Noottcchh ssiiggnnaalliinngg,, tthhee sseeggmmeennttaattiioonn ccl - PDF document

Noottcchh ssiiggnnaalliinngg,, tthhee sseeggmmeennttaattiioonn cclloocckk,, aanndd tthhee ppaatttteerrnniinngg ooff vveerrtteebbrraatteessoommiitteessJulian Lewis, Anja Hanisch and Maxine HolderAddress: Vertebrate Development Laboratory, Cancer Research UK London Research Institute, 44 Lincolns Inn Fields, London WC2A In one way or another, at one stage or another, almost everytissue in an animal body depends for its patterning on theNotch cell-cell signaling pathway [1]. The evidence frommutants is clear: disrupted Notch signaling entails disruptedpattern. The challenge is to define precisely what it is thatNotch signaling does in any given case, and when it does it.This problem is posed in a particularly striking and curiousway by the phenomena of somitogenesis - the process bywhich the vertebrate embryo lays down the regularsequence of tissue blocks that will give rise to the musculo-skeletal segments of the neck, trunk, and tail.These blocks of embryonic tissue, the somites, are arrangedsymmetrically in a neat, repetitive pattern on either side ofthe central body axis. Each somite is separated from the nextby a cleft - the segment boundary; and each somite has adefinite polarity, with an anterior portion and posteriorportion expressing different sets of genes [2]. Mutations in AAbstractThe Notch signaling pathway has multifarious functions in the organization of the developingvertebrate embryo. One of its most fundamental roles is in the emergence of the regularpattern of somites that will give rise to the musculoskeletal structures of the trunk. The partsit plays in the early operation of the segmentation clock and the later definition and differentiation of the somites are beginning to be understood. Journal of Biology 88:44 Journal of Biology88:44 (doi:10.1186/jbiol145)found online at http://jbiol.com/content/8/4/44 formation depends on the ways in which these components -ormation depends on the ways in which these components -matical modeling highlights several possibilities. Thus, onetype of linkage, where Notch activation leads to down-regulation of Notch ligand expression in the signal-receivingcell, can lead to lateral inhibition, forcing neighboring cellsto become different from one another [11] (Figure 2). Anopposite linkage, whereby Notch activation stimulatesligand expression, can have an opposite effect, inducingcontiguous cells to be similar [12]. Still other types ofcircuitry built from the same components can perform yetother tricks, including the production of temporal oscilla-tions of gene expression [13,14]. And this brings us back tosomitogenesis, where such oscillations are in fact seen.AA ggeennee--eexxpprreessssiioonn oosscciillllaattoorr mmaarrkkss oouutt tthhee ppeerriiooddiiccppaatttteerrnn ooff bbooddyy sseeggmmeennttssSomites derive from the unsegmented presomitic meso-derm (PSM) at the tail end of the embryo. PSM cells arespecified by the combined action of Wnt and fibroblastgrowth factor (FGF) signaling molecules, which areproduced at the tail end of the PSM and spread anteriorly togenerate a morphogen gradient. At the point where the levelof Wnt and FGF falls below a threshold value, somites form.Thus, as the PSM grows caudally, extending the embryo,one pair of somites after another is budded off from theanterior end of the PSM in a regular head-to-tail sequence.Each species generates its characteristic number of somitesat its own pace, ranging from one new somite pair approxi-mately every 30minutes in zebrafish to one pair every2hours in mice. This rhythmic process involves coordinatedhours in mice. This rhythmic process involves coordinatedoscillate in the PSM and that show this cyclic expression inall vertebrates belong to the Notch signaling pathway; theseoscillatory genes include, specifically, certain members ofthe Hairy/E(spl) gene family of bHLH transcriptionalregulators - in particular Hes1and Hes7in mice, her1andher7in zebrafish, and hairy1and hairy2in chick [15-22] -and (in zebrafish) the Notch ligand DeltaC, whoseexpression is controlled by them. These, and certain otheroscillatory genes, display a characteristic pattern ofexpression that can be seen in fixed specimens stained by insituhybridization. In the posterior part of the PSM, the levelof expression may be high or low, depending on the phaseof the oscillation cycle at the moment