Phillip Grote et al. (2003) International Worm Meeting "tra-4 is necessary to ensure complete hermaphrodite development and encodes a zinc finger DNA binding protein"

BARBARA CONRADT, Phillip Grote, Claudia Huber

[International Worm Meeting, 2003]

During C. elegans development, the decision to develop a female (XX) or male (XO) soma is determined by the ratio of the number of X chromosomes to the number of sets of autosomes (X:A ratio). The resulting signal is transmitted through a cascade of interacting genes and determines the activity of the most downstream factor of this cascade, the transcription factor TRA-1A (TRA, transformer). Strong loss-of-function (lf) mutations in the tra-1 gene lead to a complete transformation of the XX soma into a male soma while weak tra-1(lf) mutations result in a partial masculinization of the XX soma. A partially masculinized XX soma overall resembles a wild-type XX soma, however, frequently the hermaphrodite-specific HSN neurons are absent and the male-specific CEM neurons are present. The HSNs and CEMs are the only neurons in C. elegans that are sex-specific as a result of programmed cell death. The specification of the cell death fate of these cells therefore seems to be particularly sensitive to changes in tra-1 dosage. In a screen for mutants, in which the CEMs inappropriately survive in XX animals, we identified mutations in a previously not described gene, referred to as tra-4, which is required for complete feminization of the XX soma. bc45, a lf mutation in the tra-4 gene, causes a partial masculinization of the XX soma: 73% of the HSNs undergo programmed cell death and 80% of the CEMs escape programmed cell death. In addition, in tra-4(bc45) L1 hermaphrodites the B cells and the ventral left coelomocytes have a male appearance or position, respectively, indicating weak masculinization. Double mutant analyses suggest that tra-4 acts upstream of fem-1, fem-2, and fem-3 (fem, feminization of XX and XO animals), and downstream of her-1 (her, hermaphrodization of XO animals) and possibly tra-2. tra-4 might therefore be a negative regulator of the fem genes. The tra-4 locus encodes a protein with predicted nuclear localization signals and C2H2 type Zinc finger domains, which indicates a role in transcriptional regulation. bc45 is a missense mutation leading to the exchange of a conserved amino acid in one of the C2H2 DNA-binding motives, suggesting that bc45 might not be a null allele. The analysis of the expression pattern of a functional Ptra-4GFP::TRA-4 construct indicates that tra-4 is expressed in most if not all somatic nuclei at all developmental stages in animals of both sexes. Using various approaches, we are currently searching for targets of the TRA-4 protein.

Phillip Grote et al. (2004) East Coast Worm Meeting "The genes tra-4 and mog-7 are necessary to ensure hermaphrodite development in the soma and in the germline"

Phillip Grote, Claudia Huber, BARBARA CONRADT

[East Coast Worm Meeting, 2004]

During C. elegans development, the decision to develop into a hermaphrodite (XX) or a male (XO) is determined by the ratio of the number of X chromosomes to the number of sets of autosomes (X :A ratio). In the soma the signal induced by this ratio is transmitted through a cascade of interacting genes, which determines the activity of the most downstream factor of this cascade, the transcription factor TRA-1A ( tra , tra nsformer). The same genes are involved in determining sexual fate in the germline; however, additional genes are required for this process. The determination of the sexual fate in the germline is a complex regulatory process because self-fertilizing hermaphrodites produce sperm during the last larval stage (L4) but have to switch to the production of oocytes after becoming adults. Strong loss-of-function (lf) mutations in genes required for feminization ( tra genes) can lead to the development of completely transformed animals: XX animals develop into phenotypic males. Weak lf mutations in tra genes can cause a weak masculinization: XX animals lack the hermaphrodite-specific neurons (HSNs), have the male-specific cephalic companion neurons (CEMs) and show additional defects indicative of masculinization. In a forward genetic screen originally designed to identify factors involved in the regulation of the sexually dimorphic death of the CEMs, we identified a gene, which when mutated causes a weak masculinization of XX animals, suggesting that it is required for feminization. We therefore named this gene tra-4 . tra-4 encodes a protein with seven C2H2 type zinc-finger domains, which are known to interact with DNA and/or RNA. Furthermore we identified a homologue of tra-4 in the C. elegans genome . Hermaphrodites homozygous for a lf mutation in the tra-4 homologue fail to switch from sperm production to oocyte production after the L4 stage. We therefore refer to this gene as mog-7 ( mog , m ale- o nly g onad). We are currently investigating the roles of tra-4 and mog-7 in the regulation of sex determination in the soma and in the germline, respectively. We propose that tra-4 is necessary for female development in the soma and that mog-7 fulfills a similar function in the germline. Furthermore, while tra4 and mog-7 seem to play similar but independent roles in sex determination, they appear to have a redundant function in an essential process, since tra-4 (lf) ; mog-7 (lf) double mutants are non-viable.

