Worm Breeder's Gazette 12(3): 80 (June 15, 1992)

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 LacZ Expression in A-Type Motor Neurons

David M. Miller, Charles J. Niemeyer

Department of Cell Biology, Duke University Medical Center, Durham, NC 27710

unc-4 mutants are unable to crawl backwards due the absence of appropriate input to VA motor neurons in the ventral nerve cord; synapses from interneurons AVA, AVD, and AVE are replaced with input from AVB, an interneuron normally reserved for VB motor neurons. We have shown that this wiring defect corresponds to the unc-4 null phenotype. unc-4 encodes a homeodomain protein which suggests that it functions as a transcription factor. In the simplest model, unc-4 would be selectively activated in the VA's and therein control the expression of downstream genes which govern synaptic choice. Alternatively, unc-4 could be expressed in the interneurons (AVA, AVD, AVE or AVB) or in some other nearby cell which provides an inductive signal to the prospective synaptic partners.

We have used an unc-4 -LacZconstruct to address this question. A 5.5 kb Pstl-Clal genomic fragment spanning the known unc-4 coding region is sufficient to rescue unc-4 ( wd1 )in microinjection assays. The presumptive unc-4 promoter at the 5' end of this fragment, a 3 kb Pstl-Smal fragment, was fused in frame to Lac Z in pPD21 .28(from Andy Fire). We used dpy-20 (+JDNA (from Min Han) as a coselectable marker to generate an unc-4 ::LacZstrain in dpy-20 (e1 282).

We observe a periodic pattern of B-galactosidase staining in the ventral nerve cord. All of the VA motor neurons (VA1-VA12) are LacZ positive. The interneurons AVA, AVD, AVE, and AVB have cell bodies adjacent to the nerve ring in the head and these are not blue in the unc-4 ::LacZstrain. It seems likely, therefore, that unc-4 expression in the postsynaptic VA motor neurons controls some distinctive feature of these cells that is differentially recognized by the pool of presynaptic partners. It interesting that most VA motor neurons are born with VB sister cells in the L1 and that these VA's accept the VB pattern of input in the absence of unc-4 activity. Perhaps unc-4 is turning off a gene in the VA's that is normally expressed in the VB's and which excludes input from AVA, AVD, and AVE in favor of connections with AVB. (see unc-4 suppressors, this issue).

We also observe unc-4 -drivenLacZ expression in the embryo. Based upon the LacZ staining pattern of newly hatched L1 's,we have assigned this expression to the DA's (9 cells) and provisionally to the SAB's (3 cells) which are difficult to identify unambiguously because of variability in the position of cell bodies in the retrovesicular ganglion. We have used a quantitative reverse transcription PCR assay to verify that the endogenous unc-4 gene is transcribed in the embryo and during the L1 and early L2 stages. It appears that unc-4 is turned on twice, first in the DA's and SAB's which are generated in the embryo, and then in the late L1 stage after the birth of the VA's. John White has previously described DA's, SAB's, and VA's as "A-type" motor neurons because they are morphologically similar (anteriorly directed axons) and accept input from a common set of interneurons (AVA, AVD, and AVE). Curiously, the DA's and SAB's are not miswired in unc-4 mutants nor are three VA motor neurons VA1 , VA11 and VA12 .It may be significant that these A-type motor neurons do not have B-type motor neuron siblings whereas VA2 -VA10are born with VB sisters and are miswired in unc-4 mutants.