Ahringer JA (1996) Genes Dev "Posterior patterning by the Caenorhabditis elegans even-skipped homolog vab-7."

Ahringer JA

[Genes Dev, 1996]

Patterning of the posterior end in animals is not well understood. Homologs of Drosophila even-skipped (eve) have a similar posterior expression pattern in many animals, and in vertebrates they are linked physically to the "posterior" ends of homeotic clusters (HOM-C), suggesting a conserved role in posterior development. However, the function of this posterior expression is not known. Here I show that the Caenorhabditis elegans gene vab-7 encodes an eve homolog that is required for posterior development and expressed in a pattern strikingly similar to that of vertebrate eve genes. Using a four-dimensional recording system, I found that posterior body muscles and the posterior epidermis are patterned abnormally in vab-7 mutants, but commitment to muscle and epidermal fates is normal. Furthermore, vab-7 activity is required for the complete expression of the most posterior HOM-C gene egl-5 in muscle cells, supporting the idea that eve homologs may act with the HOM-C to determine posterior cell fates. The conservation of sequence and expression pattern between vab-7 and eve homologs in other animals argues that most eve genes have posterior mesodermal and ectodermal patterning functions.

Ahringer JA (1994) Worm Breeder's Gazette "vab-7 Encodes an even-skipped Homolog"

Ahringer JA

[Worm Breeder's Gazette, 1994]

At the worm meeting, the sequencing consortium reported some sequence homologies found in a YAC that was partially sequenced. One of these was to the homeobox of the Drosophila even-skipped (eve) gene. In Drosophila, eve is expressed in the embryo in stripes and in the nervous system and is required for segmentation and for the development of certain neurons. eve homologs have been identified in Xenopus, mouse, human, grasshopper, and coral. In Xenopus, mouse and grasshopper, eve is expressed in the posterior of the embryo during gastrulation (and not in stripes) and also in the developing nervous system. Experiments in Xenopus point to a role for eve in axis formation. To determine whether any known mutations were in this eve homolog, I looked for candidates in the region corresponding to the YAC (Y52D3 ,just left of tra-1 ).The gene vab-7 was the best candidate because vab-7 (e1562)animals have posterior defects and are uncoordinated (suggesting a possible nervous system defect). I sequenced the eve homeobox from vab-7 (e1562)and found a mutation that introduces a stop codon near the beginning of the homeobox. Further, a cosmid containing the eve gene rescues vab-7 mutants. vab-7 RNA is expressed primarily in embryos, L1 'sand L2 's.Because vab-7 mutants have posterior defects, vab-7 RNA may be expressed in the posterior as it is in other organisms; in situs are in progress to test this. I have isolated a vab-7 cDNA from Pete Okkema's embryonic cDNA library and am sequencing it and the genomic region to complete the sequence of the gene. To analyze the function of vab-7 ,I am first isolating more alleles. The only existing allele of vab-7 is probably not a null mutation (which is surprising given it is a nonsense mutation in the beginning of the homeobox). It is usually homozygous viable, but is sometimes lethal; mutants die with a Nob phenotype similar to that of pal-1 and nob-1 mutants. The phenotype of vab-7 (e1562)/tDf2is stronger than that of vab-7 (e1562)homozygotes, but they often become fertile adults. Therefore, I should be able to isolate null mutations in a noncomplementation screen. So far, I have two probable new alleles which appear to be stronger than e1562 but can be maintained as homozygotes. Because the posterior defects of vab-7 mutants are visible at hatching, vab-7 activity is probably required during embryogenesis, and a null mutant may be embryonic lethal. In Drosophila, eve helps to regulate the genes in the homeobox clusters, and it responds to both maternal and zygotic products. In C. elegans, vab-7 may also be part of the connection between maternal products and the HOM-C genes. As a first test of this, I plan to examine the expression of lin-39 ,mab-5 ,and egl-5 in vab-7 mutants.

