C. elegans male tail has 9 pairs of sensory rays specialized for locating the vulva of hermaphrodites. Each of these sensory rays composed of two neuronal cells, a structural cell and the hypodermis develops into a tapered shape. Mutations in six genes,
ram-1 to
ram-5 and
mab-7 , result in sensory rays of an amorphous shape. The rays appear swollen. One of these ram genes,
ram-4 encodes a Group 3 cuticular collagen. Mutations of this gene result in abnormal morphology of all ray cell components. Such observation suggests that
ram-4 plays an important role in the differentiation of all these cell types. The regional expression of
ram-4 has been studied previously. The expression of
ram-4 is restricted to male animals in the posterior hypodermal syncytium at the onset of tail retraction. This expression domain expanded anteriorly upon reaching adulthood. This specific expression profile implicates that there may be an induction signal activating the transcription of
ram-4 gene or a repressing signal restricting the anterior expansion of the
ram-4 expression domain. To understand how this temporal and spatial regulation takes place, deletion analysis on
ram-4 promoter was conducted. A 741bp 5' flanking sequence of the
ram-4 gene carrying 3 GATA sites was linked to a cfp-lacZ reporter gene to define the cis-acting elements responsible for transducing the activating or repressing signal. The reporter assay results showed that deletion of 241bp of the 5' sequence of this
ram-4 promoter sequence led to an expanded
ram-4 expression domain towards the anterior part of the animal reaching the mid-body, while further deletion of the promoter to only 274bp greatly reduced the transcriptional activity. When all 3 GATA sites were deleted leaving only 85bp promoter sequence,
ram-4 expression was completely abolished. These observations argue that there are both activating and repressing components to control the proper expression of
ram-4. To identify the origin of this activation signal, the same
ram-4 cfp-lacZ reporter gene was introduced into different mutant genetic backgrounds. Among a number of mutants tested,
ram-4 promoter activity was found to withdraw into the posterior part of the male tail in
mab-5 mutants. In contrast, anterior expansion of the expression domain was found in
lin-22 mutant animals. Based on these results, we hypothesize that
ram-4 transcription is regulated by
mab-5 and
lin-22 antagonistically along the anteroposterior axis. Characterization of the target cis-acting element will be coupled with this genetic analysis to define the spatial regulation of
ram-4 gene activity.