Otx/otd homeobox genes specify distinct sensory neuron identities in C. elegans

The mechanisms by which the diverse functional identities of neurons are generated are poorly understood. C. elegans responds to thermal and chemical stimuli using 12 types of sensory neurons. The Otx/otd homolog ttx-1 specifies the identities of the AFD thermosensory neurons. We show here that ceh-36 and ceh-37, the remaining two Otx-like genes in the C. elegans genome, specify the identities of AWC, ASE, and AWB chemosensory neurons, defining a role for this gene family in sensory neuron specification. All C. elegans Otx genes and rat Otx1 can substitute for ceh-37 and ceh-36, but only ceh-37 functionally substitutes for ttx-1. Functional substitution in the AWB neurons is mediated by activation of the same downstream target lim-4 by different Otx genes. Misexpression experiments indicate that although the specific identity adopted upon expression of an Otx gene may be constrained by the cellular context, individual Otx genes preferentially promote distinct neuronal identities.     

DAF-7/TGF-beta expression required for the normal larval development in C. elegans is controlled by a presumed guanylyl cyclase DAF-11.

In C. elegans development, unfavorable growth conditions lead a larva to an arrested and enduring form called a dauer. To elucidate components upstream of DAF-7/TGF-beta in this control pathway, we isolated a mutant that was defective in daf-7 promoter::gfp reporter expression and showed an arrested (dauer-constitutive) phenotype. It has a new mutation in the daf-11 gene encoding a transmembrane guanylyl cyclase. We show that daf-11 gene and a related gene daf-21 act upstream of daf-7, and cilium-related genes che-2 and che-3 are placed between daf-11 and daf-7, in the genetic pathway controlling dauer formation. Expression of daf-11 cDNA by cell specific promoters suggests that daf-11 acts cell autonomously in ASI chemosensory neurons for daf-7 expression.     

C. elegans Demonstrates Distinct Behaviors within a Fixed and Uniform Electric Field

C. elegans will orient and travel in a straight uninterrupted path directly towards the negative pole of a DC electric field. We have sought to understand the strategy worms use to navigate to the negative pole in a uniform electric field that is fixed in both direction and magnitude. We examined this behavior by quantifying three aspects of electrotaxis behavior in response to different applied field strengths: the mean approach trajectory angles of the animals’ tracks, turning behavior (pirouettes) and average population speeds. We determined that C. elegans align directly to the negative pole of an electric field at sub-preferred field strength and alter approach trajectories at higher field strengths to maintain taxis within a preferred range we have calculated to be ~ 5V/cm. We sought to identify the sensory neurons responsible for the animals’ tracking to a preferred field strength. eat-4 mutant animals defective in glutamatergic signaling of the amphid sensory neurons are severely electrotaxis defective and ceh-36 mutant animals, which are defective in the terminal differentiation of two types of sensory neurons, AWC and ASE, are partially defective in electrotaxis. To further elucidate the role of the AWC neurons, we examined the role of each of the pair of AWC neurons (AWC^OFF and AWC^ON), which are functionally asymmetric and express different genes. nsy-5/inx-19 mutant animals, which express both neurons as AWC^OFF, are severely impaired in electrotaxis behavior while nsy-1 mutants, which express both neurons as AWC^ON, are able to differentiate field strengths required for navigation to a specific field strength within an electric field. We also tested a strain with targeted genetic ablation of AWC neurons and found that these animals showed only slight disruption of directionality and turning behavior. These results suggest a role for AWC neurons in which complete loss of function is less disruptive than loss of functional asymmetry in electrotaxis behavior within a uniform fixed field.     

Graded expression of ceh-14 reporters in the hypodermis is induced by a gonadal signal

ceh-14, a LIM class homeobox gene from Caenorhabditis elegans, is the orthologue of the vertebrate Lhx3/Lhx4 genes. ceh-14 reporter constructs are expressed in several different cell types: head and tail neurons, spermatheca and hypodermis. An intriguing aspect of the hypodermal expression pattern is that it takes the form of a gradient which is strongest in the central body region in L4 to young adult hermaphrodites. Promoter deletion analyses revealed that important regulatory elements for hypodermal expression are located within the transcribed region of ceh-14. Since a large part of the hypodermis is a syncytium, we hypothesized that this expression is triggered in a non-cell-autonomous fashion, a possible source being the underlying gonad. In males, which have a different gonadal organisation, the ceh-14 reporter constructs are expressed in a gradient that is strongest in the tail. By laser ablation of the gonadal precursor cells we found that ceh-14 reporter construct expression is eliminated in the hermaphrodite hypodermis, suggesting that the gonad plays a role in the generation of the gradient. Several signaling pathways are known in the gonad and the vulva, thus we crossed the mutations lin-3, egl-17 and lin-12 with the ceh-14 reporter lines. However, the expression of the reporter constructs is not affected in these mutant backgrounds. This suggests that another, presently unknown, signal triggers the graded hypodermal expression. Received: 31 March 2000 / Accepted: 16 July 2000     

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.     

