejection of worm proteins and/or internal organ(s) from the vulva. Finally, we find that Avid is preventable by removing worms from food, suggesting that Avid results from the intake, digestion, and/or absorption of food. Our results show that Avid is a significant cause of death in worm populations maintained under laboratory conditions and that its prevention often correlates with worm longevity. We propose that Avid is a powerful marker of worm healthspan whose underlying molecular mechanisms may be conserved.

Age-associated vulval integrity is an important marker of nematode healthspan.

Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt) and extends lifespan. Here we report that transaldolase (tald-1) deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.

Gholamali Jafari

Mucosal Immunology and Biology Research Center,
Developmental Biology and Genetics Core,
Massachusetts General Hospital,
Mucosal Immunology and Biology Research Center, Developmental Biology and Genetics Core, Massachusetts General Hospital

Harvard Medical School

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging