Emily T. Shifley

Oscillatory lunatic fringe activity is crucial for segmentation of the anterior but not posterior skeleton

The Notch pathway plays multiple roles during vertebrate somitogenesis, functioning in the segmentation clock and during rostral/caudal (R/C) somite patterning. Lunatic fringe (Lfng) encodes a glycosyltransferase that modulates Notch signaling, and its expression patterns suggest roles in both of these processes. To dissect the roles played by Lfng during somitogenesis, a novel allele was established that lacks cyclic Lfng expression within the segmentation clock, but that maintains expression during R/C somite patterning (LfngΔFCE1). In the absence of oscillatory Lfng expression, Notch activation is ubiquitous in the PSM of LfngΔFCE1 embryos. LfngΔFCE1 mice exhibit severe segmentation phenotypes in the thoracic and lumbar skeleton. However, the sacral and tail vertebrae are only minimally affected in LfngΔFCE1 mice, suggesting that oscillatory Lfng expression and cyclic Notch activation are important in the segmentation of the thoracic and lumbar axial skeleton (primary body formation), but are largely dispensable for the development of sacral and tail vertebrae (secondary body formation). Furthermore, we find that the loss of cyclic Lfng has distinct effects on the expression of other clock genes during these two stages of development. Finally, we find that LfngΔFCE1 embryos undergo relatively normal R/C somite patterning, confirming that Lfng roles in the segmentation clock are distinct from its functions in somite patterning. These results suggest that the segmentation clock may employ varied regulatory mechanisms during distinct stages of anterior/posterior axis development, and uncover previously unappreciated connections between the segmentation clock, and the processes of primary and secondary body formation.

Manic fringe is not required for embryonic development, and fringe family members do not exhibit redundant functions in the axial skeleton, limb, or hindbrain.

Tight regulation of Notch pathway signaling is important in many aspects of embryonic development. Notch signaling can be modulated by expression of fringe genes, encoding glycosyltransferases that modify EGF repeats in the Notch receptor. Although Lunatic fringe (Lfng) has been shown to play important roles in vertebrate segmentation, comparatively little is known regarding the developmental functions of the other vertebrate fringe genes, Radical fringe (Rfng) and Manic fringe (Mfng). Here we report that Mfng expression is not required for embryonic development. Further, we find that despite significant overlap in expression patterns, we detect no obvious synergistic defects in mice in the absence of two, or all three, fringe genes during development of the axial skeleton, limbs, hindbrain, and cranial nerves. Developmental Dynamics 238:1803–1812, 2009.

Lunatic fringe protein processing by proprotein convertases may contribute to the short protein half-life in the segmentation clock.

During vertebrate segmentation, oscillatory activation of Notch signaling is important in the clock that regulates the timing of somitogenesis. In mice, the cyclic activation of NOTCH1 requires the periodic expression of Lunatic fringe (Lfng). For LFNG to play a role in the segmentation clock, its cyclic transcription must be coupled with post-translational mechanisms that confer a short protein half-life. LFNG protein is cleaved and released into the extracellular space, and here we examine the hypothesis that this secretion contributes to a short LFNG intracellular half-life, facilitating rapid oscillations within the segmentation clock. We localize N-terminal protein sequences that control the secretory behavior of fringe proteins and find that LFNG processing is promoted by specific proprotein convertases including furin and SPC6. Mutations that alter LFNG processing increase its intracellular half-life without preventing its secretion. These mutations do not affect the specificity of LFNG function in the Notch pathway, thus regulation of protein half-life affects the duration of LFNG activity without altering its function. Finally, the embryonic expression pattern of Spc6 suggests a role in terminating LFNG activity during somite patterning. These results have important implications for the mechanisms that contribute to the tight control of Notch signaling during vertebrate segmentation.

Posterior skeletal development and the segmentation clock period are sensitive to Lfng dosage during somitogenesis

The segmental structure of the axial skeleton is formed during somitogenesis. During this process, paired somites bud from the presomitic mesoderm (PSM), in a process regulated by a genetic clock called the segmentation clock. The Notch pathway and the Notch modulator Lunatic fringe (Lfng) play multiple roles during segmentation. Lfng oscillates in the posterior PSM as part of the segmentation clock, but is stably expressed in the anterior PSM during presomite patterning. We previously found that mice lacking overt oscillatory Lfng expression in the posterior PSM (Lfng(∆FCE)) exhibit abnormal anterior development but relatively normal posterior development. This suggests distinct requirements for segmentation clock activity during the formation of the anterior skeleton (primary body formation), compared to the posterior skeleton and tail (secondary body formation). To build on these findings, we created an allelic series that progressively lowers Lfng levels in the PSM. Interestingly, we find that further reduction of Lfng expression levels in the PSM does not increase disruption of anterior development. However tail development is increasingly compromised as Lfng levels are reduced, suggesting that primary body formation is more sensitive to Lfng dosage than is secondary body formation. Further, we find that while low levels of oscillatory Lfng in the posterior PSM are sufficient to support relatively normal posterior development, the period of the segmentation clock is increased when the amplitude of Lfng oscillations is low. These data support the hypothesis that there are differential requirements for oscillatory Lfng during primary and secondary body formation and that posterior development is less sensitive to overall Lfng levels. Further, they suggest that modulation of the Notch signaling by Lfng affects the clock period during development.

Disruption of somitogenesis by a novel dominant allele of Lfng suggests important roles for protein processing and secretion.

Vertebrate somitogenesis is regulated by a segmentation clock. Clock-linked genes exhibit cyclic expression, with a periodicity matching the rate of somite production. In mice, lunatic fringe (Lfng) expression oscillates, and LFNG protein contributes to periodic repression of Notch signaling. We hypothesized that rapid LFNG turnover could be regulated by protein processing and secretion. Here, we describe a novel Lfng allele (LfngRLFNG), replacing the N-terminal sequences of LFNG, which allow for protein processing and secretion, with the N-terminus of radical fringe (a Golgi-resident protein). This allele is predicted to prevent protein secretion without altering the activity of LFNG, thus increasing the intracellular half-life of the protein. This allele causes dominant skeletal and somite abnormalities that are distinct from those seen in Lfng loss-of-function embryos. Expression of clock-linked genes is perturbed and mature Hes7 transcripts are stabilized in the presomitic mesoderm of mutant mice, suggesting that both transcriptional and post-transcriptional regulation of clock components are perturbed by RLFNG expression. Contrasting phenotypes in the segmentation clock and somite patterning of mutant mice suggest that LFNG protein may have context-dependent effects on Notch activity. Summary: A radical/lunatic fringe chimeric allele predicted to affect protein secretion disrupts segmentation clock function and somite patterning with context-dependent effects.