when the embryo wasfixed. In the anterior part of the PSM, meanwhile, one sees astripy pattern, in which bands of cells that express theoscillatory gene strongly alternate with bands of cells thatdo not (Figure3). This pattern reflects the gradual slowing 2009,Volume 8, Article 44Lewis 88:44 Figure 1receptor whose ligand Delta is also expressed on the cell surface.Binding of Delta to Notch activates cleavage of Notch at themembrane, thereby releasing the Notch intracellular domain (NICD), with Delta, is endocytosed into the Delta-expressing cell. DeltaNotch DeltaNICDNICD NECDNECD in nucleus Cleavage Figure 2both the Notch receptor and its ligand, Delta, but the cell on the leftHes/her gene is activated inexpression is maintained, and genes specifying differentiation are gene DeltaNotch Differentiation Low Notch Differentiation posterior cells, with the consequence that one sees laid out along the antero-posterior axis of the PSM an ordered array of cells in different phases of the oscillator cycle [15,23]. Disturbances of oscillator behavior are thus clearly displayed in a disturbed spatial pattern of gene expression in the anterior PSM - a great convenience for experimental analysis. N N o o t t c c h h s s i i g g n n a a l l i i n n g g k k e e e e p p s s c c e e l l l l c c l l o o c c k k s s s s y y n n c c h h r r o o n n i i z z e e d d Since, as we noted earlier, any mutation that blocks Notch signaling leads to disrupted somite segmentation, an obvious suggestion is that the oscillation depends on Notch signaling and fails to occur when Notch signaling fails. However, the detailed consequences of mutations in the Notch pathway do not quite fit this simple explanation. A different interpretation is instead suggested by a closer examination of the behavior of one of the oscillatory genes, coding for the Notch ligand DeltaC, in zebrafish with mutations in the Notch pathway [24]. The individual PSM cells in these mutants still express DeltaC, but in an uncoordinated way: tissue fixed for analysis by in situ hybridization shows a pepper-and-salt mixture of cells expressing DeltaC at different levels, as though the cells are still oscillating individually, but no longer in synchrony with their neighbors (Figure 4). Moreover, both in zebrafish and in mice, the first few somites of embryos with Notch pathway mutations develop almost normally [25-27], implying that Notch signaling is not absolutely necessary for somite segmentation and that the consequences of failure of Notch signaling make themselves felt only gradually, after the onset of somitogenesis. These findings led to the suggestion that the primary function of Notch signaling is not to drive the oscillations of individual cells, but only to coordinate them and keep them synchronized; and that the cells begin oscillation in synchrony at the start of somitogenesis, and take several cycles to drift out of synchrony when Notch signaling is defective [24]. This proposal - that Notch signaling from cell to cell in the PSM serves to maintain synchrony but is not necessary for oscillation of individual cells - has been supported by several subsequent experiments. For example, zebrafish embryos can be treated at different stages of somitogenesis http://jbiol.com/content/8/4/44 Journal of Biology 2009,Volume 8, Article 44Lewis et al. 44.3 Journal of Biology 2009, 8 8 : : 44 F F i i g g u u r r e e 3 3 Somitogenesis and the segmentation clock. ( ( a a ) ) The pattern of expression of one of the oscillatory genes - deltaC - during somitogenesis in the zebrafish. Two specimens are shown, fixed and stained by in situ hybridization (ISH) at different phases of their somitogenesis cycle. ( ( b b ) ) Diagram showing how the observed pattern of gene expression reflects the cyclic behavior of the individual cells. Each cell contains a gene-expression oscillator - a clock - which slows down as the cell moves from the posterior to the anterior part of the PSM, giving rise to a pattern of stripes of cells in different phases of their oscillation. The oscillation is halted as cells emerge from the PSM, leaving them arrested i n different states (blue versus white shading), thereby demarcating the somite boundaries (black lines). The extent of the PSM is defined by an Fgf + Wn t signal gradient, with its origin at the tail end of the embryo. Caudal growth Individual cell clocks in different phases of the clock cycle Anterior