Phillip Grote et al. (2001) International C. elegans Meeting "Specification of the Sexually Dimorphic Programmed Cell Death of the CEM Neurons"

Phillip Grote, Claudia Huber, BARBARA CONRADT

[International C. elegans Meeting, 2001]

Sexual dimorphism in the nervous system of C. elegans is created by differential numbers of cell divisions, by differential cell fates or by programmed cell death (PCD). The hermaphrodite specific neurons (HSNs), two motoneurons necessary for egg laying, and the male-specific cephalic companion cells (CEMs), four sensory neurons of so far unknown function, are the only neurons that are sex-specific as a result of sexually dimorphic PCD. The HSNs and CEMs are born in both sexes; however, the HSNs are subsequently eliminated by PCD in males and the CEMs in hermaphrodites (1). The survival of the HSNs in hermaphrodites is specified by a direct interaction between the terminal, global regulator of somatic sexual fate, the Zn finger DNA binding protein TRA-1A ( tra , tra nsformer) (2), and the egl-1 locus ( egl , eg g- l aying defective), which encodes the primary activator of PCD in somatic tissues (3). The TRA-1A protein, which acts to promote female development in somatic tissues, binds to a TRA-1A binding site in the egl-1 locus thereby causing the repression of egl-1 transcription in the HSNs and HSN survival in hermaphrodites (3). To determine the role of the tra-1 and egl-1 genes in the specification of the death of the CEMs in hermaphrodites, we analyzed the survival of the CEMs in hermaphrodites (XX) homozygous mutant for loss-of-function (lf) mutations in either tra-1 or egl-1 . We found that both genes are required for the death of the CEMs in hermaphrodites. Furthermore, an egl-1 (lf) mutation blocks the ability of a tra-1 gain-of-function (gf) mutation to induce the CEMs to undergo PCD in males (X0). This suggests that TRA-1A acts as an activator of egl-1 in the CEMs. The TRA-1A binding site required for the ability of TRA-1A to repress egl-1 transcription in the HSNs, is, however, not required for the ability of TRA-1A to induce the CEMs to die. This indicates that TRA-1A acts as an indirect activator of egl-1 transcription or through a different binding site within the egl-1 locus. To determine how TRA-1A might specify the death of the CEMs and to identify additional factors required for this process, we are using two approaches. First, we are determining the region of the egl-1 locus that is required to rescue the death of the CEMs in egl-1 (lf) hermaphrodites. So far, we have identified a 690 bp region 3 kb downstream of the egl-1 transcription unit that is required to cause CEM death in hermaphrodites. Second, using a CEM-specific GFP reporter ( pkd-2 :: gfp ) (4), we performed a screen for mutations that cause the CEMs to survive in hermaphrodites and have identified at least two new loci that can mutate to cause CEM survival in hermaphrodites. (1) Sulston, JE and Horvitz, HR (1977) Dev Biol , 56(1):110-156 (2) Zarkower, D and Horvitz, HR (1992) Cell , 70(2):237-249 (3) Conradt, B and Horvitz, HR (1999) Cell , 98(3):317-327 (4) Barr, MM and Sternberg, PW (1999) Nature , 401(6751):386-389

Peixoto H et al. (2019) Molecules "Bark Extract of the Amazonian Tree Endopleura uchi (Humiriaceae) Extends Lifespan and Enhances Stress Resistance in Caenorhabditis elegans."

Wang X, Silva E, Braun M, Peixoto H, Roxo M, Valente K, Wink M

[Molecules, 2019]

<i>Endopleura uchi</i> (Huber) Cuatrec (Humiriaceae), known as uxi or uxi-amarelo in Brazil, is an endemic tree of the Amazon forest. In traditional medicine, its stem bark is used to treat a variety of health disorders, including cancer, diabetes, arthritis, uterine inflammation, and gynecological infections. According to HPLC analysis, the main constituent of the bark extract is the polyphenol bergenin. In the current study, we demonstrate by in vitro and in vivo experiments the antioxidant potential of a water extract from the stem bark of <i>E. uchi</i>. When tested in the model organism <i>Caenorhabditis elegans,</i> the extract enhanced stress resistance via the DAF-16/FOXO pathway. Additionally, the extract promoted an increase in the lifespan of the worms independent from caloric restriction. It also attenuated the age-related muscle function decline and formation of polyQ40 plaques, as a model for Huntington's disease. Thus, these data support anti-aging and anti-oxidant properties of <i>E. uchi</i>, which has not yet been described. More studies are needed to assess the real benefits of <i>E. uchi</i> bark for human health and its toxicological profile.