Ahringer JA (1996) European Worm Meeting "vab-7 and posterior patterning in C. elegans"

Ahringer JA

[European Worm Meeting, 1996]

Homologs of Drosophila even-skipped (eve) have a similar posterior expression pattern in many animals, and in vertebrates they are physically linked to the "posterior" ends of HOX clusters, suggesting a conserved role in posterior development. However, the function of this posterior expression is not known. I found that the C. elegans gene vab-7 encodes an eve homolog that is required for posterior development and expressed in a pattern strikingly similar to that of vertebrate eve genes. In vab-7 mutants, posterior body muscles and the posterior epidermis are abnormally patterned but commitment to muscle and epidermal fates is normal. Furthermore, vab-7 activity is required for the complete expression of the most posterior HOM-C gene, egl-5, in muscle cells, supporting the idea that eve homologs may act with the HOM-C to determine posterior cell fates. The conservation of sequence and expression pattern between vab-7 and even-skipped homologs in other animals argues that most eve genes have posterior mesodermal and ectodermal patterning functions. To try to connect maternal gene expression to zygotic patterning I have been investigating the cues that direct early vab-7 expression. I found that vab-7 expression does not depend on position in the embryo, but rather is a property of the C blastomere. This suggests that a maternal factor controlling vab-7 expression may be localised during early cleavages. One candidate being tested is the maternal product of the pal-1 gene (a caudal homolog) which is expressed in a pattern consistent with that predicted for a vab-7 regulator (Hunter and Kenyon, pers. comm.).

Suparna Sanyal et al. (2001) International C. elegans Meeting "A Dopamine Receptor in C. elegans"

Dave Merz, Joseph G Culotti, Hubert H Van Tol, Richard F Wintle, Eve Bigras, Suparna Sanyal, Terence Hebert

[International C. elegans Meeting, 2001]

A large body of evidence suggests that dopamine (DA) might function as a neurotransmitter in C. elegans . Neurons containing DA, the enzymes necessary for its synthesis and metabolism, as well as distinct transporters required for its uptake into neurons and synaptic vesicles have all been identified in C. elegans . Here we report the first DA receptor to be identified in C. elegans . The C. elegans DA receptor gene encodes two alternatively spliced forms of DA receptors. The longer form of this receptor (CeDA1 long ) contains a 58 amino acid sequence in its third cytoplasmic loop that is spliced out in the shorter form (CeDA1 short ). Radioligand binding studies using membrane preparations of COS-7 cells transfected with the cDNA of this gene (long or short forms) show that DA binds to these receptors in a saturable and concentration-dependent manner. Using agonist-mediated GTP-gamma-[ 35 S] stimulation studies we found that DA, epinephrine (E) and norepinephrine (NE) can induce GTP-gamma-[ 35 S] binding to membranes of COS-7 cells transiently expressing either the long or short form of this receptor. DA was found to be about 10-fold more potent in the GTP-gamma-[ 35 S] binding assay as compared to E or NE. Other amines such as tyramine, histamine, octopamine and serotonin were found to be ineffective in this assay. HPLC analysis of worm extracts showed the presence of dopamine, its precursors and metabolites, however norepinephrine and epinephrine could not be detected at significant levels. These findings strongly suggest that dopamine is most likely the endogenous ligand for the CeDA1 long and CeDA1 short receptors. Both CeDA1 long and CeDA1 short displayed sensitivity to cholera toxin (CTX). Cells pretreated with CTX for 24 hours prior to assay failed to stimulate GTP-gamma-[ 35 S] binding following incubation with DA, NE or E. Neither of the receptor subtypes showed pertussis toxin (PTX) sensitivity in the same assay, suggesting that these receptors are likely coupled to Gs but not Gi/o. To test this hypothesis, we transfected CHO-K1 cells with the cDNAs of these receptors and assessed their ability to stimulate intracellular cAMP presumably through Gs activation. Both receptor types increased cAMP in a concentration-dependent manner with DA, NE and E. The ability of these clones to functionally couple to the human inwardly rectifying potassium channel Kir 3.2 in Xenopus oocyte was also tested following co-expression with Go-/Gi- or Gs-alpha subunit cDNAs. All known mammalian dopamine receptors have been found to couple to Kir 3.2. Neither receptor subtype could stimulate Kir 3.2 using the endogenous oocyte G proteins or when co-expressed with C. elegans Go-alpha subunits in response to DA or NE; suggesting that these receptors were not coupled to PTX-sensitive G proteins. However, co-expression of bovine Gs-alpha resulted in significant stimulation of Kir 3.2 current by both DA and NE for both receptor subtypes. We have generated GFP transcriptional reporter constructs with the promoter region of this receptor gene fused to GFP so as to identify the distribution and localization of this protein following injections of this construct into the worm. We have also isolated a C.elegans mutant in which this gene has been deleted. Dopamine is known to modulate a variety of behaviors including egg laying, food sensing and motility. Both, analysis of gene expression and behavioral studies are still under progress