PSM Oscillations slowing Posterior PSM Oscillations at maximum speed Formed somites Oscillations halted DeltaC ISH phase B DeltaC ISH phase A 1/2 cycle 1 cycle (a)(b) evidence comes from experiments where PSM cells aretransplanted into a wild-type zebrafish embryo from antransplanted into a wild-type zebrafish embryo from anindividual PSM cells and the influence of Notch signalingcan also be demonstrated through study of cells from thePSM of a transgenic mouse embryo containing aluminescent Hes1reporter. These cells show oscillatingexpression of the reporter gene even when they are disso-ciated and thus unable to communicate via Notch [31], butin that condition the oscillations are much less regular thanin the intact tissue.WWhhaatt iiss tthhee uullttiimmaattee ppaacceemmaakkeerr ooff tthhee sseeggmmeennttaattiioonncclloocckk??All these findings support the view that Notch is needed tomaintain synchrony between the oscillations of theindividual cells, which are somewhat noisy and imperfecttimekeepers when left to their own devices. But what istimekeepers when left to their own devices. But what is()Loss of Hes7in the mouse, or of her1and her7in thezebrafish, disrupts segmentation all along the body axis;and it has been shown experimentally that these genes areindeed subject to negative regulation by their own products 2009,Volume 8, Article 44Lewis 88:44 Figure 4signaling fails, the individual cells (in zebrafish at least) continue to oscillate but fall out of synchrony, and somite patterning breaks down. Synchrony lost boundaries Figure 5her1/7 her1/7 gene itself, but only after a delay fortranscription (T whose period is determined by the total delay in the feedback loop. gene DelayDelay p vertebrate hindbrain, likewise, it is involved in organizingthe boundaries between rhombomeres [42]. As for segmentpolarity, the creation of a difference between the cells of theanterior and posterior parts of each somite could be seen assimilar to the creation of differences between adjacent cellsthrough lateral inhibition - a well known function of Notchsignaling in many different systems [1].NNoottcchh ssiiggnnaalliinngg iiss ddiissppeennssiibbllee ffoorr bboouunnddaarryy ffoorrmmaattiioonniinn zzeebbrraaffiisshhIt is in the anterior part of the PSM, where the oscillation ofcyclic genes slows down and then halts, that cells are assignedto anterior or posterior somite compartments and clefts form,finally demarcating one somite from the next. Thus, theformation of the segment boundary and the specification ofantero-posterior polarity are both processes that occurrelatively late in the history of each somite, after its precursorcells have graduated to the anterior part of the PSM from theposterior as the embryo grows and extends. If the earlyfunction of Notch signaling in maintaining synchrony in theposterior PSM is disrupted, any failure in these later functionsis likely to be imperceptible amid the general chaos. One can,however, test for the later functions by imposing a block ofNotch signaling part way through somitogenesis. Forexample, one can take a zebrafish that has already formedfive somites and immerse it in a DAPT solution to blockNotch signaling from that time point onwards. The result isstriking: the next approximately 12 somites proceed to formin the normal way, with regularly spaced boundaries, andonly after that does one begin to see segmentation defects[28,29]. This shows that Notch signaling is not needed, in thezebrafish at least, for the creation of somite boundaries, andit quantitatively matches predictions based on theproposition that the only function of Notch signaling is tomaintain synchrony in the posterior PSM [29].CClleefftt ffoorrmmaattiioonn ccoorrrreellaatteess wwiitthh tthhee aappppeeaarraannccee ooffsshhaarrpp bboouunnddaarriieess ooff ggeennee eexxpprreessssiioonnFindings in the mouse, however, are not so clear, and thereare differing schools of thought. In a series of papers[43-49], Saga and colleagues have argued that Notchgene expression that is necessary to mark the future cleftgene expression that is necessary to mark the future cleftconclusions emerge from study of a pair of transcriptionalregulators - Mesp2, and the less well characterized Mesp1 -that are expressed in the anterior PSM. They seem to operateas orchestrators of the process by which the output of thesomite oscillator is translated into the spatially repeatingpattern of