Claudia Walser et al. (2006) European Worm Meeting "Distinct Roles of the Pumilio and FBF Translational Repressors during C. elegans Vulval Development"

Gopal Battu, ALEX HAJNAL, Claudia Walser, Erika Frohli Hoier

[European Worm Meeting, 2006]

Claudia Walser, Gopal Battu, Erika Frhli Hoier and Alex Hajnal. Members of the conserved PUF (Pumilio and FBF repeat) protein family regulate various aspects of germ line development by selectively binding to the 3 untranslated region of their target mRNAs and repressing translation. FBF-1 and FBF-2 regulate the sperm/oocyte switch and mitosis versus meiosis decision by repressing fem-3 and gld-1 translation, respectively (Zhang et al., 1997; Crittenden et al., 2002). We found that puf-8, fbf-1 and fbf-2 also act in the soma where they negatively regulate vulval development.. Loss-of-function mutations in puf-8 cause excess vulval differentiation when combined with mutations in negative regulators of the EGFR/RAS/MAPK pathway, and they suppress the Vulvaless phenotype caused by mutations that reduce EGFR/RAS/MAPK signaling. A PUF-8::GFP translational reporter is initially expressed in all six vulval precursor cells (the VPCs P3.p through P8.p) but gets restricted to the distal, uninduced (3) VPCs after vulval fate specification. Moreover, the fusion of the distal VPCs to the hypodermal syncytium is retarded in puf-8(lf) mutants. Thus, PUF-8 acts cell-autonomously to limit the temporal competence of the vulval precursor cells to respond to the extrinsic patterning signals. fbf-1 and fbf-2, on the other hand, function as redundant inhibitors of vulval cell fate specification in a distinct pathway that involves the repression of gld-1 translation. fbf-1 fbf-2 double mutants show ectopic 1 cell fate marker expression in adjacent VPCs. We conclude that the FBFs ensure that only one VPC (P6.p) is selected for the 1 cell fate.. Therefore, the PUF family translational repressors regulate various aspects of cell fate specification in the soma, and they may play a conserved role in modulating signal transduction during animal development.

Swenson SI et al. (1995) International C. elegans Meeting "THE STEROID RECEPTOR HOMOLOGUE GENE, nhr-1, IN C. ELEGANS AND C. BRIGGSAE"

Purac A, Eagen RM, Honda BM, Swenson SI, Davison PJ

[International C. elegans Meeting, 1995]

We are studying the nhr-1 (nuclear hormone receptor) gene, one of a number of steroid receptor homologues identified in C. elegans. Sequencing of the cDNA has shown interesting features for this putative "orphan" receptor, including an unusual sequence in the P box of the DNA binding domain, which might indicate a very different DNA binding specificity and function, and a very long 3' untranslated region. The nhr-1 gene has been mapped to the right arm of the X chromosome, very close to sup-10. We are now characterizing the genomic organization of nhr-1 in C. elegans. We have also initiated work with C. briggsae in order to identify conserved, functionally important regions. The presence of an apparent homologue in C. briggsae was of special interest to us, given the unusual properties of nhr-1 (different P box, the long 3'UT region), and the apparent viability of homozygous deficiencies in this region around sup-10 (C. Cummins and P. Anderson). The C. elegans nhr-1 gene appears to contain 12 exons spread over 9.5 kb, including several relatively large introns, 1.3 to 2.3 kb in size. Work to date with genomic clones from C. briggsae shows conserved exons which share approximately 81-84% homology at the nucleotide level with the nhr-1 gene in C. elegans. This conservation between species, in the absence of a lethal phenotype for the homozygous deficiency, suggests that the nhr-1 gene may nevertheless have an evolutionarily relevant function. We wish to thank Ann Sluder, Gary Ruvkun, Claudia Cummins and Dave Baillie et al. for their assistance, and Alan Coulson et al. for help with mapping clones. Supported by a Research Reorientation Associateship (NSERC Canada) to SS, and an NSERC Operating grant to BMH.