Pretot RF et al. (1997) International C. elegans Meeting "CEH-26, A PROSPERO (PROS) ORTHOLOGUE"

Burglin TR, Pretot RF

[International C. elegans Meeting, 1997]

In Drosophila the homeodomain encoding gene pros has been shown to be required for certain neuronal identities. Neuroblasts that give rise to Ganglion Mother Cells (GMCs) sequester prospero protein only to one of their daughters (i.e., GMC). Furthermore pros has been shown to be required for activation of the homeobox genes even-skipped (eve) and fushi-tarazu (ftz) in Ganglion Mother Cells. Deficiencies in ftz and eve expression in pros mutants lead to pathfinding defects of CNS and PNS axons in embryos. The C. elegans genome project sequenced an open reading frame with significant similarity to the Drosophila pros gene both within and carboxy-terminal to the homeodomain. We have called this ORF ceh-26. We isolated ceh-26 cDNAs and cloned them into E. coli expression vectors to express fusion proteins (GST and MBP) against which we have raised antibodies. Whole mount antibody staining is nuclear and observed from comma stage to the adult animal. Consistent signals in nuclei of the head (m5, g2 in the pharynx; excretory cell, ALA, ASG, Cepsh D/V, URYD/V and others not yet identified), some nuclei of tail neurons and one cell in the postdeirids (probably PDEso) are observed. We are now particularly interested in observing, whether Ceh-26 protein is segregated asymmetrically. However, there are differences to Drosophila: no expression in the ventral nerve cord, and expression is not turned of in differentiated neurons. Antisense RNA injection (in collaboration with Craig Mello) showed an early larval lethal phenotype, which is possibly due to excretory cell defects; this is reminiscent of the ceh-6 knock-out.

Suparna Sanyal et al. (2002) East Coast Worm Meeting "A DOPAMINE RECEPTOR MODULATING MECHANOSENSORY RESPONSES IN C.elegans."

Scott M Summers, Joseph G Culotti, Terence Hebert, Rokhand Arvan, Hubert H M Van Tol, Suparna Sanyal, Dave Merz, Richard F Wintle, William R Schafer, Eve Bigras

[East Coast Worm Meeting, 2002]

Various dopamine-modulated behaviors, including motor response, reward and information processing, are subject to sensitization and habituation. Mal-adaptation of these processes are thought to play a role in learning and memory processes as related to addictions and psychosis. The underlying mechanisms of these processes are still poorly understood, and genetic approaches with C. elegans may allow the study of these mechanisms for dopamine mediated responses. A large body of evidence suggests that dopamine functions as a neurotransmitter in C. elegans modulating various behaviors, including egg laying, food sensing, and motility. Here we report the identification and functional characterization of a C. elegans dopamine receptor. The dar-1 gene encodes two alternatively spliced forms of the receptor recognizing catecholamines, with dopamine displaying the highest potency. Other amines did not activate the receptor. The absence of (nor)epinephrine in C. elegans indicates that dopamine is the likely endogenous ligand for this receptor. Functional coupling of the receptor was dependent on Gas. The dar-1 gene displays a restricted neural expression pattern including several neurons in the nerve ring and mechanosensory neurons like ALM and PLM. A deletion mutant for dar-1 (dar-1(ev748)) displayed altered behavioral responses to mechanosensory stimuli, but not previously established dopamine-related behaviors. The dar-1 mutant showed a greatly enhanced facilitation of accelerations in response to tap habituation training. This phenotype was rescued by a dar-1 wild-type transgene indicating that the DAR-1 receptor is required for modulation of mechanosensory behavioral plasticity.

