Worm Breeder's Gazette 16(3): 25 (June 1, 2000)
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.
Columbia University, College of Physicians & Surgeons, Center for Neurobiology and Behavior, New York, NY 10032
The LIM homeobox (Lhx) gene ttx-3 is required for correct thermotactic behavior (1,2). In ttx-3 mutants, the AIY interneuron, a component of the thermoregulatory neural circuit, is structurally and functionally defective. We have previously described that a ttx-3 reporter construct which contains 3.1 kb of the ttx-3 promoter and the first three exons translationally fused to GFP is exclusively expressed in an interneuron of the thermotactic circuit, AIY (1). A translational GFP fusion construct that encompasses a rescuing genomic clone of the ttx-3 gene is expressed in AIY, and additionally in ADL, ASI, a pair of cholinergic ventral ganglion neurons, and a pharyngeal neuron. We have set out to test what cellular role the ttx-3 gene plays and present here our analysis of the execution of the AIY cell fate in ttx-3 mutants.
Previously we have shown that in ttx-3 mutants maintenance of postembryonic ttx-3 expression is lost in AIY due to autoregulation. Using GFP reporter gene constructs, we have now found that expression of other AIY cell fate markers including a 7-TM receptor, sra-11, a homeobox gene, ceh-23, and a secreted protein, C36B7.7 (kindly provided by T. Ishihara) is also downregulated in AIY in ttx-3 mutants. A GFP fusion to the octopamine/serotonin receptor ser-2 (kindly provided by T. Niacaris and L. Avery), which we identified as being expressed in AIY as well, is also downregulated. Additionally, AIY loses its cholinergic phenotype in ttx-3 mutants as detected by VAchT antibody staining. These results suggest that AIY fails to differentiate correctly in ttx-3 mutants. So far it is unclear whether ttx-3 directly controls the expression of the genes described above or functions through intermediary transcription factors. We are addressing this question by delineating and comparing the ttx-3 -dependent regulatory elements in the distinct AIY cell fate markers.
Although ttx-3(ks5) mutants behave as behavioral nulls in ttx assays (1,2), comparison of expression levels of the AIY cell fate markers in the ks5 allele with those in three new ttx-3 alleles that we obtained in screens for thermoregulatory defective mutants, mg158, ot22, and ot23, suggests that ks5 has less severe molecular defects than the others.
Previously it was shown that mutations in the Lhx gene lim-4 lead to a switch of the fate of the AWB sensory neuron to AWC (3). Although many if not all aspects of the correct fate of AIY are lost in ttx-3 mutants, our preliminary tests for a cell fate switch of AIY into any of its lineal, structural or functional homologs suggest that AIY has not taken over the identity of AIM, AIZ, ASE or AWC. In contrast to AWB in lim-4 and AIY in ttx-3 mutants, the DVB motor neuron maintains its correct fate in animals mutant for the Lhx gene lim-6 (O. H., unpubl.). Hence, in Lhx mutants the identity of a neuron may be maintained, lost or switched.
In ttx-3 mutants, the apparently undifferentiated AIY interneuron still extends an axon along the ventral nerve cord; this main axon often terminates prematurely and also displays outgrowth of additional neurites ("neurite sprouts"). What aspects of differentiation failure may cause the occurrence of these axonal defects ? We find that animals defective in axon pathfinding (e.g. unc-76, unc-73, unc-34, unc-33 etc.) show similar axonal defects as ttx-3 mutants, i.e. premature termination of axonal outgrowth and extension of additional neurites. The incidence of the axonal defects in ttx-3 mutants is not enhanced by unc-34, suggesting that the neurite outgrowth defects in both mutant backgrounds may have a common cause and that ttx-3 may also be involved in determining the axonal outgrowth patterns of the AIY neuron.
References:
(1) Hobert et al., 1997, Neuron 19, 345
(2) Mori and Oshima, 1995, Nature 376, 344
(3) Sagasti et al., 1999, Genes Dev 13, 1794