The UNC-73/Trio RhoGEF-2 domain is required in separate isoforms for the regulation of pharynx pumping and normal neurotransmission in C. elegans

Abstract

In both Caenorhabditis elegans and Drosophila, UNC-73/Trio functions in axon guidance by signaling through the Rac GTPase to regulate cytoskeletal rearrangements necessary for growth cone migrations. Here, we show that the complex C. elegans unc-73 gene encodes at least eight differentially expressed UNC-73 intracellular protein isoforms. Previously reported mutations affecting UNC-73 isoforms encoding the Rac-specific RhoGEF-1 domain cause uncoordinated movement, correlating with defects in axon guidance. Mutations in isoforms encoding the Rho-specific RhoGEF-2 domain, which we describe here, result in L1 stage larval lethality with no associated axon guidance defects. Isoform-specific rescue experiments reveal separate functions for the various RhoGEF-2-containing UNC-73 isoforms, which would not likely be discovered by conventional genetic screening. UNC-73 D1 and D2 appear to function redundantly in pharynx muscle to regulate the rate and strength of pharynx pumping, and in the HSN neurons and vulval muscles to control egg laying. Isoforms C1, C2, E, and F act redundantly within the nervous system to regulate the speed of locomotion. The multiple UNC-73 isoforms containing Rac- and Rho-specific RhoGEF domains therefore have distinct physiological functions. In addition to its previously identified role involving RhoGEF-1 in migrating cells and growth cones, our data indicate that UNC-73 signals through RhoGEF-2 to regulate pharynx and vulva musculature and to modulate synaptic neurotransmission.

Figure 1.

Structures of the predicted unc-73 transcripts. The unc-73-containing cosmid, C11B5, and a deleted version, ΔC11B5, are represented at the top of the figure. The locations of several identified mutations in the unc-73 gene are indicated on or above the cosmid. The mutations in blue result in an Unc phenotype, and those in green are associated with L1 lethality. Exons of the predicted unc-73 transcripts (A, B, C1, C2, D1, D2, E, and F) are shown to scale as filled (coding) or open (noncoding) boxes with the regions encoding the RhoGEF-1 and RhoGEF-2 domains in blue and green, respectively. See Figure 2 for exon numbering. SL1 is a trans-spliced 5′ leader sequence. The red lines above the transcripts represent the probes used for the accompanying Northern blots. The size markers drawn on the left side of the PH-2 blot also apply to the remaining unmarked blots. In some cases, the small differences in size between the six smaller transcripts do not allow the individual transcripts to be resolved on the blot and, as a result, multiple transcripts appear as one band on the Northern blot. Note that there is a splice variation at intron 25, represented by the cDNA yk1532c04, which is not shown on this figure but is described in the Supplemental Material.

Figure 2.

C. elegans versus C. briggsae percentage identity plot for the unc-73 region. The genomic sequences of C. elegans and C. briggsae are compared by plotting the percentage identities for short regions of unc-73 as horizontal lines on a graph using the Pipmaker algorithm at http://bio.cse.psu.edu/pipmaker. The horizontal axis represents the length of the unc-73 gene. The vertical axis indicates the level of identity between 50% and 100%. The numbered boxes above the plot delineate the unc-73 exons. Open boxes indicate untranslated regions. The plot indicates the high sequence conservation between the two genomes in the regions encoding the unc-73 exons. Possible promoter elements are indicated on the plot by the “clouds” of short sequence identity 5′ of the first exons of the different transcripts. The two exons (A and B) of the predicted gene F55C7.2 (Supplemental Material) are also indicated. The C. elegans DNA clones C09D1 and F55C7 and the C. briggsae clone CB032N22 (accession nos. AF003131, U80436, and AC087076, respectively) were used for the comparison.

Figure 3.

