Notch signaling, the segmentation clock, and the patterning of vertebrate somites
© BioMed Central Ltd 2009
Published: 22 May 2009
The Notch signaling pathway has multifarious functions in the organization of the developing vertebrate embryo. One of its most fundamental roles is in the emergence of the regular pattern of somites that will give rise to the musculoskeletal structures of the trunk. The parts it plays in the early operation of the segmentation clock and the later definition and differentiation of the somites are beginning to be understood.
In one way or another, at one stage or another, almost every tissue in an animal body depends for its patterning on the Notch cell-cell signaling pathway . The evidence from mutants is clear: disrupted Notch signaling entails disrupted pattern. The challenge is to define precisely what it is that Notch signaling does in any given case, and when it does it. This problem is posed in a particularly striking and curious way by the phenomena of somitogenesis – the process by which the vertebrate embryo lays down the regular sequence of tissue blocks that will give rise to the musculoskeletal segments of the neck, trunk, and tail.
These blocks of embryonic tissue, the somites, are arranged symmetrically in a neat, repetitive pattern on either side of the central body axis. Each somite is separated from the next by a cleft – the segment boundary; and each somite has a definite polarity, with an anterior portion and posterior portion expressing different sets of genes . Mutations in components of the Notch signaling pathway play havoc with this whole pattern: although somites may eventually form, the segment boundaries are irregular and randomly positioned, and the regular antero-posterior polarity of individual somites is lost. Genetic screens for mutations that disrupt segmentation in this way chiefly identify Notch pathway components as the critical players. Notch signaling is clearly central to somitogenesis [3–6]. But precisely how?
Notch pathway components can be wired together in different ways for different outcomes
A gene-expression oscillator marks out the periodic pattern of body segments
Notch signaling keeps cell clocks synchronized
What is the ultimate pacemaker of the segmentation clock?
The need for Notch signaling may extend beyond the control of the clock
Failure of synchronization is sufficient to explain the disruption of segmentation in Notch pathway mutants. But that is not necessarily the end of the story. To acknowledge that Notch signaling has this critical function, and that that is enough to explain the mutant phenotypes, is not the same as saying that synchronization is the only function of whose period is determined by the total delay in the feedback loop.
Notch signaling in somitogenesis. At least two additional functions have been proposed. One is in the final step at which a segment boundary is created by physical separation of one nascent somite from the next; the other is in creating or maintaining the difference between anterior and posterior halves of each somite. Each of these possible further roles for Notch signaling – in boundary formation and in segment polarity – seems attractive on the basis of analogies with other systems. Thus, in the Drosophila wing disc, Notch signaling plays a critical part in organizing the dorso-ventral compartment boundary ; and in the vertebrate hindbrain, likewise, it is involved in organizing the boundaries between rhombomeres . As for segment polarity, the creation of a difference between the cells of the anterior and posterior parts of each somite could be seen as similar to the creation of differences between adjacent cells through lateral inhibition – a well known function of Notch signaling in many different systems .
Notch signaling is dispensible for boundary formation in zebrafish
It is in the anterior part of the PSM, where the oscillation of cyclic genes slows down and then halts, that cells are assigned to anterior or posterior somite compartments and clefts form, finally demarcating one somite from the next. Thus, the formation of the segment boundary and the specification of antero-posterior polarity are both processes that occur relatively late in the history of each somite, after its precursor cells have graduated to the anterior part of the PSM from the posterior as the embryo grows and extends. If the early function of Notch signaling in maintaining synchrony in the posterior PSM is disrupted, any failure in these later functions is likely to be imperceptible amid the general chaos. One can, however, test for the later functions by imposing a block of Notch signaling part way through somitogenesis. For example, one can take a zebrafish that has already formed five somites and immerse it in a DAPT solution to block Notch signaling from that time point onwards. The result is striking: the next approximately 12 somites proceed to form in the normal way, with regularly spaced boundaries, and only after that does one begin to see segmentation defects [28, 29]. This shows that Notch signaling is not needed, in the zebrafish at least, for the creation of somite boundaries, and it quantitatively matches predictions based on the proposition that the only function of Notch signaling is to maintain synchrony in the posterior PSM .
