Caenorhabditis elegans male tail comprises nine pairs of sensory rays. Each ray is composed of a structural cell, two neuronal cells and a sheathing hypodermis. Mutation of tbx-2 encoding a T-box transcription factor (TF) is associated with a ray-loss phenotype. tbx-2::gfp transcriptional reporter reveals its expression in structural cells beginning at late L4 stage and subsiding in 1 day-old adults. I have demonstrated that tbx-2 expression is under its own negative regulation via two binding sites, namely, TBS1 and TBS2, in its intron 1. This feedback circuitry is crucial for controlling its transient expression at the onset of the ray cell assembly event. The ability of TBX-2 to bind to these two regulatory motifs and its specificity were confirmed by a gel mobility shift assays. The...[ Read more ]

Caenorhabditis elegans male tail comprises nine pairs of sensory rays. Each ray is composed of a structural cell, two neuronal cells and a sheathing hypodermis. Mutation of tbx-2 encoding a T-box transcription factor (TF) is associated with a ray-loss phenotype. tbx-2::gfp transcriptional reporter reveals its expression in structural cells beginning at late L4 stage and subsiding in 1 day-old adults. I have demonstrated that tbx-2 expression is under its own negative regulation via two binding sites, namely, TBS1 and TBS2, in its intron 1. This feedback circuitry is crucial for controlling its transient expression at the onset of the ray cell assembly event. The ability of TBX-2 to bind to these two regulatory motifs and its specificity were confirmed by a gel mobility shift assays. These findings constitute the first report of a T-box gene whose expression is governed by a negative auto-feedback mechanism.

To extend my study of regulatory control beyond tbx-2 during organ assembly, an integrative approach combining experimental work, in silico analysis and candidate gene approach were adopted to identify the downstream targets and upstream regulators of tbx-2. Expression of pqn-47, revealed by transcriptional reporter assay, was shown to be downregulated by tbx-2 in a dosage-dependent fashion. Thus, pqn-47 could be one of the downstream targets of TBX-2 to execute sensory organ assembly. Deletion analysis of pqn-47 promoter has mapped TBX-2-responsive sequences to confer their direct regulatory relationship. In addition, ceh-43, a distal-less family factor, was observed to act as a transcriptional activator of tbx-2. Seven putative CEH-43 binding sites (CBS) were identified within the tbx-2 genomic sequence. Site-directed mutagenesis of these sites revealed that CEH-43 acts through one of these sites to activate tbx-2 expression in structural cells.

Through these approaches, I have defined the hierarchical regulatory components required for ray assembly. The findings are of significance in recognizing how coordinated regulation of TFs drives normal organogenesis events in animal.