Worm Breeder's Gazette 14(5): 74 (February 1, 1997)
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.
| 1 | Dept. Genetics and Cell Biology, University of Minnesota |
| 2 | Dept. Genetics, Washington Univ. School of Medicine |
We have identified the unc-3 gene through deficiency end-point
mapping using fluorescence in situ hybridization (FISH) followed by
identification of RFLPs associated with particular unc-3 alleles in the
defined region. Two alleles have been especially useful. mn419,
isolated in a TR679 mutator background, is associated with a Tc1
insertion; reversion to wild-type locomotion coincides with excision of
the Tc1 element. cn4146 (kindly provided by R. Hosono; WBG 10:1) is a
rearrangement in the unc-3 region most easily explained as an inversion
accompanied by a 3-kb deletion that includes most of the unc-3 coding
region, and is the best candidate for an unc-3 null mutation. The major
2.3-kb transcript from this region is most abundant in embryos and L1
larvae, and is predicted to encode a product homologous to the
transcription factors EBF/OLF-1 in rodents (Hagman et al., Genes Dev.
7:760; Wang and Reed, Nature 364:121) and collier in Drosophila
(Crozatier et al., Curr. Biol. 6:707). This group of very similar
transcription factors has a 200 amino acid DNA-binding domain as well as
a predicted helix-loop-helix domain; UNC-3 shares 80% identity in the
DNA binding domain and 70% identity in the HLH region with the other
family members.
The major unc-3 transcript is derived from an 11-kb genomic
sequence (excluding regulatory regions) that extends into a gap in
cosmid coverage of the physical map. We isolated a l genomic clone
extending into the gap that rescues unc-3. We engineered a derivative
of this phage clone with an in-frame GFP positioned in the loop of the
predicted HLH domain; this construct rescues the unc-3 locomotion defect
in 5/5 integrated lines. GFP can be detected in embryos by
approximately the 400-cell stage and is localized in nuclei. At the
time of hatching, expressing cells can be identified as the DA and DB
motor neurons, the ASI amphid neuron, and several other more
weakly-expressing cells in the head. By the L2 stage the newly-emerged
excitatory motor neuron classes VA, VB, and AS also express GFP, as well
as a few other unidentified cells in the tail. Expression in all five
excitatory motor neuron classes continues into adulthood. The DD and VD
inhibitory motor neurons and the VC motor neurons do not appear to
express GFP.
The observed GFP expression pattern corresponds well with the
phenotype of unc-3 mutants. EM serial section reconstruction of unc-3
mutants showed a highly disorganized ventral nerve cord (J. White, The
Worm Book), accounting for the locomotory defect in unc-3 animals.
Therefore it appears unc-3 is required for proper development of
excitatory motor neurons in the ventral nerve cord. In addition, unc-3
mutants have a propensity towards dauer formation in the presence of
food, and display a synthetic dauer constitutive phenotype in
combination with several other mutations, including unc-31 (Bargmann et
al., CSHSQB 60:529). Bargmann and Horvitz observed (WBG 11:1) that
laser ablation of ASI in an unc-31 background resulted in constitutive
dauer formation (Daf-c); therefore it appears that mutation in unc-3
results in an ASI defect that contributes to the synthetic Daf-c
phenotype in unc-31; unc-3 animals.
We have begun to examine defects in the ASI amphid neuron in a
collection of seven unc-3 alleles by assaying dauer formation and FITC
dye-filling. All unc-3 alleles tested have a synthetic Daf-c phenotype
with unc-31 at 25o; however, the penetrance varies, allowing us to
classify the alleles as strong or weak. In wild-type animals ASI is
stained by the fluorescent dye FITC, taken up from the environment.
Examination of FITC staining in unc-3 mutants indicates that their ASI
neurons are defective in dye-filling and that the penetrance of this
phenotype also varies with different alleles. Interestingly, although
our unc-3::GFP construct rescues locomotion very well in a cn4146
background, it does not rescue the ASI dye-filling defect in the same
background, suggesting that the functions of unc-3 in motor neurons and
in ASI may be separable.