LIM homeobox gene-dependent expression of biogenic amine receptors in restricted regions of the C. elegans nervous system

Biogenic amines regulate a variety of behaviors. Their functions are predominantly mediated through G-protein-coupled 7-transmembrane domain receptors (GPCR), 16 of which are predicted to exist in the genome sequence of the nematode Caenorhabditis elegans. We describe here the expression pattern of several of these aminergic receptors, including two serotonin receptors (ser-1 and ser-4), one tyramine receptor (ser-2), and two dopamine receptors (dop-1 and dop-2). Moreover, we describe distinct but partially overlapping expression patterns of different splice forms of the ser-2 tyramine receptor locus. We find that each of the aminergic receptor genes is expressed in restricted regions of the nervous system and that many of them reveal significant overlap with the expression of regulatory factors of the LIM homeobox (Lhx) gene family. We demonstrate that the expression of several of the biogenic amine receptors is abrogated in specific cell types in Lhx gene mutants, thus establishing a role for these Lhx genes in regulating aspects of neurotransmission. We extend these findings with other cell fate markers and show that the lim-4 Lhx gene is required for several but not all aspects of RID motor neuron differentiation and that the lim-6 Lhx gene is required for specific aspects of RIS interneuron differentiation. We also use aminergic receptor gfp reporter fusions as tools to visualize the anatomy of specific neurons in Lhx mutant backgrounds and find that the development of the elaborate dendritic branching pattern of the PVD harsh touch sensory neuron requires the mec-3 Lhx gene. Lastly, we analyze a mutant allele of the ser-2 tyramine receptor, a target of the ttx-3 Lhx gene in the AIY interneuron class. ser-2 mutants display none of the defects previously shown to be associated with loss of AIY function.     

A novel WD40 protein, CHE-2, acts cell-autonomously in the formation of C. elegans sensory cilia.

To elucidate the mechanism of sensory cilium formation, we analyzed mutants in the Caenorhabditis elegans che-2 gene. These mutants have extremely short cilia with an abnormal posterior projection, and show defects in behaviors that are mediated by ciliated sensory neurons. The che-2 gene encodes a new member of the WD40 protein family, suggesting that it acts in protein-protein interaction. Analysis of mutation sites showed that both the amino-terminal WD40 repeats and the carboxyl-terminal non-WD40 domain are necessary for the CHE-2 function. CHE-2-tagged green fluorescent protein is localized at the cilia of almost all the ciliated sensory neurons. Expression of che-2 in a subset of sensory neurons of a che-2 mutant by using a heterologous promoter resulted in restoration of the functions and cilium morphology of only the che-2-expressing neurons. Thus, che-2 acts cell-autonomously. This technique can be used in the future for determining the function of each type of che-2-expressing sensory neuron. Using green fluorescent protein, we found that the extension of cilia in wild-type animals took place at the late embryonic stage, whereas the cilia of che-2 mutant animals remained always short during development. Hence, the abnormal posterior projection is due to the inability of cilia to extend, rather than degeneration of cilia once correctly formed. Expression of che-2 in a che-2 mutant under a heat shock promoter showed that the extension of cilia, surprisingly, can occur even at the adult stage, and that such cilia can function apparently normally in behavior.     

The maintenance of neuromuscular function requires UBC-25 in Caenorhabditis elegans

Caenorhabditis elegans gene ubc-25 encodes a novel type of an E2 ubiquitin transferase domain (UBCc) protein, which is highly conserved in multicellular animals, but which is not present in the genomes of fungi or plants. To identify the cellular localization of UBC-25 during the development of C. elegans, we used a ubc-25::gfp reporter gene construct. These experiments showed that ubc-25 expression starts during embryogenesis and that it is restricted to neurons and muscle cells in all later stages of development as well as in adult animals. RNA interference with ubc-25 caused late-onset paralysis of most muscular functions such as locomotion, egg laying, and defecation. We therefore propose that ubc-25 in C. elegans is required for the maintenance (homeostasis) of neuromuscular functions by contributing to a tissue specific protein modification pathway, and we speculate that the adult onset phenotype results from the accumulation of target proteins which fail to be degraded.     

