Worm Breeder's Gazette 14(2): 81 (February 1, 1996)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

unc-4 regulates neurotransmitter (FMRF-amide-like)expression in VC motor neurons

Karin Schinkmann1, Kyle Butkiewicz1, Chris Li1, Mike Nonet2, Nikla Emambokus3, David Miller3

1 Department of Biology, Boston University, Boston, MA
2 Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO
3 Department of Cell Biology, Vanderbilt University Medical Center, Nashville, TN

unc-4 encodes a homeodomain protein [1] that determines the pattern of synaptic input to VA motor neurons in the ventral cord [2]. unc-4 mutations lead to an uncoordinated phenotype whereby animals can crawl forward but not backward. Electron microscopic examination of unc-4(e120) revealed that VA motor neurons, which are necessary for backward movement, receive synaptic input normally reserved for their sister cells, the VB motor neurons [2]. Experiments with unc-4-lacZ reporter genes detected expression in all A-type motor neurons (SAB, DA, VA) and demonstrated that unc-4 activity in the VAs in L1/L2 larvae is sufficient to establish connections with the appropriate interneurons [3]. Thus, one unc-4 function is to distinguish the fates of VA and VB sisters cells with respect to presynaptic input.

Here we describe new evidence that unc-4 is also required for specifying the fates of VC motor neurons. The VCs express FMRFamide (Phe-Met-Arg-Phe-NH2)-related neuropeptides in N2 animals and innervate vulval muscles. Although the VCs are generated in the L1, they do not become FMRFamide-like immunoreactive (FLI) until L4/early adulthood [4]. While screening for altered patterns of FMRFamide-like immunoreactivity, we observed that the VCs are not FLI in unc-4(e120, wd1, and e2322ts) mutants. Transgenic rescue of unc-4(wd1) animals with the unc-4(+) cosmid, C07E2, restores immunoreactivity to the VCs, suggesting that unc-4 activity is necessary for FMRFamide-like expression in VC cells. Transgenic lines carrying unc-4-GFP constructs show GFP in the VCs as well as in A-type motor neurons which may indicate that endogenous UNC-4 is also expressed in the VCs. Interestingly, unc-4-GFP is not detected in the VCs until late L3/! early L4 whereas VA motor neurons are GFP-positive in L1/L2 larvae (see figure below). A reexamination of staining of the nuclear-localized unc-4-lacZ reporter (pNC4-23Lz) confirms VC expression in late larvae/adults; this expression was previously not reported [3].

unc-4 could exert its actions in the VC cells in one of several ways. For instance, UNC-4 could directly activate transcription of a FMRFamide-related peptide coding gene; at least five flp (FMRFamide-like peptide) genes are present in C. elegans [5; Worm Sequencing Project; A. Stretton, pers. comm.]. Alternatively, unc-4 could regulate a broader range of downstream genes such that VC motor neurons are transformed to a different cell type in unc-4 mutants. Unfortunately, the EM reconstruction of unc-4(e120) did not include the midventral region occupied by the VC cells [2]. It should be possible, however, to use our unc-4-GFP constructs to determine if VC axonal morphologies or synapses onto vulval muscles are affected by unc-4 mutations.

[1] Miller et al. (1992) Nature 355: 841. [2] White et al., (1992) Nature 355: 838. [3] Miller and Niemeyer (1995) Development 121: 2877. [4] Schinkmann and Li (1992) J. Comp. Neurol. 316: 251. [5] Rosoff et al. (1992) J. Neurosci. 12: 2356.

Lefthand panels: VC4 projecting single unbranched axon into L4 vulva
Righthand panel: Elaborate branching pattern of VC4 and VC5 axons in adult vulva