Body-Building

The Faculty of 1000 is aWeb-based literature awareness tool published by BioMed Central. For more information visit www.facultyof1000.com. Since Aristotle, humans have pondered how body patterns form. Almost invariably, each person has the same skeletomuscular arrangements. Some bodily structures, such as vertebra and ribs, seem to come about by reiteration of a common process. These segments derive from somites, which are serially repeated precursors that in turn develop by sequentially 'budding off' from the developing embryo's undifferentiated presomitic mesoderm (PSM). Somitogenesis occurs like clockwork, and in 1976, an oscillatory mechanism was proposed to account for it.1 In 1997, molecular evidence of an oscillator in vertebrate PSM was uncovered, found in a chicken homologue of the Drosophila segmentation gene hairy. This gene--coding for part of the Notch signaling pathway--was cloned and found to be expressed in cycles, with a period corresponding to the time it takes to form a single somite.2 Genetic and other analyses have since found that other proteins in the Notch pathway, including Lunatic fringe (Lfng) are expressed cyclically as well. Thus, a clock's existence has been evinced, with Notch seen as a key player. Yet, until recently researchers could not discern how an oscillatory mechanism operates. Two groups have independently reported uncovering enhancer elements for murine Lfng, which is capable of driving cyclical expression in the PSM.3,4 These papers demonstrate that the oscillation of a segmentation clock gene is controlled primarily at the transcriptional level, opening the door to understanding how that clock drives somitogenesis. The work is "fairly important," remarks developmental biologist Olivier Pourquie of the Stowers Institute for Medical Research in Kansas City, Mo. "They make some significant progress toward implicating Notch signaling more deeply in the core mechanism of the oscillator." Susan Cole, a postdoctoral researcher under Thomas Vogt, and their Princeton University colleagues created a series of transgenes using noncoding sequences from Lfng to drive expression of the bacterial lacZ reporter gene. By examining embryos that are transgenic for the reporter constructs, they isolated a 4.3-kb fragment capable of recapitulating the oscillatory expression pattern of endogenous Lfng in the PSM. The researchers then compared the murine regulatory se-quences with their human counterparts; they discovered several conserved regions of high nucleotide identity between the two species. Of these, two 110 base pairs each were found necessary to direct Lfng expression. The regions contain E-box sequences known to bind basic helix-loop-helix transcription factors, many of which have been implicated in the Notch signaling pathway. At the same time, David Ish-Horowicz's group at Cancer Research UK was working to determine how Lfng levels oscillate. Using cDNA probes that recognize intronic sequences, the researchers found a diverse pattern of nascent Lfng mRNA in the chick nuclei, indicating that transcription is dynamic rather than constant. Patterns of cytoplasmic mRNA, visualized with exonic probes, matched that of the nuclear accumulations. This, coupled with data showing that the mRNA is inherently unstable, implied that Lfng message levels are regulated at the transcriptional level. The London researchers began looking for sequences capable of dynamically controlling transcription in the murine PSM, using methods similar to that of their American counterparts. By using Lfng upstream sequences linked to lacZ reporters, they identified several sequences that seemed to be responsible for oscillation--among them a putative binding site for the transcription factor CBF1, believed to be the sole direct mediator of Notch signaling. "When we knocked out the E-boxes, there was no expression in the PSM at all," Cole notes. When Ish-Horowicz's group mutated the CBF1 binding sites, LacZ expression was greatly reduced; they also found that in CBF1-/- mice, Lfng expression is very weak and does not cycle. Cole suggests that perhaps the E-boxes turn Lfng on, and CBF1 refines the expression into the perfect cyclic pattern. Josh P. Roberts (tcwriter@msn.com) is a freelance writer in Minneapolis. References 1. J. Cooke, E.C. Zeeman, "A clock and wavefront model for control of the number of repeated structures during animal morphogenesis," Journal of Theoretical Biology, 58:455-76, 1976. 2. I. Palmeirim et al., "Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis," Cell, 91:639-48, 1997. 3. A.V. Morales et al., "Periodic Lunatic fringe expression is controlled during segmentation by a cyclic transcriptional enhancer responsive to notch signaling," Developmental Cell, 3:63-74, July 2002. 4. S.E. Cole et al., "Clock regulatory elements control cyclic expression of Lunatic fringe during somitogenesis," Developmental Cell, 3:75-84, July 2002.

Body-Building

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