A Caenorhabditis elegans homeobox gene expressed in the male tail, a link between pattern formation and sexual dimorphism?

 ceh-7 is a small Caenorhabditis elegans homeobox gene. We have shown that this gene is transcribed. Examination of the expression pattern of ceh-7 using reporter constructs revealed that it is expressed in a few cells of the male tail, which form a ring around the rectum. The most posterior member of the C. elegans Hox cluster, egl-5, an Abd-B homologue, has previously been shown to be required for the proper development of several blast cells in the male tail. We have examined the expression of ceh-7 in mutant backgrounds of egl-5 and also mab-5, an Antp/Ubx/Abd-A homologue. We find that ceh-7 is not expressed in egl-5 mutants, but is still expressed in mab-5 mutants. Received: 16 March 1998 / Accepted: 4 September 1998     

pha-2 encodes the C. elegans ortholog of the homeodomain protein HEX and is required for the formation of the pharyngeal isthmus

The pha-2 mutant was isolated in 1993 by Leon Avery in a screen for worms with visible defects in pharyngeal feeding behavior. In pha-2 mutant worms, the pharyngeal isthmus is abnormally thick and short and, in contrast to wild-type worms, harbors several cell nuclei. We show here that pha-2 encodes a homeodomain protein and is homologous to the vertebrate homeobox gene, Hex (also known as Prh). Consistent with a function in pharyngeal development, the pha-2 gene is expressed in the pharyngeal primordium of Caenorhabditis elegans embryos, particularly in pm5 cells that form the bulk of the isthmus. We show that in the pha-2 mutant there is a failure of the pm5 cells to elongate anteriorly while keeping their nuclei within the nascent posterior bulb to form the isthmus during the 3-fold embryonic stage. We also present evidence that pha-2 regulates itself positively in pm5 cells, that it is a downstream target of the forkhead gene pha-4, and that it may also act in the isthmus as an inhibitor of the ceh-22 gene, an Nkx2.5 homolog. Finally, we have begun characterizing the regulation of the pha-2 gene and find that intronic sequences are essential for the complete pha-2 expression profile. The present report is the first to examine the expression and function of an invertebrate Hex homolog, that is, the C. elegans pha-2 gene.     

Chemotaxis mutants of Spirochaeta aurantia.

Five Spirochaeta aurantia chemotaxis mutants were isolated. One mutant (the che-101 mutant) never reversed, one (the che-200 mutant) flexed predominantly, two (the che-300 and che-400-1 mutants) exhibited elevated reversal frequencies, and one (the che-400 mutant) exhibited chemotactically unstimulated behavior similar to that of the wild-type strain. The che-101 and che-400 mutants were essentially nonchemotactic, whereas the che-200, che-300, and che-400-1 mutants showed impaired chemotactic responses. Protein methylation in response to attractant addition appeared normal in all of the mutants. Compared with the wild type, all of the mutants exhibited significantly altered membrane potential responses to the attractant xylose.     

The ABC transporter PGP-2 from Caenorhabditis elegans is expressed in the sensory neuron pair AWA and contributes to lysosome formation and lipid storage within the intestine

The functional role of the ABC transporter PGP-2 from the nematode Caenorhabditis elegans has been studied by combining phenotype analyses of pgp-2 deletion mutants or pgp-2 RNAi treated worms with reporter gene studies using a pgp-2::GFP construct. pgp-2 mutants showed a strong reduction of lipid stores. In addition, we found that in the case of the pgp-2 mutant or after pgp-2 RNAi the worms were unable to perform pinocytosis and to acidify intestinal lysosomes. Especially under cholesterol-restricted conditions, the viability of the mutant was reduced. Surprisingly, the chemosensory AWA neurons in the head region were identified as expression sites by reporter gene studies. These neurons are known to be involved in attraction behaviour towards odorants associated with potential food bacteria. Our results imply that PGP-2 is involved in a signalling process that connects sensory inputs to intestinal functions, possibly by influencing acidification of intestinal lysosomes, which in turn may affect pinocytosis and lipid storage.     

Conserved regulation of the Caenorhabditis elegans labial/Hox1 gene ceh-13

Caenorhabditis elegans contains a set of six cluster-type homeobox (Hox) genes that are required during larval development. Some of them, but unlike in flies not all of them, are also required during embryogenesis. It has been suggested that the control of the embryonic expression of the worm Hox genes might differ from that of other species by being regulated in a lineal rather than a regional mode. Here, we present a trans-species analysis of the cis-regulatory region of ceh-13, the worm ortholog of the Drosophila labial and the vertebrate Hox1 genes, and find that the molecular mechanisms that regulate its expression may be similar to what has been found in species that follow a regulative, non-cell-autonomous mode of development. We have identified two enhancer fragments that are involved in different aspects of the embryonic ceh-13 expression pattern. We show that important features of comma-stage expression depend on an autoregulatory input that requires ceh-13 and ceh-20 functions. Our data show that the molecular nature of Hox1 class gene autoregulation has been conserved between worms, flies, and vertebrates. The second regulatory sequence is sufficient to drive correct early embryonic expression of ceh-13. Interestingly, this enhancer fragment acts as a response element of the Wnt/WG signaling pathway in Drosophila embryos.