Dual electrochemical and physiological apoptosis assay detection of in vivo generated nickel chloride induced DNA damage in Caenorhabditis elegans.

Environmental nickel exposure is known to cause allergic reactions, respiratory illness, and may be responsible for some forms of cancer in humans. Nematodes are an excellent model organism to test for environmental toxins, as they are prevalent in many different environments. Nickel exposure has previously been shown to impact nematode life processes. In this study, Caenorhabditis elegans nematodes exposed to NiCl2 featured high levels of programmed cell death (PCD) in a concentration-dependent manner as measured by counting apoptotic corpses in the nematode germ line. A green fluorescent protein (GFP) reporter transgene was used that highlights cell corpse engulfment by fluorescence microscopy. Analysis of the reporter in a p53 mutant strain putatively indicates that the PCDs are a result of genomic DNA damage. In order to assay the potential genotoxic actions of NiCl2, DNA was extracted from nematodes exposed to increasing concentrations of NiCl2 and electrochemically assayed. In vivo damaged DNA was immobilized on pyrolytic graphite electrodes using the layer-by-layer (LbL) technique. Square-wave voltammograms were obtained in the presence of redox mediator, ruthenium trisbipyridine (Ru(bpy)3(2+)), that catalytically oxidizes guanines in DNA. Oxidative peak currents were shown to increase as a function of NiCl2 exposure, which further suggests that the extracted DNA from nematodes exposed to the nickel was damaged. This report demonstrates that our electrochemical biosensor can detect damage at lower Ni concentrations than our physiological PCD assay and that the results are predictive of physiological responses at higher concentrations. Thus, a biological model for toxicity and animal disease can be assayed using an electrochemical approach.

Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs.

Poly(A) tail length is a readout of an mRNA's translatability and stability, especially in developmental systems. PolyAdenylation Test (PAT) assays attempt to quickly measure the average poly(A) tail length of RNAs of experimental interest. Here we present sPAT, splint-mediated PAT, a procedure that uses a DNA splint to aid in the ligation of an RNA-tag to the poly(A) tail of an mRNA. In comparison to other PAT methodologies, including ePAT, sPAT is highly sensitive to low-abundance mRNAs, gives a more accurate profile of the poly(A) tail distribution, and requires little starting material. To demonstrate its strength, we calibrated sPAT on defined poly(A) tails of synthetic mRNAs, reassessed developmentally regulated poly(A) tail-length changes of known mRNAs from established model organisms, and extended it to the emerging evolutionary developmental nematode model Pristionchus pacificus. Lastly, we used sPAT to analyze the contribution of the two cytoplasmic poly(A) polymerases GLD-2 and GLD-4, and the deadenylase CCR-4, onto Caenorhabditis elegans gld-1 mRNA that encodes a translationally controlled tumor suppressor whose poly(A) tail length measurement proved elusive.

Assaying environmental nickel toxicity using model nematodes.

Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water), we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegans and P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species.

Wnt signaling in Pristionchus pacificus gonadal arm extension and the evolution of organ shape.

Changes in organ morphology have been essential to the evolution of novel body forms and in permitting organisms to invade new ecological niches. Changes in the arrangement of cells and tissues and in the regulation of morphological movements are fundamental to evolutionary transitions of organ shape and function. However, little is known about the genetic and developmental control of these changes. We use interspecific differences in the migration and extension of the nematode hermaphrodite gonadal arms to study the generation of morphological novelty. We show that the extending Pristionchus pacificus gonadal arms display a ventral migration that is unique to the Diplogastridae in comparison to the Rhabditidae, including Caenorhabditis elegans, and other nematodes. This results in the distal gonad residing along the ventral side of the body in P. pacificus in contrast to lying on the dorsal side of the body as in C. elegans. We show that at the cellular level this morphogenetic movement is regulated by signals from the developing vulva and the sister gonadal arm. We further show that in P. pacificus Wnt signaling is essential for this regulation. We show genetic and molecular evidence that suggest the Wnt ligands Ppa-mom-2 and Ppa-cwn-2 are components of the signaling mechanism. Supporting these findings, the hermaphrodite gonad of Ppa-bar-1 mutant animals mimics the shape of the C. elegans hermaphrodite gonad; the arms fail to extend ventrally. Thus, this genetic analysis of gonad migration provides insight into the mechanisms underlying the generation of morphological novelty and organ shape.

Gonadogenesis in Pristionchus pacificus and organ evolution: development, adult morphology and cell-cell interactions in the hermaphrodite gonad.