Structures of the predicted UNC-73 protein isoforms. (A) The predicted UNC-73 isoforms are drawn to scale. An antibody raised to the portion of UNC-73 indicated by the red line was used to immunoprecipitate UNC-73 from soluble lysates of mixed stage wild-type and unc-73(e936) animals. The immunoprecipitated proteins were separated and blotted, and then probed with the same UNC-73 antibody. The A, B, C1, C2, and F bands on the Western are competed away by the UNC-73 antigen, but the indicated nonspecific band is not (data not shown). The C1 isoform appears to run as a doublet. (B) A Western blot of C. elegans lysates from ev802 strains containing constructs encoding the indicated UNC-73 isoforms fused to GFP. The strains containing C1, C2/F, and E fused to GFP also have a construct encoding D1 (not fused to GFP) to rescue ev802 lethality. The blot is probed with a polyclonal antibody to GFP.

Figure 4.

Expression patterns of the unc-73 isoforms. The patterns of expression of the unc-73 isoforms B through F were determined using constructs that each specifically encode an individual unc-73 isoform tagged with GFP at the C terminus. The constructs were injected into wild-type animals, and the larvae and adults of the resulting stable lines were examined. All animals are oriented with the posterior to the left and anterior to the right. (A) UNC-73 B is strongly expressed in the axons of neurons. A cross-sectional side view of a late-larval stage animal reveals the nerve ring as two bright patches of fluorescence around the pharynx (arrowheads). (B) Expression of D1 driven by the myo-2 promoter in [myo-2p::D1::GFP] ev802/unc-11 dpy-5 animals is limited to the muscles of the pharynx. The C1 isoform is expressed in a limited set of neurons in the midbody of the animal (C) and the head (D). Arrowheads indicate the ventral cord. Also visible in the head are the neuronal support socket and/or sheath cells. The C2/F isoform has the same pattern of expression as C1. (E) D1 expression in the muscles of the pharynx and two neurons in the head. A few unidentified axons are faintly visible in a cross-section of the nerve ring (arrowheads). (F) The processes of D1-expressing neurons are visible in the ventral cord. The muscles of the vulva seen in the shape of an “X”, and the HSNR cell body (arrowhead) also express D1. The D2 isoform has the same pattern of expression as D1. (G) UNC-73 E is expressed in the axons and cell bodies of most if not all neurons and the excretory canals. The bright region in the head is a result of expression in axons of the nerve ring and the cell bodies of neurons in the ganglia surrounding the nerve ring.

Figure 5.

The pharynx pumping rate is reduced in unc-73(ev802) animals. Plots of the pharynx pumping rate in pumps per minute. (A) Rates for L1 stage ev802 (n = 14), unc-73(ev802)/unc-11 dpy-5 (n = 30), and unc-11 dpy-5 (n = 14) animals. The pumping rate is reduced in ev802 and unc-11 homozygotes. (B) Rates for young-adult N2 (wild type) (n = 20), [D1] ev802 (n = 20), [D2] ev802 (n = 22), and [myo-2p::D1] ev802 (n = 22) animals. The reduced pumping rate in ev802 animals is rescued to wild-type levels with the presence of the D1, D2 or myo-2p::D1 isoform transgenes.

Figure 6.

The rate of movement is reduced in unc-73(ev802) animals. The rates of movement for various strains are measured as body bends per 20-sec interval as nematodes travel uninterrupted on agar plates free of bacteria. The movement rates of young adults (A) and L1 stage animals (B) are shown. The lethargy of ev802, [D1] ev802, [D2] ev802, and [myo-2p::D1] ev802 animals is rescued to wild-type levels with the presence of either the C1 or E isoform transgenes. C1 loses the ability to rescue the lethargy if it contains a mutation in the RhoGEF-2 domain.

Figure 7.

The UNC-73 D1 isoform is required for normal egg laying. The percentage of animals with an egg-laying defect (Egl) is plotted for each strain. An animal is defined as Egl if one or more of its eggs hatch inside its body within a period of 3 d of growth from the L4 stage. [myo-2p::D1] ev802 animals have a severe Egl phenotype. This defect is partially rescued by the addition of UNC-73 E. A Q1944A mutation in the D1 RhoGEF domain also results in an Egl phenotype.