Sprunger SA et al. (1991) International C. elegans Meeting "Molecular Analysis of mlc-3, the C. elegans Alkali Myosin Light Chain Gene."

Anderson P, Sprunger SA

[International C. elegans Meeting, 1991]

Our goal is the molecular genetic study of the structure, function, and interaction of muscle proteins. To that end, we have undertaken the molecular analysis of mlc-3, which encodes the single C. elegans'alkali' (or 'essential') myosin light chain (MLC). Claudia Cummins cloned mlc-3 by homology to the Drosophila melanogaster alkali MLC. The deduced mlc-3 amino acid sequence is more than 40% identical to other vertebrate and invertebrate MLCs. mlc-3 is a single-copy gene. Unlike the regulatory MLCs, which are encoded by the mlc-1 and mlc-2two-gene family, all the alkali MLCs are produced from mlc-3, as indicated by the absence of additional mlc-3- hybridizing bands on very low stringency Southern blots. mlc-3 has two polyadenylation sites. Two distinct groups of mlc-3 transcripts are observed on Northern blots as two thick bands of roughly 1.05 and 0.75 kb. These two groups of mRNAs differ in size due to the utilization of two polyadenylation sites separated by 0.25 kb, as shown by cDNA sequences and by Northern blots with transcript-specific probes. mlc-3 has multiple transcription initiation sites. Use of these sites creates additional heterogeneity in mlc-3 transcript size. Primer- extension analysis suggests that mlc-3 mRNAs can initiate at any of six different sites clustered 50 to 100 bp upstream of the AUG start codon. These putative transcription initiation sites are similar to those found in mlc-1, unc-54, act-4, and the vit genes, with a concensus sequence of TCA(C/G)T. None of the mlc-3 transcripts appear to be trans-spliced. Physical mapping has placed mlc-3 on LGIII, between ced-4 and daf-4. We are currently using a PCR technique to isolate mutant animals that do not produce any functional mlc-3 gene product. Our method is described in detail in the abstract by Rushforth et al. in this volume.

Gengyo-Ando K et al. (1988) Worm Breeder's Gazette "head piece of paramyosin and progress of tropomyosin gene cloning."

Kagawa H, Sugimoto K, Gengyo-Ando K

[Worm Breeder's Gazette, 1988]

Complete nucleotide sequence data of paramyosin gene, unc-15 will appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Databases under the accession number X08068. By the friendly information from St Louis, Larry Shriefer and Bob Waterston, a head piece of paramyosin was extended a little bit. Finally, the gene is composed of 10 exons encoding a protein of 866 amino acid with molecular mass of 99,890. The gene had no TATA-box and promoter like sequences at 5' upstream as other muscle genes of the worm. Manuscript entitled 'The paramyosin gene (unc-15) of Caenorhabditis r cloning, nucleotide sequence and models for thick filament assembly' collaboration with Andrew D. McLachlan, Sydney Brenner and Jonathan Karn was submitted to J. Mol. Biol. cDNA clone of tropomyosin gene was cloned from a cDNA library (48hr sample) which was constructed by Julie Ahringer and Judith Kimble by using a tropomyosin gene fragment of C. elegans as a probe. The fragment of probe was cloned by screening an exon expression plasmid library (1985 C. elegans meeting abstract) with anti-tropomyosin antibody. Cloned 1.6kb fragment encoded the 6th to 241th amino acid residue of tropomyosin. The amino acid sequence was very conserved. To clone a complete fragment of the gene of genomic lambdoid phage library (kindly supplied by Claudia Cummins and Phil Anderson) was screened by using 1.6kb of cDNA clone as a probe. Ten positive clones were processed and mapped on the cosmid library by Alan Coulson and John Sulston. Unfortunately, all clones was contained the identical sequence to other part of unrelated cDNA (Worm gazette, Vol. 10, No. 1) which located the upstream of the cDNA clone of tropomyosin. The junction sequence of the both fragment was as follows. We are not sure why tropomyosin gene was not contained in the genomic library. [See Figure 1] Tropomyosin gene will be cloned from a genomic library which will construct with the genomic restriction fragment of the correct size. Original gene of cloned fragment of other part leaves in question.

John Kratz et al. (2007) International Worm Meeting "Characterizing the C. elegans PHB domain proteins."

