Functional study of dpy-24 and its heterochronic regulation in distal tip cell migration in Caenorhabditis elegans
Date Issued
2009
Date
2009
Author(s)
Cho, Chun-Yi
Abstract
Cell migration plays an essential role during animal development. In the hermaphrodite of Caenorhabditis elegans, two distal tip cells (DTCs) undergo three sequential phases of linear migration during larval stages and lead the formation of bi-lobed U-shaped gonad arm. Previous studies have shown that the initiation of the ventral-to-dorsal phase Ⅱ migration occurs in the late L3 stage and is controlled by the transcriptional up-regulation of the dorsal guidance receptor UNC-5. On the other hand, the heterochronic genes control the temporal identity of DTCs: DAF-12/nuclear hormone receptor, DRE-1/F-box protein, and LIN-29/zinc finger transcription factor function redundantly to promote phase Ⅱ and phase Ⅲ migration in the L3 stage. In contrast, LIN-42/Period prevents phase Ⅱ and phase Ⅲ to occur precociously in L2. Our laboratory has previously isolated and characterized a dpy-24 mutation, which results in precocious DTC dorsal turn in early L3 stage. Previous studies have shown that dpy-24 represses the transcription of unc-5 to prevent precocious DTC dorsal turn. DPY-24 contains one PR domain and five zinc fingers and is detected in DTCs prior to, but not during or after, dorsal phase Ⅱ migration. In late L3, lin-29, dre-1, and daf-12 function redundantly to down-regulate the dpy-24 level to promote DTC dorsal turn. However, how DPY-24 represses unc-5 transcription and how the DPY-24 level is maintained during phase Ⅰ and is down-regulated during the phase Ⅰ to Ⅱ transition have not been explored. My structural and functional analysis of DPY-24 reveals that the PR and zinc finger domains and the region outside of these domains are required for its complete function in regulating DTC migration. Furthermore, I demonstrated that DPY-24 zinc fingers are able to bind to unc-5 promoter in EMSA (electrophoretic mobility shift assay), suggesting that DPY-24 may repress unc-5 transcription directly. In addition, DAF-12 and LIN-29 each can bind to the unc-5 promoter and when both are mutated, no unc-5 transcription is observed. Therefore, DAF-12 and LIN-29 may activate unc-5 transcription by directly binding to its promoter. As for temporal regulation of dpy-24, I generated transgenic worms carrying dpy-24::GFP reporters and found that DAF-12 and LIN-29 repress dpy-24 transcription, and DRE-1 decreases DPY-24 protein stability. Furthermore, dpy-24 expression is also repressed through its 3’UTR. Finally, dpy-24 transcription during phase Ⅰ is activated by LIN-42 and maintained by a positive feedback loop. These results elucidate the molecular mechanism of temporal regulation during cell migration. In phase Ⅰ, LIN-42 activates dpy-24 transcription, which in turn blocks lin-29 and unc-5 transcription and hence prevents DTC from dorsalward turning. In phase Ⅱ, DPY-24 is down-regulated by LIN-29 and DAF-12 at the transcriptional level and by DRE-1 at the post-translational level, which relieves the repression of unc-5 by DPY-24 and allows LIN-29 and DAF-12 to activate unc-5 transcription, leading to DTC dorsal turn.
Subjects
DTC (distal tip cell)
cell migration
temporal regulation
heterochronic gene
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