We have generated an extensive library of C. elegans promoters as Invitrogen Gateway entry clones to facilitate easy cloning of tissue-specific reporters. As a lab that focuses on males, we have analyzed the expression of many of the promoters in the male. In addition to an extensive list of published promoters (Table 1) (Gruninger et al., 2006; Gruninger et al., 2008; LeBoeuf et al., 2007; Liu et al., 2007; Reiner et al., 2006), we have a large library of promoters in entry clones that have not been published (Table 2), and we wish to make these plasmids available to the worm community. We have also included a list of Gateway destination vectors we have generated (Table 3). Plasmids available are listed in the tables below.

Contact Dr. Rene Garcia to obtain plasmids of interest.

To study the expression pattern of innexins in the male worm, we crossed innexin reporter strains (Altun et al., 2009) with him-5(e1490) strains and analyzed GFP expression using a compound microscope. As describe in the table, innexins are expressed in different tissues and cells of the male worm. These results will be compared to the male connectome project where a lot of gap junctions have been observed in the nervous system.

In the study of C. elegans navigational behaviors, it helps to be able to quantify worm movements on spatial gradients that can be manufactured with precision, speed, and ease. It has been straightforward to produce such gradients with stimuli like temperature (Ryu and Samuel, 2002), but less so with chemical stimuli (Pierce-Shimomura et al., 1999; Iino and Yoshida, 2009). Here, we describe a simple way to make linear gradients of soluble chemicals for studies of worm chemotaxis.

1. Elevate one side of a square Petri plate as shown, such that the bottom of one side is level with the top of the opposite side (Fig. 1a).
2. Fill with melted agar with a chemical concentration a, and wait until it hardens, creating a triangular wedge of agar (Fig. 1b).
3. Lay the plate flat. Now fill with melted agar with a chemical concentration b, and wait until it hardens. Agar hardens at a lower temperature (<30°C) than it melts (>60°C), so the first triangular wedge is not disrupted as the second wedge is poured (Fig. 1c).

Diffusion will first equilibrate the soluble chemical in the vertical direction (Fig. 1d). The diffusion equation, x² = 2Dt, tells us how long we need to wait. Using the diffusion coefficient for a small molecule in water (D = 10^-5 cm² s^-1), one estimates that the molecules diffuse ~1 cm in ~12 hr. So, after ~1 day, the surface of the agar plate will have a linearly varying concentration of chemoattractant, a on one side, b on the other. The diffusion equation also tells us when the gradient disappears in the horizontal direction (after ~40 days with 9 cm plates).

A tiny amount of methylene blue in one of the agar solutions helps to visualize the gradient. Fig. 2a shows a photograph of the edge of a 24 cm x 24 cm agar plate shortly after the second wedge was poured. Fig 2b is a photograph of the surface of the same plate taken the following day.

We have used this linear gradient assay to study chemotaxis towards NaCl. We use a high-resolution tracking system to follow the movements of individual worms navigating linear gradients on 24 cm x 24 cm square agar plates. Each panel of Fig. 3 shows each track of individual wild-type worms navigating linear NaCl gradients spanning 0 to 50 mM (Fig. 3a) or 50 to 100 mM (Fig. 3b, c).  For presentation purposes, all tracks in each experiment are depicted as starting from a common point (red circles in each panel). Worms grown on standard NGM plates (50 mM NaCl) will crawl towards 50 mM NaCl whether initially placed at 25 mM NaCl (Fig. 3a) or at 75 mM NaCl (Fig. 3b). On the other hand, worms grown on high salt NGM plates (100 mM NaCl) will crawl towards 100 mM NaCl (Fig. 3c). Thus, worms develop a preference for the salt concentration that corresponds to their cultivation conditions, and are capable of either moving up gradients or down gradients in pursuit of their preferred salt concentration.

For us, the linear gradient assay has opened new doors in the study of the different navigational modes in NaCl chemotaxis (positive chemotaxis up gradients, negative chemotaxis down gradients) as well as experience-dependent plasticity. As any soluble chemical can be used, the method should facilitate numerous studies in C. elegans chemotaxis.

C. elegans is known to be attracted to various chemicals including volatile odorants and water-soluble salts. Chemotaxis to salts is conventionally tested on agar plates with salt concentration gradient. Most popular ways to make the gradient plate is either: 1) to spot a drop of concentrated salt on a chemotaxis plate (either once or twice at separate time points) and allow diffusion for several hours to overnight (Ward, 1973; Iino and Yoshida, 2009), or 2) to place a small agar block containing high concentration of salt on a chemotaxis plate and allow diffusion (Tomioka et al., 2006). Both of these procedures lead to concentration gradient ranging from, say, 10-20 mM at the peak to several millimolars at the periphery, corresponding to the salt concentration of basal buffer in the chemotaxis agar. The standard procedure therefore tests the worm’s behavior only in this low salt concentration range.

Here we show that by simply including salt in the background agar, chemotaxis at a much broader range of salt concentration can be tested. In Figure 1, background agar includes 25 mM potassium phosphate (pH 6.0), 1 mM CaCl₂, 1 mM MgSO₄, and 50 mM NaCl. On top of the agar plate with this composition, two cylindrical agar blocks were placed (each 15 mm in diameter and 5 mm in thickness, excised from an agar plate using a cork borer) that included either 0 mM (left side) or 150 mM (right side) of NaCl in addition to the chemotaxis buffer for 16 hours. This allows creating a concentric salt gradient on the test plate with highest concentration at the center of the high salt plug, lowest at the opposite side, and background concentration around the center. Animals washed from culture plates were then placed off center on the test plates to observe chemotaxis.

One remarkable observation that came out of this assay is that worms are not merely attracted to salt, but they are attracted to the salt concentration at which they have been raised. In addition, when worms are starved at various salt concentrations and then tested on the same assay plates, they now avoid the salt concentration that they have experienced. Therefore, the behavior on salt concentration gradient is based on salt concentration memory and depends on the food availability they experienced (a brief report on this observation was presented at the EAWM, 2010). This manner of behavioral control is reminiscent of that already known for thermotaxis, except that we have not so far observed obvious tracks along the line with same salt concentration.