The nematode gonad is an exemplary system for the study of organogenesis and fundamental problems in developmental and cellular biology. Nematode gonads vary dramatically across species (Chitwood, B.G., Chitwood, M.B., 1950. Introduction to Nematology." University Park Press, Baltimore; Felix, M.A., Sternberg, P.W., 1996. Symmetry breakage in the development of one-armed gonads in nematodes. Development 122, 2129-2142). As such, comparative developmental biology of gonadogenesis offers the potential to investigate changes in developmental and cellular processes that result in novel organ morphologies and thus may give insights into how these changes can affect animal bauplane. Pristionchus pacificus is a free-living nematode that diverged from the model nematode Caenorhabditis elegans around 200-300 million years ago. The morphology and development of P. pacificus is highly homologous to that of C. elegans. However, many differences in morphology and the underlying molecular signaling networks are easy to identify, making P. pacificus ideal for a comparative approach. Here, we report a detailed description of the P. pacificus hermaphrodite gonad using electron and fluorescent microscopy that will provide a basis for both phenotypic studies of genetic mutations and in vivo molecular studies of cloned genes involved in P. pacificus gonad development. We report that the morphology of the P. pacificus gonad is distinct from that of C. elegans. Among these differences are germ line patterning differences, heterochronic differences, novel gonadal arm-migrations, novel cellular composition of some somatic tissues (e.g., the number of cells that comprise the sheath and different spermathecal regions are different), the absence of a somatic tissue (e.g., the spermathecal valve cells), a novel architecture for the sheath, and changes in the cellular and sub-cellular morphology of the individual sheath cells. Additionally, we report a set of cell ablations in P. pacificus that indicate extensive cell communication between the somatic gonadal tissues and the germ line. Individual ablation experiments in P. pacificus show significant differences in the effects of individual somatic tissues on germ line patterning in comparison to C. elegans.

The evolution of developmental mechanisms.

Over the past two to three decades, developmental biology has demonstrated that all multicellular organisms in the animal kingdom share many of the same molecular building blocks and many of the same regulatory genetic pathways. Yet we still do not understand how the various organisms use these molecules and pathways to assume all the forms we know today. Evolutionary developmental biology tackles this problem by comparing the development of one organism to another and comparing the genes involved and gene functions to understand what makes one organism different from another. In this review, we revisit a set of seven concepts defined by Lewis Wolpert (fate maps, asymmetric division, induction, competence, positional information, determination, and lateral inhibition) that describe the characters of many developmental systems and supplement them with three additional concepts (developmental genomics, genetic redundancy, and genetic networks). We will discuss examples of comparative developmental studies where these concepts have guided observations on the advent of a developmental novelty. Finally, we identify a set of evolutionary frameworks, such as developmental constraints, cooption, duplication, parallel and convergent evolution, and homoplasy, to adequately describe the evolutionary properties of developmental systems.

Evolution of discrete Notch-like receptors from a distant gene duplication in Caenorhabditis.

SUMMARY Caenorhabditis elegans possesses two Notch-like receptors, LIN-12 and GLP-1, which have both overlapping and individual biological functions. We examined the lin-12 and glp-1 genes in closely related nematodes to learn about their evolution. Here we report molecular and functional analyses of lin-12 orthologs from two related nematodes, C. briggsae (Cb) and C. remanei (Cr). In addition, we compare these lin-12 findings with similar studies of Cb-glp-1 and Cr-glp-1 orthologs. Cb-LIN-12 and Cr-LIN-12 retain the same number and order of motifs as Ce-LIN-12. Intriguingly, we find that LIN-12 conservation differs from that of GLP-1 in two respects. First, individual motifs are conserved to a different degree for the two receptors. For example, the transmembrane domain is 16-32% identical among LIN-12 orthologs but 65-70% identical among GLP-1 orthologs. Second, certain amino acids are conserved in a receptor-specific manner, a phenomenon most prevalent in the CC-linker. We suggest that LIN-12 and GLP-1 have been molded by selective constraints that are receptor specific and that the two proteins may not be entirely interchangeable. To analyze the functions of the lin-12 orthologs, we used RNA-mediated interference (RNAi). Cb-lin-12(RNAi) or Cr-lin-12(RNAi) progeny are nearly 100% Lag, a larval lethality typical of C. elegans lin-12 glp-1 double mutants, but not the primary defect observed in Ce-lin-12 null mutants or Ce-lin-12(RNAi). Therefore, LIN-12 functions are similar, but not identical, among the Caenorhabditis species. We suggest that ancestral functions may have been divided between LIN-12 and GLP-1 receptors in a process contributing to the retention of both genes after gene duplication (i.e., subfunctionalization).