Thomas Benzing, Shifang Zhang, John Kratz, Marty Chalfie

[International Worm Meeting, 2007]

The Prohbitin homology domain (PHB d) is found in a large number of membrane-embedded proteins (currently 1780 in the SMART database) in organisms ranging from bacteria to mammals. PHB-d proteins have been found in association with ER, mitochondria and cell membranes; they have been implicated in a wide variety of processes including control of cell proliferation, melanogenesis, osmotic homeostasis, and mechanosensation. Previous work (Huber et al., PNAS 103:7019-17086, 2006) has shown that two non-homologous PHB-d proteins, mammalian Podocin and worm MEC-2, function similarly in different systems. Both bind and regulate membrane proteins (TRPC and DEG/ENaC ion channels respectively), homo-oligomerize, and bind cholesterol. These properties led to the hypothesis that PHB-d proteins regulate membrane bound proteins by controlling their lipid environment. To test whether other PHB-d proteins share these characteristics, we have begun to characterize the complete family of C. elegans PHB-d proteins. Thirteen worm proteins contain PHB domains: MEC-2, STO-1-6, STL-1, UNC-1, UNC-24, PHB-1, PHB-2 and C42C1.15. Eight of the worm proteins (MEC-2, STO-1-6 and UNC-1) are closely related to mammalian Stomatin, a PHB-d protein found in many cell types including sensory neurons. Three (UNC-1, UNC-24 and MEC-2) have been previously shown to be expressed in neurons and to regulate ion channels. We generated GFP promoter fusions to characterize the expression of the remaining genes. The sto genes are expressed almost exclusively in neurons, suggesting that they could regulate ion channels or other membrane proteins. Most sto genes (sto-1, sto-2, sto-3 and sto-5) are expressed in only a few cells; sto-4 is expressed in many neurons and sto-6 may be pan-neuronal. Single knockout strains of the 6 sto genes display no apparent phenotype; they may be redundant or exert subtle effects. The PHB-d genes known to function in mitochondria (phb-1, phb-2 and stl-1) are expressed widely, including in pharynx and body wall muscle where phb-1 and phb-2 have been shown to be important for mitochondrial morphology. We are currently testing oligomerization and cholesterol binding of these proteins.

Okkema PG et al. (1986) Worm Breeder's Gazette "molecular cloning of tra-2."

Okkema PG, Kimble JE

[Worm Breeder's Gazette, 1986]

We have cloned the tra-2 gene by Tc1 tagging. The tra-2 gene product is thought to regulate the fem genes (or their products) to allow female development in both soma and germ line of XX animals (Hodgkin et al.,1985,CSHSQB,50,585-593). To isolate Tc1 insertions into tra-2, we selected for intragenic revertants of the tra-2(gf) allele q122. We backcrossed q122 (a gain-of-function allele of tra-2 that was induced by EMS on a Bristol chromosome) and a closely linked marker, e120, into two strains in which Tc1 actively transposes (TR403 or EM1002). Because XX animals heterozygous for q122 develop as females and XO animals develop as males, it is possible to construct male/female mating strains. Large populations of stable male/female strains (q122 e120/ mnCl in TR403 or EM1002) were grown on Petri plates to permit mating. Five spontaneous recessive tra-2 alleles located in cis to q122 were isolated by shifting these animals to liquid culture to prevent mating and to select for self-fertility. Two of these alleles, q149 and q150, were backcrossed several times against N2. In addition, recombinants between each of these tra-2 alleles and linked markers on both sides of tra-2 were isolated to eliminate Tc1s which are not closely linked to tra-2. A radioactively labelled Tc1 probe was used to hybridize N2 DNA, q122 e120/mnCl (in Bristol) DNA, and DNAs isolated from a strain carrying the recombined q149 or q150 chromosome in Southern blots. A single novel 7.0kb BglII fragment was detected in q150 DNA. The fragment carrying this novel Tc1 was cloned, and a unique sequence flanking the q150 Tc1 insertion was subcloned to probe DNAs from other spontaneous tra-2 alleles. Hybridization with the unique probe showed that q149 contains a short deletion and that sc146, a spontaneous tra-2 allele isolated in TR679 and kindly sent to us by Corrine Basch and Bob Edgar, contains a rearrangement. Interestingly, this TR679 derived spontaneous allele is not a Tc1 insertion. The same probe was then used to isolate 30kb of DNA in the region of tra-2 from a C. library provided by Claudia Cummins and Phil Anderson. We are currently mapping the tra-2 gene more precisely within this 30kb region and attempting to identify the tra-2 transcript.