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The Regulation of Lunatic fringe during Somitogenesis

Shifley, Emily T.

Abstract Details

2009, Doctor of Philosophy, Ohio State University, Molecular Genetics.

Somitogenesis is the morphological hallmark of vertebrate segmentation. Somites bud from the presomitic mesoderm (PSM) in a sequential, periodic fashion and give rise to the rib cage, vertebrae, and dermis and muscles of the back. The regulation of somitogenesis is complex. In the posterior region of the PSM, a segmentation clock operates to organize cohorts of cells into presomites, while in the anterior region of the PSM the presomites are patterned into rostral and caudal compartments (R/C patterning). Both of these stages of somitogenesis are controlled, at least in part, by the Notch pathway and Lunatic fringe (Lfng), a glycosyltransferase that modifies the Notch receptor. To dissect the roles played by Lfng during somitogenesis, we created a novel allele that lacks cyclic Lfng expression within the segmentation clock, but that maintains expression during R/C somite patterning (LfngΔFCE1). LfngΔFCE1/ΔFCE1 mice have severe defects in their anterior vertebrae and rib cages, but relatively normal sacral and tail vertebrae, unlike Lfng knockouts. Segmentation clock function is differentially affected by the ΔFCE1 deletion; during anterior somitogenesis the expression patterns of many clock genes are disrupted, while during posterior somitogenesis, certain clock components have recovered. R/C patterning occurs relatively normally in LfngΔFCE1/ΔFCE1 embryos, likely contributing to the partial phenotype rescue, and confirming that Lfng plays separate roles in the two regions of the PSM. These results reveal that the oscillatory regulation of the Notch pathway plays an important role in the segmentation clock during the development of the anterior skeleton.

As part of the segmentation clock, Lfng mRNA is periodically transcribed and LFNG protein levels have also been observed to cycle. Lfng mRNA and LFNG protein molecules must therefore have rapid turnover rates in the PSM. We hypothesize that cyclic Lfng transcription is coupled with signals in the Lfng 3’UTR that confer a short half-life on Lfng mRNA. To test this, we examined the expression patterns of transgenes containing conserved sections of the Lfng 3’UTR and determined which sections cooperatively confer a short RNA half-life in the PSM. LFNG protein acts in the Golgi, but is also cleaved and released into the extracellular space. We hypothesized that this cleavage/secretion contributes to short LFNG intracellular half-life, facilitating its rapid oscillations. To test this, we localized N-terminal protein sequences that control the secretory behavior of the fringe proteins and found that LFNG processing is promoted by furin-like protein convertases. Mutations that alter LFNG processing increase its intracellular half-life in vitro without affecting the specificity of its function in the Notch pathway. To determine the importance of LFNG cleavage/secretion in vivo, we created a novel allele that tethers LFNG to the Golgi (LfngRL). LfngRL/+ mice show significant segmentation and patterning defects suggesting that the short intracellular half-life of LFNG is important for somitogenesis. Thus, the cyclic activity of Lfng in the segmentation clock is achieved through multiple mechanisms, including tight regulation of mRNA and protein levels. Overall, we find that Lfng plays an important role in spatially and temporally regulating Notch signaling during vertebrate segmentation.

Susan Cole, PhD (Advisor)
Christine Beattie, PhD (Committee Member)
Mark Seeger, PhD (Committee Member)
Michael Weinstein, PhD (Committee Member)
161 p.

Recommended Citations

Citations

  • Shifley, E. T. (2009). The Regulation of Lunatic fringe during Somitogenesis [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1242245140

    APA Style (7th edition)

  • Shifley, Emily. The Regulation of Lunatic fringe during Somitogenesis. 2009. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1242245140.

    MLA Style (8th edition)

  • Shifley, Emily. "The Regulation of Lunatic fringe during Somitogenesis." Doctoral dissertation, Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1242245140

    Chicago Manual of Style (17th edition)