Roger Pocock et al. (2003) International Worm Meeting "tbx-8 and tbx-9 are essential for embryonic morphogenesis and act upstream of the posterior patterning gene vab-7"

Roger Pocock, Michael Mitsch, Julie Ahringer, Sara Maxwell, Alison Woollard

[International Worm Meeting, 2003]

T-box genes are a family of transcriptional regulators unified by a conserved T-box DNA binding domain. There are 20 T-box genes in C.elegans, more than in any other species analysed so far but relatively little is known about their function. Using RNA interference, we have inactivated each of the worm T-box genes and have found obvious phenotypes in only a few cases. The remaining T-box genes give no obvious phenotype when inactivated singly by RNAi. Phylogenetic analysis reveals that several pairs of T-box genes are very similar to each other. Therefore, these gene pairs are likely to be the result of recent duplications and may share overlapping functions. Double RNAi experiments have revealed this to be the case with at least one of the T-box gene pairs, tbx-8 and tbx-9, which have been found to have essential, overlapping functions during embryonic morphogenesis. The earliest defect observed is in the process of dorsal hypodermal intercalation, which together with hypodermal ventral enclosure is crucial for embryonic elongation to occur. The dorsal hypodermal migration of cells is aberrant in tbx-8/9 RNAi embryos resulting in defects in elongation and morphogenesis. These embryos also have defects in muscle cell positioning and intestinal development. tbx-8 and tbx-9 are expressed in gut, muscle and hypodermis. Their expression domains largely overlap with both genes being highly expressed in the nuclei of dorsal hypodermal cells undergoing intercalation. tbx-9 single RNAi produces worms which have a bobbed-tail phenotype that is similar to the vab-7 mutant phenotype (vab-7 is an even-skipped homologue required for posterior patterning). This suggests that these genes may be acting in the same pathway. We find that tbx-8 and tbx-9 are both necessary and sufficient for vab-7 expression, and thereby function upstream of vab-7 to activate its expression in particular posterior cells. Furthermore, we find that vab-7 in turn represses another T-box gene, mab-9, in the posterior of embryos. Thus, there are complex interactions between vab-7 and T-box genes in C. elegans. Positive regulation of eve homeobox genes by T-box genes has been reported in other systems yet this is the first demonstration that T-box genes can also be subject to repression by eve. The potential evolutionary association between the role of tbx-8 and tbx-9 in dorsal hypodermal intercalation in the worm and the role of T-box genes in convergent extension movements of cells during morphogenesis of other organisms is an interesting parallel.

Pretot RF et al. (1996) Worm Breeder's Gazette "ceh-26, a putative prospero (pros) homologue"

Pretot RF, Burglin TR

[Worm Breeder's Gazette, 1996]

In Drosophila the homeodomain encoding pros gene has been shown to be required for activation of the homeobox genes even-skipped (eve) and fushi-tarazu (ftz ) in ganglion mother cells (GMCs). During mitosis of the neuroblasts, the protein is segregated in a cortical crescent that becomes part of the resulting GMC. Only in established interphase, pros protein migrates into the nucleus, where it presumably acts as a transcription factor. The genome project sequenced an open reading frame (K12H4.1, which we call now ceh-26 ) with sequence similarity to pros*. The homeodomain of ceh-26 is atypical, with 3 extra amino acids between helix 2 and helix 3. In addition, there is a conserved domain following the homeodomain (see Fig. 1). Using the open-reading-frame (ORF) predicted by Genefinder, we succeeded in generating cDNAs using PCR. However, sequencing showed various PCR induced point mutations. With these PCR cDNAs, we isolated cDNAs from the Chris Martin and the Pete Okkema libraries. One consistent difference to the sequence from the genome project, an extra base, was found in all cDNAs looked at. Overall, the ORF prediction was confirmed, except for one splice donor. This difference in the splice pattern is necessary to compensate for the extra base to produce the correct ORF. The cDNAs are now being used to generate fusion proteins. Upon obtaining antibodies, we will be able to determine, whether there is a similar phenomenon of asymmetric protein localization in C. elegans.

Catherine M Dempsey et al. (2005) International Worm Meeting "A genetic screen for the targets of dopamine in the egg-laying paradigm."

