Antibody Staining for Nematodes with Heat-induced Antigen Retrieval (HIAR).

Immunocytochemistry remains a valuable and necessary tool for biologists working with nematodes, even those nematode model organisms with advanced molecular genetic tools and transgenics. Because of the highly idiosyncratic nature of successful immunostaining procedures, innovations can still be found for this long-established technique. Heat-induced antigen retrieval (HIAR) is well known from other systems, but seems not to have been applied to antibody staining in nematodes. For some antigens, adding HIAR to an established antibody staining protocol for nematodes can reveal strong and reliable staining that without HIAR is poor or completely absent.

Cuticle integrity and biogenic amine synthesis in Caenorhabditis elegans require the cofactor tetrahydrobiopterin (BH4).

Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host-pathogen interactions.

Patterning of sexually dimorphic neurogenesis in the caenorhabditis elegans ventral cord by Hox and TALE homeodomain transcription factors.

BACKGROUND: Reproduction in animals requires development of distinct neurons in each sex. In C. elegans, most ventral cord neurons (VCNs) are present in both sexes, with the exception of six hermaphrodite-specific neurons (VCs) and nine pairs of male-specific neurons (CAs and CPs) that arise from analogous precursor cells. How are the activities of sexual regulators and mediators of neuronal survival, division, and fate coordinated to generate sex-specificity in VCNs? RESULTS: To address this, we have developed a toolkit of VCN markers that allows us to examine sex-specific neurogenesis, asymmetric fates of daughters of a neuroblast division, and regional specification on the anteroposterior axis. Here, we describe the roles of the Hox transcription factors LIN-39 and MAB-5 in promoting survival, differentiation, and regionalization of VCNs. We also find that the TALE class homeodomain proteins CEH-20 and UNC-62 contribute to specification of neurotransmitter fate in males. Furthermore, we identify that VCN sex is determined during the L1 larval stage. CONCLUSIONS: These findings, combined with future analyses made possible by the suite of VCN markers described here, will elucidate how Hox-mediated cell fate decisions and sex determination intersect to influence development of neuronal sex differences.

Melanisation of Teladorsagia circumcincta larvae exposed to sunlight: a role for GTP-cyclohydrolase in nematode survival.

Trichostrongylid nematode parasites of livestock inhabit two very different niches during their life-cycle; within the host and free-living in the environment. UV radiation plays a significant role in the survival of free-living, pre-parasitic nematode larvae, with different species exhibiting differing levels of sensitivity. In many eukaryotes, melanisation is a key protective mechanism against UV damage, however there is little information about this process in parasitic nematodes. Caenorhabditis elegans cat-4 mutants, which are deficient in the enzyme guanosine triphosphate-cyclohydrolase I (GTP-CH), have both depleted levels of melanin in their cuticles and an increased sensitivity to anthelmintic drugs. Some parasitic nematodes have very high levels of GTP-CH transcript in their pre-parasitic stages, suggesting an important role for this biopterin synthetic enzyme. Here, we show that the Tci-cat-4 gene, which encodes GTP-CH in Teladorsagia circumcincta, has a role in melanisation and is also capable of rescuing C. elegans cat-4 mutants. In addition, following exposure of T. circumcincta L3s to sunlight, there is a 32% increase in GTP-CH enzyme activity (P=0.019), and a 21% increase in levels of melanin (P=0.031) compared with unexposed larvae. These data suggest that one explanation for the high level of GTP-CH present in pre-parasitic stages of trichostrongylid nematodes is to facilitate melanisation in response to UV exposure.

A comparison of experience-dependent locomotory behaviors and biogenic amine neurons in nematode relatives of Caenorhabditis elegans.

BACKGROUND: Survival of an animal depends on its ability to match its responses to environmental conditions. To generate an optimal behavioral output, the nervous system must process sensory information and generate a directed motor output in response to stimuli. The nervous system should also store information about experiences to use in the future. The diverse group of free-living nematodes provides an excellent system to study macro- and microevolution of molecular, morphological and behavioral character states associated with such nervous system function. We asked whether an adaptive behavior would vary among bacterivorous nematodes and whether differences in the neurotransmitter systems known to regulate the behavior in one species would reflect differences seen in the adaptive behavior among those species. Caenorhabditis elegans worms slow in the presence of food; this 'basal' slowing is triggered by dopaminergic mechanosensory neurons that detect bacteria. Starved worms slow more dramatically; this 'enhanced' slowing is regulated by serotonin. RESULTS: We examined seven nematode species with known phylogenetic relationship to C. elegans for locomotory behaviors modulated by food (E. coli), and by the worm's recent history of feeding (being well-fed or starved). We found that locomotory behavior in some species was modulated by food and recent feeding experience in a manner similar to C. elegans, but not all the species tested exhibited these food-modulated behaviors. We also found that some worms had different responses to bacteria other than E. coli. Using histochemical and immunological staining, we found that dopaminergic neurons were very similar among all species. For instance, we saw likely homologs of four bilateral pairs of dopaminergic cephalic and deirid neurons known from C. elegans in all seven species examined. In contrast, there was greater variation in the patterns of serotonergic neurons. The presence of presumptive homologs of dopaminergic and serotonergic neurons in a given species did not correlate with the observed differences in locomotory behaviors. CONCLUSIONS: This study demonstrates that behaviors can differ significantly between species that appear morphologically very similar, and therefore it is important to consider factors, such as ecology of a species in the wild, when formulating hypotheses about the adaptive significance of a behavior. Our results suggest that evolutionary changes in locomotory behaviors are less likely to be caused by changes in neurotransmitter expression of neurons. Such changes could be caused either by subtle changes in neural circuitry or in the function of the signal transduction pathways mediating these behaviors.

Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans.

In humans, liver phenylalanine hydroxylase (PAH) has an established catabolic function, and mutations in PAH cause phenylketonuria, a genetic disease characterized by neurological damage, if not treated. To obtain novel evolutionary insights and information on molecular mechanisms operating in phenylketonuria, we investigated PAH in the nematode Caenorhabditis elegans (cePAH), where the enzyme is coded by the pah-1 gene, expressed in the hypodermis. CePAH presents similar molecular and kinetic properties to human PAH [S(0.5)(L-Phe) approximately 150 microM; K(m) for tetrahydrobiopterin (BH(4)) approximately 35 microM and comparable V(max)], but cePAH is devoid of positive cooperativity for L-Phe, an important regulatory mechanism of mammalian PAH that protects the nervous system from excess L-Phe. Pah-1 knockout worms show no obvious neurological defects, but in combination with a second cuticle synthesis mutation, they display serious cuticle abnormalities. We found that pah-1 knockouts lack a yellow-orange pigment in the cuticle, identified as melanin by spectroscopic techniques, and which is detected in C. elegans for the first time. Pah-1 mutants show stimulation of superoxide dismutase activity, suggesting that cuticle melanin functions as oxygen radical scavenger. Our results uncover both an important anabolic function of PAH and the change in regulation of the enzyme along evolution.

Evolution of neuronal patterning in free-living rhabditid nematodes I: Sex-specific serotonin-containing neurons.

As a first step toward understanding the evolution of neuronal patterning and function in a group of simple animals, we have examined serotonin-containing neurons in 17 species of free-living rhabditid nematodes and compared them with identified neurons of Caenorhabditis elegans. We found many serotonin-immunoreactive (serotonin-IR) neurons that are likely homologs of those in C. elegans; this paper focuses on sex-specific neurons such as the egg laying hermaphrodite-specific neurons (HSNs), VCs, and male CAs, CPs, and ray sensory neurons known to function in mating. These cells vary in number and position in the species examined but are consistent with a current molecularly based phylogeny. Two groups (Oscheius and Pristionchus) appear independently to have lost a serotonin-IR HSN. Oscheius furthermore has no serotonin-IR innervation of the vulval region, in contrast to every other species we examined. We also saw variation in the location of somas of putative HSN, consistent with evolutionary changes in HSN migration. In C. elegans, the HSN soma migrates during embryogenesis from the tail to the central body, where it innervates its major postsynaptic targets, the vulval muscles. For other species, we observed putative HSN homologs along the anterior-posterior axis from the head to the tail, but typically HSNs were located near the vulva, which also varies in anterior-posterior position among the species we examined. The varying positions of the HSN somas in other species are reminiscent of phenotypes seen in various C. elegans mutants with altered HSN migration, suggesting possible mechanisms for the evolutionary differences we observed.

Function and evolution of the serotonin-synthetic bas-1 gene and other aromatic amino acid decarboxylase genes in Caenorhabditis.

BACKGROUND: Aromatic L-amino acid decarboxylase (AADC) enzymes catalyze the synthesis of biogenic amines, including the neurotransmitters serotonin and dopamine, throughout the animal kingdom. These neurotransmitters typically perform important functions in both the nervous system and other tissues, as illustrated by the debilitating conditions that arise from their deficiency. Studying the regulation and evolution of AADC genes is therefore desirable to further our understanding of how nervous systems function and evolve. RESULTS: In the nematode C. elegans, the bas-1 gene is required for both serotonin and dopamine synthesis, and maps genetically near two AADC-homologous sequences. We show by transformation rescue and sequencing of mutant alleles that bas-1 encodes an AADC enzyme. Expression of a reporter construct in transgenics suggests that the bas-1 gene is expressed, as expected, in identified serotonergic and dopaminergic neurons. The bas-1 gene is one of six AADC-like sequences in the C. elegans genome, including a duplicate that is immediately downstream of the bas-1 gene. Some of the six AADC genes are quite similar to known serotonin- and dopamine-synthetic AADC's from other organisms whereas others are divergent, suggesting previously unidentified functions. In comparing the AADC genes of C. elegans with those of the congeneric C. briggsae, we find only four orthologous AADC genes in C. briggsae. Two C. elegans AADC genes - those most similar to bas-1 - are missing from C. briggsae. Phylogenetic analysis indicates that one or both of these bas-1-like genes were present in the common ancestor of C. elegans and C. briggsae, and were retained in the C. elegans line, but lost in the C. briggsae line. Further analysis of the two bas-1-like genes in C. elegans suggests that they are unlikely to encode functional enzymes, and may be expressed pseudogenes. CONCLUSIONS: The bas-1 gene of C. elegans encodes a serotonin- and dopamine-synthetic AADC enzyme. Two C. elegans AADC-homologous genes that are closely related to bas-1 are missing from the congeneric C. briggsae; one or more these genes was present in the common ancestor of C. elegans and C. briggsae. Despite their persistence in C. elegans, evidence suggests the bas-1-like genes do not encode functional AADC proteins. The presence of the genes in C. elegans raises questions about how many 'predicted genes' in sequenced genomes are functional, and how duplicate genes are retained or lost during evolution. This is another example of unexpected retention of duplicate genes in eukaryotic genomes.