Worm Breeder's Gazette 16(1): 52 (October 1, 1999)
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||Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064|
|2||Fred Hutchinson Cancer Research Center, Seattle, Washington|
The basic Helix-Loop-Helix (bHLH) transcription factor NeuroD has been implicated in neuronal fate specification, differentiation and survival . Here we report the expression and function of cnd-1, a C. elegans NeuroD gene. We isolated cnd-1(ju29) in a screen for mutations affecting the localization of Punc-25 SNB-GFP, a presynaptic marker expressed in GABAergic DD and VD motor neurons. ju29 animals kink when moving backward. ju29 was mapped to the dpy-17lon-1 interval on chromosome III by standard linkage analysis. A 10 kb subclone of cosmid ZC129 was capable of rescuing the uncoordinated phenotype. This subclone contains one predicted open reading frame that is most similar to NeuroD. The bHLH domain of cnd-1 shares approximately 55% identity and 72% similarity to vertebrate NeuroD proteins . cnd-1(ju29) contains a G to A transition in the splice acceptor site of exon 3, changing TTTCAG to TTTCAA.
Using GFP reporter constructs, we first detected CND-1::GFP expression in eight cells of 28-cell stage embryos. Double labeling with LIN-26 antibodies in later stage embryos indicated that CND-1 is expressed within a population of neuroblasts. CND-1::GFP expression diminished below detection levels in later stage embryos, and was not seen in postembryonic neurons. Expression of a cnd-1 promoter driven GFP was reduced in cnd-1(ju29) embryos, indicating that CND-1 may regulate its own expression.
We focused our functional analysis on the progeny of two CND-1 expressing neuroblasts, ABprppap and ABplppap which give rise to two classes of embryonic ventral cord motor neurons (VCNs), the cholinergic DAs 1-5 and GABAergic DDs 1-6. We observed several defects in cnd-1(ju29) L1s: 1) Reduction in the number of VCNs, apparently a result of premature precursor differentiation, 2) Reduction in the number of VCNs expressing GABAergic and cholinergic markers, 3) Partial fate transformations as indicated by simultaneous expression of GABAergic and cholinergic markers within the same cell, and 4) Defects in terminal differentiation including axonal morphology and synaptic connectivity. The particular cells affected by the ju29 mutation varied from animal to animal. The phenotypes of animals heterozygous for the deficiencies sDf121 over ju29 appeared similar to those of ju29 homozygotes, suggesting that ju29 may cause a complete loss of function.
Our results support the hypothesis that a major function of cnd-1 is to specify cell fate. However, unlike observations made for the vertebrate retina where loss of NeuroD function favors the production of one type of neuron over another , the VCNs of cnd-1 mutants seem to adopt GABAergic or cholinergic fates at random, and may even adopt mixed attributes of both. CND-1 may function as a neural fate determination modulator by regulating the correct spatial expression of transcription factors like unc-30, unc-3 and unc-4, that are responsible for specific differentiated characteristics of VCNs.