the somites [45] - a process that is disrupted inMesp2mutants [46]. Mesp2 is expressed dynamically ineach forming somite, beginning as a one-somite-wide stripe,h forming somite, beginning as a one-somite-wide stripe,particular, somite boundaries form at interfaces where cellswith high expression of but low Notch activationrestriction of the restriction of the thus essential for the establishment of the anterior-posteriorpolarity of each new somite. However, these observationsdo not amount to firm proof: correlation need not implycausation, and Mesp2, acting independently of Notchactivity, could be the critical factor. The pattern of Mesp2expression is indeed altered in Notch pathway mutants[43], but it is hard to be sure whether this reflects a functionof Notch signaling in the anterior PSM where Mesp2isexpressed, or merely the aftermath of the disorder created 2009,Volume 8, Article 44Lewis 88:44 2009,Volume 8, Article 44Lewis 88:44 between cells with differing levels of Notch activation; thesethese clefts form later than normal and are crooked andthese clefts form later than normal and are crooked andNNoottcchh ssiiggnnaalliinngg iiss uusseedd rreeppeeaatteeddllyy iinn tthhee ssoommiittee cceelllllliinneeaaggeeThe formation of the somites is not the end of theinvolvement of Notch signaling in the development of thesomitic cell lineage. For example, skeletal muscle tissue,which arises from the somites, also depends on this path-way to control the differentiation of myoblasts and satellitecells and their incorporation into multinucleate musclefibers [51-54]. Like that other ubiquitous communicationdevice, the mobile phone network, the Notch signalingpathway has been recruited for many different purposes -for the simple delivery of instructions from one individualto another, for competitions and collaborations, for thesynchronization of individual actions, and for the playingof the tunes to which cells dance.RReeffeerreenncceess1.Bray SJ: 2.Hughes D, Keynes R, Tannahill D: 3.Gridley T: 4.Holley SA: 5.Saga Y, Takeda H: 6.Weinmaster G, Kintner C: 7.Kopan R, Ilagan MX: 8.Krejci A, Bernard F, Housden BE, Collins S, Bray SJ: 9.Ong CT, Cheng HT, Chang LW, Ohtsuka T, Kageyama R, Stormo10.Bray S: 11.Collier JR, Monk NA, Maini PK, Lewis JH: 12.Lewis J: 13.Lewis J: 14.Monk NAM: 15.Palmeirim I, Henrique D, Ish-Horowicz D, Pourquie O: 16.Bessho Y, Sakata R, Komatsu S, Shiota K, Yamada S, Kageyama R:17.Gajewski M, Sieger D, Alt B, Leve C, Hans S, Wolff C, Rohr KB,18.Henry CA, Urban MK, Dill KK, Merlie JP, Page MF, Kimmel CB,19.Holley SA, Geisler R, Nusslein-Volhard C: 20.Holley SA, Julich D, Rauch GJ, Geisler R, Nusslein-Volhard C: 21.Jouve C, Palmeirim I, Henrique D, Beckers J, Gossler A, Ish-22.Oates AC, Ho RK: 23.Giudicelli F, Ozbudak EM, Wright GJ, Lewis J: 24.Jiang YJ, Aerne BL, Smithers L, Haddon C, Ish-Horowicz D, Lewis25.Conlon RA, Reaume AG, Rossant J: 26.Huppert SS, Ilagan MX, De Strooper B, Kopan R: 27.van Eeden FJ, Granato M, Schach U, Brand M, Furutani-Seiki M, anndd ppaatttteerrnniinngg iinn tthhee zzeebbrraaffiisshh,, DDaanniioo rreerriioo..Development1996,112233::153-164.28.Riedel-Kruse IH, Muller C, Oates AC: 29.Ozbudak EM, Lewis J: 30.Horikawa K, Ishimatsu K, Yoshimoto E, Kondo S, Takeda H:31.Masamizu Y, Ohtsuka T, Takashima Y, Nagahara H, Takenaka Y,Proc Natl Acad Sci USA32.Bessho Y, Hirata H, Masamizu Y, Kageyama R: 33.Hirata H, Bessho Y, Kokubu H, Masamizu Y, Yamada S, Lewis J,34.Lewis J, Ozbudak EM: 35.Aulehla A, Wehrle C, Brand-Saberi B, Kemler R, Gossler A,36.Dale JK, Malapert P, Chal J, Vilhais-Neto G, Maroto M, Johnson T,37.Dequeant ML, Glynn E, Gaudenz K, Wahl M, Chen J, Mushegian A,38.Aulehla A, Wiegraebe W, Baubet V, Wahl MB, Deng C, Taketo M,39.Giudicelli F, Lewis J: 40.Ozbudak EM, Pourquie O: 41.Irvine KD: 42.Cheng YC, Amoyel M, Qiu X, Jiang YJ, Xu Q, Wilkinson DG:43.Morimoto M, Takahashi Y, Endo M, Saga Y: 44.Saga Y: 45.Oginuma M, Niwa Y, Chapman DL, Saga Y: 46.Saga Y, Hata N, Koseki H, Taketo MM: 47.Takahashi Y, Inoue T, Gossler A, Saga Y: 48.Takahashi Y, Koizumi K, Takagi A, Kitajima S, Inoue T, Koseki H,SagaY: 49.Koizumi K, Nakajima M, Yuasa S, Saga Y, Sakai T, Kuriyama T, Shi-50.Feller J, Schneider A, Schuster-Gossler K, Gossler A: 51.Conboy IM, Rando TA: 52.Hirsinger E, Malapert P, Dubrulle J, Delfini MC, Duprez D, Hen-53.Schuster-Gossler K, Cordes R, Gossler A: 54.Vasyutina E, Lenhard DC, Birchmeier C: 2009,Volume 8, Article 44Lewis 88:44