Cleft formation correlates with the appearance of sharp boundaries of gene expression
Findings in the mouse, however, are not so clear, and there are differing schools of thought. In a series of papers [43–49], Saga and colleagues have argued that Notch signaling is indeed needed to create a sharp boundary of gene expression that is necessary to mark the future cleft between one nascent somite and the next [43, 44]. Their conclusions emerge from study of a pair of transcriptional regulators – Mesp2, and the less well characterized Mesp1 – that are expressed in the anterior PSM. They seem to operate as orchestrators of the process by which the output of the somite oscillator is translated into the spatially repeating pattern of the somites  – a process that is disrupted in Mesp2 mutants . Mesp2 is expressed dynamically in each forming somite, beginning as a one-somite-wide stripe, rapidly narrowing to a half-somite-wide stripe (which marks the future anterior compartment of the somite), then disappearing completely as the somite buds off from the PSM. In the brief window during which it is expressed, Mesp2 seems to be responsible for allocating anterior or posterior identity to the cells of the somite through activation or repression of various targets that distinguish the anterior from the posterior cells, and for regulating some of the genes required for border formation [47, 48]. In particular, somite boundaries form at interfaces where cells with high expression of Mesp2 but low Notch activation confront cells in an opposite state, with high Notch activation but no expression of Mesp2. These observations strongly suggest that some sort of feedback loop involving Mesp2 and Notch signaling organizes the formation of an interface between cells with high Notch activation and cells with low Notch activation, and that this interface is necessary to define the segment boundary. Moreover, the same studies suggest that Notch signaling is involved in the restriction of the Mesp2 expression domain from the whole presumptive somite to just its anterior half [48, 49], and thus essential for the establishment of the anterior-posterior polarity of each new somite. However, these observations do not amount to firm proof: correlation need not imply causation, and Mesp2, acting independently of Notch activity, could be the critical factor. The pattern of Mesp2 expression is indeed altered in Notch pathway mutants , but it is hard to be sure whether this reflects a function of Notch signaling in the anterior PSM where Mesp2 is expressed, or merely the aftermath of the disorder created by prior failure of Notch signaling in the posterior PSM.
Notch signaling is required to give each somite its antero-posterior polarity
Feller et al.  tested the role of Notch signaling in the mouse PSM in a different way and came to a somewhat different view. When they artificially expressed NICD, the intracellular transcriptional regulator domain of Notch, throughout the entire PSM, they found that many somite boundaries still formed, despite the absence of any interface between cells with differing levels of Notch activation; these boundaries, however, were irregularly spaced, and the resulting irregular blocks of somite tissue lacked the normal antero-posterior polarity. The same was seen when Notch signaling, instead of being artificially activated, was inactivated by mutations in Notch1, or Dll1 (Delta1), or Pofut1 (coding for an enzyme that fucosylates Notch and is required for Notch function). In fact, a similar outcome is seen in zebrafish Notch pathway mutants – clefts eventually appear in the mesoderm, dividing it up into somites, but these clefts form later than normal and are crooked and irregularly spaced. The somitic mesoderm, it seems, has a propensity to split up into tissue blocks and will do so even if the segmentation clock is broken and Notch signaling defective. The role of the clock is to control the pattern of this splitting, ensuring that the clefts are regularly spaced, and to confer on each somite a regular antero-posterior polarity. For this last step, it seems that Notch signaling is required directly and not merely to keep the segmentation clocks of the individual cells ticking synchronously in the run-up to overt segmentation; for in the mice where NICD is expressed throughout the tissue, each somite has a double-posterior character, whereas when Notch fails each somite has a double-anterior character .
Notch signaling is used repeatedly in the somite cell lineage
The formation of the somites is not the end of the involvement of Notch signaling in the development of the somitic cell lineage. For example, skeletal muscle tissue, which arises from the somites, also depends on this pathway to control the differentiation of myoblasts and satellite cells and their incorporation into multinucleate muscle fibers [51–54]. Like that other ubiquitous communication device, the mobile phone network, the Notch signaling pathway has been recruited for many different purposes – for the simple delivery of instructions from one individual to another, for competitions and collaborations, for the synchronization of individual actions, and for the playing of the tunes to which cells dance.
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