C. elegans male, left lateral view. (A) Diagram of the major anatomical features of the male. (B) Micrograph of a male using differential interference optics. (C–E) Magnified views of the following regions: the one-armed male gonad (C), the tail structures, ventral view (D), and the larval tail prior to differentiation (E). Scale bar represents 0.1 mm. Reprinted from Wormatlas (www.wormatlas.org) with permission.

Life cycle of the C. elegans hermaphrodite at 22°C. An adult hermaphrodite with developing oval-shaped embryos inside is shown at the top of the life cycle. Once the embryos reach the 28–30-cell stage, they are laid into the environment and continue to develop. When embryogenesis is complete, L1 larvae hatch out of the eggshell and begin post-embryonic development in the presence of adequate nutritional sources. The animal continues larval development through the L2–L4 stages until reaching adulthood. Note the dark-colored gonad that is expanding during this time period. Prior to the L1/L2 molt, if there is a lack of food or there are too many animals or the temperature is too high then the animals will enter the alternative life cycle and become dauer larvae until these unfavorable conditions improve. The size of the animals at each stage of development is indicated in parentheses, and the amount of time spent at each stage in development is indicated along the arrows.

Methods used for RNA interference (RNAi) in C. elegans. A plasmid with opposing T7 promoter sequences containing “your favorite gene (YFG)” is constructed. The plasmid can be used in two ways. The top of the figure shows dsRNA made by in vitro transcription that is used for either soaking or injecting the animals. The bottom of the figure shows how animals can be fed bacteria that are producing dsRNA in vivo.

Mapping using single nucleotide polymorphism (SNP) markers. (A) A particular restriction site (grey circle) is not present in the N2 Bristol strain but is present in the CB4686 Hawaiian strain. DNA from each strain can be distinguished by PCR amplification of the genomic region followed by restriction digest (snip) and gel electrophoresis. (B) The method for mapping using snip-SNPs is shown. In this example, Bristol hermaphrodites harboring a recessive mutation are crossed with the Hawaiian males and wild-type heterozygous hermaphrodite cross progeny are selected. The chromosomes segregate during self-fertilization and the second generation offspring are separated based on phenotype into mutant and wild-type populations. DNA from the two populations is amplified at various SNP sites as in (A) with only one site shown here. If the mutation is linked to the SNP site, there will be a bias for Bristol DNA and not CB4686 in the mutant population. If the mutation is elsewhere in the genome or unlinked, the chromosomes with the SNP site with assort randomly and both Bristol and CB4868 DNA will be present and detected by snip-SNP analysis in the mutant and wild-type populations.The techniques and reagents available for studying C. elegans have made it possible to define the role that the HLH-8 protein plays in nematode development, but there is also clinical relevance to the work. In humans, Twist mutations cause birth defects that include craniofacial and digit malformation from defective bone development [90]. Despite the absence of bones in C. elegans, they do have a conserved Twist pathway that is useful for other aspects of mesoderm development. At this stage, multiple genes are known downstream of HLH-8 that have human homologs that cause similar craniofacial syndromes. Therefore, the information learned from the nematode will be relevant for understanding human mesoderm development and birth defects.

C. elegans hermaphrodite, left lateral view. (A) Micrograph of a hermaphrodite using differential interference optics. Two oval-shaped embryos can be seen near the vulval opening of the animal. (B) Diagram of the major anatomical features of the hermaphrodite. At the anterior (on the left) of the animal the distal gonad bends behind the intestine whereas at the posterior (on the right) the entire U-shaped gonad is visible (shown in shades of blue). The dotted lines labeled 2B–F correspond with the cross sections B–F in Figure 3. Scale bar represents 0.1 mm. Reprinted from Wormatlas (www.wormatlas.org) with permission.

Anatomical features in the body cavity of C. elegans. (A) This diagram shows the major body plan of the animal with two concentric tubes and the intervening pseudocoelomic cavity. Alae are exterior structures derived from the hypodermis. (B–F) Cross-sections that correspond with the approximate position of the dotted lines on the hermaphrodite in Figure 1B. DNC, dorsal nerve cord. VNC, ventral nerve cord. Reprinted from Wormatlas (www.wormatlas.org) with permission.

Flowchart for studying a favorite protein in C. elegans.

Isolating and following C. elegans genomic deletions. (A) An abbreviated schematic for finding a strain that contains a deletion of interest is shown. Animals from hundreds of plates are harvested for DNA isolation and the templates are pooled together for PCR in a 96-well format. PCR primers (lines with half arrowheads) are designed to flank a gene or gene region of interest. A pool of templates that contains animals with a deletion will be apparent by the smaller PCR fragment shown on the gel on the right (lane of interest labeled with an asterisk). (B) Animals containing a deletion can be distinguished from wild-type animals by designing 3 PCR primers that amplify different sizes of PCR products based on the genotype of the animal. Hypothetical PCR products from wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) animals are shown on the gel on the right. WT, wild-type animals. mut, mutant animals.