In organisms ranging from vertebrates, to flies and to C. elegans, the LIM homeobox class of transcriptional regulators has been shown to be important in the determination of aspects of terminal differentiation of neurons. While there has been much information detailing expression patterns and implicating these transcription factors in the terminal differentiation of neurons, little light has been shed on the identity of the downstream targets that are ultimately responsible for these characteristics that are imparted by this class of transcription factors. Previously, our lab has identified a transcriptional cascade in the AIY interneuron involving
ttx-3 , one of the C. elegans LIM homeobox proteins, and two other homeobox genes,
ceh-10 and
ceh-23 (Altun-Gultekin, et al. , Development in press). In this cascade,
ceh-10 is required to initiate expression of
ttx-3 , which then positively regulates its own expression as well as the expression of
ceh-23 . In addition to
ceh-23 , the AIY terminal differentiation markers
sra-11 ,
ser-2 ,
kal-1 , and
unc-17 , which code for two G-protein coupled receptors, a cell surface protein, and an acetylcholine vesicular transport protein, respectively, were also described as being either direct or indirect targets of
ttx-3, with
sra-11 also under the regulatory control of
ceh-23 . Intriguingly, by studying the expression patterns of these transcriptional regulators along with the expression pattern of their downstream targets, it became clear that while this regulatory cascade is critical for AIY cell fate specification, expression of
ceh-10 or
ttx-3 in other cellular contexts does not lead to an AIY-like differentiation pattern. For example, while
ceh-10 is also expressed in the CAN neuron, its expression is not sufficient to turn on the expression of
ttx-3 . Likewise,
ttx-3 expression in the ASI and AIA neurons is not sufficient to lead to expression of
ceh-23 . Additionally, as with AIY, CAN neuron fate specification, which includes the expression of
ceh-23 and
kal-1 , requires
ceh-10 expression. However, in CAN these cell fate markers are induced without utilizing
ttx-3 as an intermediary. This complexity of control in neuronal fate specification has led us to investigate the modulatory nature of the promoters of these downstream genes to elucidate why some promoter elements are used in specific cellular contexts. In order to address this question, as well as to determine whether these downstream genes are direct or indirect targets of
ttx-3 , we have made deletions and point mutations in the transcriptional regulatory regions of these genes (
ttx-3 ,
ceh-23 ,
kal-1 ,
sra-11 ,
ser-2 ,
unc-17 ) fused to GFP as a reporter. By assaying for reporter expression in the AIY class of neurons, we have identified a 12-nucleotide presumptive TTX-3 binding site that is common to the regulatory regions of the
kal-1 and
ttx-3 genes and is necessary for
kal-1 and
ttx-3 GFP reporter expression in AIY. Through gel shift analysis, we intend to show whether this sequence is in fact a TTX-3 binding site and what other factors might be involved in cooperative binding to this proposed AIY enhancer element. In addition, through further promoter analyses with the other presumptive downstream targets of
ttx-3 , we hope to further define a consensus TTX-3 binding site. We then intend to use this consensus sequence to search the C. elegans genome for additional candidate downstream targets of
ttx-3 .