Scott M Mackenzie, Catherine M Dempsey, Ji Y Sze

[International Worm Meeting, 2005]

Serotonin and dopamine act antagonistically to modulate egg-laying behaviour in C. elegans. Serotonin signals via the G-coupled serotonin receptor SER-1 in the vulval muscle to stimulate egg-laying, but the mode of the inhibitory action of dopamine remains largely unknown. To date three dopamine receptors have been identified in C. elegans, one D1 like (dop-1) and two D2 like (dop-2, dop-3). No dopamine receptor has yet been implied in the egg-laying paradigm (1,2). MOD-1 was originally characterised as an ionotropic serotonin receptor controlling the rate of locomotion of C. elegans in response to food. Surprisingly, MOD-1 appears to be essential for the inhibitory action of dopamine on egg-laying. mod-1 (ok103) deletion mutants continue to lay eggs in the presence of dopamine and following prolonged starvation (3). Inhibitory dopaminergic signaling via MOD-1 is restricted to the egg-laying paradigm as mod-1 (ok103) mutants are still susceptible to paralysis induced by dopamine. In order to determine the role and pathway of MOD-1 in this dopamine signalling pathway we are conducting a genetic screen for mutants resistant to dopamine inhibition of egg-laying. Our screen will use the following approach: F2 progeny of singled ethylmethane sulfonate mutagenized animals are exposed to 5 mg/ml serotonin and 3 mg/ml dopamine. mod-1 (ok103) mutants continue to lay eggs under such treatment whereas egg-laying in wild-type animals is inhibited. We have recovered several mutants that show similar dopamine resistance as mod-1 (ok103) mutants. 1. Sanyal, S., Wintle, R. F., Kindt, K. S., Nuttley, W. M., Arvan, R., Fitzmaurice, P., Bigras, E., Merz, D. C., Hebert, T. E., van der Kooy, D., Schafer, W. R., Culotti, J. G. & Van Tol, H. H. M. (2004) Embo Journal 23, 473-482. 2. Chase, D. L., Pepper, J. S. & Koelle, M. R. (2004) Nature Neuroscience 7, 1096-1103. 3. Dempsey, C. M. Mackenzie S. M., Gargus A., Blanco G. & Sze J. Y. (2005).. Genetics (In press).

Ahringer JA et al. (1997) International C. elegans Meeting "REGULATION OF vab-7 BY PAL-1, AND PROGRESS TOWARDS A GAL4 SYSTEM FOR C. elegans"

Neades C, Ahringer JA

[International C. elegans Meeting, 1997]

The C. elegans zygotic patterning gene vab-7 is a member of the even-skipped family of homeodomain containing proteins (Ahringer, 1996). vab-7, like eve genes in most animals, is expressed in posterior cells of the embryo destined to produce muscles and epidermis. These cells are primarily descendants of the C blastomere, which occupies a posterior location in the embryo. We found that the early expression of vab-7 at the posterior does not depend on cell position or cell contacts, but rather on the production of a C-like blastomere. Expression requires the activity of pal-1, a caudal homolog (Waring and Kenyon, 1991) with maternal product required for proper development of the C blastomere (Hunter and Kenyon, 1996). Further, ectopic expression of PAL-1 is sufficient to drive expression of vab-7 in cells where it not normally expressed. This provides a link between maternal gene activity and zygotic patterning gene expression in C. elegans. We are also trying to develop a targeted gene expression system for C. elegans, based on the widely used GAL4 system of Drosophila (Brand and Perrimon, 1993), to allow any cloned gene to be expressed in any pattern for which a promoter is available. The gene to be expressed is cloned downstream of a basal promoter with UAS binding sites for the yeast transcriptional regulator GAL4 (expression construct). This construct should not be expressed in the absence of GAL4 protein. In a second construct, the GAL4 protein is expressed in a particular pattern by placing its coding region downstream of a characterised promoter (GAL4 driver construct). To express the gene of interest, one does a cross between the two construct bearing strains and the protein of interest is expressed in the pattern desired under the control of the GAL4 protein. We have made a variety of constructs and are in the process of testing them for activity. Results (negative or positive) will be reported at the meeting.