Max Planck-Institut für Entwicklungsbiologie, Tübingen, Germany
Present address: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA
*Author for correspondence: scott.holley{at}yale.edu
Accepted 11 December 2001
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SUMMARY |
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Supplementary data available at http://www.eb.tuebingen.mpg.de/papers/holley_dev_2002.html
Key words: Zebrafish, deadly seven, notch1, her1, Somite, Segmentation, Oscillator, Morpholino
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INTRODUCTION |
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after eight (aei; dld Zebrafish Information Network), deadly seven (des), fused somites (fss), beamter (bea) and white tail/mindbomb (wit) are the five genes that are necessary for normal somite formation isolated in our zebrafish genetic screen (van Eeden et al., 1996; Jiang et al., 1996
). We have shown previously that aei codes for the notch ligand deltaD (Dornseifer et al., 1997
; Holley et al., 2000
). Moreover, we have shown that aei/deltaD is required to create the oscillating pattern of her1, but that its mRNA expression does not oscillate (Holley et al., 2000
). However, none of the genes shown to be necessary to produce the oscillating pattern of mRNA expression actually oscillate themselves. Thus, it is not clear if these genes [aei/deltaD in the zebrafish and Delta-like1 (Dll1) in the mouse] constitute core components of the oscillator or if they simply are necessary to produce the oscillator readout. Furthermore, the analysis of the oscillating genes hairy and lfng in the chick, and Hes1 and Lfng in the mouse suggest that neither of these genes functions within the oscillator mechanism (Palmeirim et al., 1997
; McGrew et al., 1998
; Forsberg et al., 1998
; Aulehla and Johnson, 1999
; Jouve et al., 2000
). Thus, it is likewise not clear if any of the known oscillating genes are central components of the oscillator.
We show that des encodes for notch1 (Bierkamp and Campos-Ortega, 1993). Like aei/deltaD, des/notch1 expression does not oscillate, but its protein is required for the oscillation of both her1 and deltaC expression. Using morpholino oligonucleotides (mo), we performed a series of gene knockdown experiments to ascertain the functions of the oscillating genes her1 and deltaC during somitogenesis. We find that both genes are required to create the oscillating pattern of her1 and deltaC expression. Further analysis of double-mutant and double-knockdown embryos indicates that the epistatic relationship between the notch pathway and her1 changes along the anterior-posterior axis of the PSM. This demonstrates that these notch pathway genes have at least two functions during somitogenesis and that these genes operate within a notch pathway
her1
notch pathway regulatory circuit (Takke and Campos-Ortega, 1999
). Because this circuit is comprised of genes that are necessary to create the oscillations in gene expression, these data suggest a model in which both the notch pathway and her1 comprise part of the oscillator that regulates zebrafish somitogenesis.
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MATERIALS AND METHODS |
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Mapping
Radiation hybrid mapping was performed as previously described (Geisler et al., 1999). For mapping of des, PCR reactions for specific SSLPs were performed as for the radiation hybrid mapping but at half the volume per reaction.
Allele sequencing
For each allele of des, PCR products derived from three independent reverse transcriptase (RT) reactions were sequenced using the ABI system and analyzed using the Lasergene software package. Total RNA was isolated from mutant embryos using TriStar reagent (Angewandte Gentechnologie Systeme GmbH) according to kit protocol. RT-PCR was performed using the SuperScript kit (GIBCO BRL). From each RT reaction, the notch1 mRNA was amplified in nine overlapping 1 kb fragments. Current allele designations relate to the originals (van Eeden et al., 1996) as follows: desAXO1B, destx201; desH35B, desth35b; desP37A, destp37; desM145B, destm145.
Morpholino injections
Morpholinos (Gene-Tools, http://www.gene-tools.com) were injected at the one-cell stage at a concentration of 50 µM (deltaCmo1, 5'-agccatctttgccttcttgtctgct-3'), 50 µM (deltaCmoC, 5'-agtcatctttggcttcttgtgttct-3'), 250 µM (deltaCmo2, 5'-cgatagcagactgtgagagtagtcc-3'), 100 µM (deltaDmo 5'-aaacagctatcattagtcgtcccat-3'), 100 µM (notch1mo, 5'-ttcaccaagaaacggttcataactc-3'), 1 mM (her1mo1, 5'-cgacttgccatttttggagtaacca-3'), 1 mM (her1moC, 5'-cgatttgacatttttggactaatca-3') and 100 µM (her1mo2, 5'-tggctgaaaatcggaagaagacgat-3') in 1xDanieau (Nasevicius and Ekker, 2000).
In situ hybridization
In situ hybridization experiments were performed as previously described (Holley et al., 2000).
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RESULTS |
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The oscillations in gene expression are dependent upon both her1 and deltaC
Because we do not have zebrafish mutants that correspond to either of the oscillating genes, her1 and deltaC, we have used a reverse genetic approach to ascertain the function of these genes in generating the oscillating pattern. Morpholino oligonucleotides specifically inhibit the translation of their target mRNAs (Nasevicius and Ekker, 2000), and here we show that injection of morpholinos specific to either deltaD or notch1, can recapitulate the phenotype of aei and des, respectively, with over 90% penetrance (Fig. 3A,B,F,L).
Injection of morpholinos specific to either her1 or deltaC leads to irregular somite border formation (Fig. 3C,D), and examination of gene expression indicates that both genes are necessary to generate the oscillating pattern of her1 and deltaC expression (Fig. 3G,H,M,N; Fig. 4B,D). The expression patterns that are observed in deltaCmo embryos are somewhat similar to the patterns observed in the existing mutants (Fig. 3). However, the expression patterns seen in her1mo embryos are unique. In her1mo embryos, her1 is expressed throughout the PSM and shows no variation in levels of expression between neighboring cells (Fig. 4B; see http://www.eb.tuebingen.mpg.de/papers/holley_dev_2002.html). This pattern reveals no evidence of oscillations in gene expression, indicating that Her1 protein is required to generate the oscillations in expression of her1 mRNA. deltaC is expressed weakly in the posterior and intermediate PSM of her1mo embryos and more strongly in the anterior PSM. Again, there is no heterogeneity in the levels of expression of deltaC among neighboring cells in these embryos, except for the refinement seen in the anteriormost PSM (Fig. 4D, Fig. 5I; see http://www.eb.tuebingen.mpg.de/papers/holley_dev_2002.html). Therefore, her1 function also is necessary to generate the oscillations of deltaC expression.
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The first set of epistasis experiments uses the fss phenotype as a reference. fss is unique among the known zebrafish genes in that it functions not in creating the oscillating pattern but in maintaining this pattern in the anterior PSM. In fss embryos, one or two her1 (and deltaC) stripes are present, but the anteriormost stripe is always missing (Fig. 5C) (van Eeden et al., 1998; Holley et al., 2000
). Analysis of fss;des/notch1 and fss;aei/deltaD double mutants indicated that the salt and pepper expression of her1 in the anterior PSM of aei/deltaD and des/notch1 embryos is dependent entirely upon fss function (Fig. 5B-D) (van Eeden et al., 1998
; Holley et al., 2000
). This indicates that fss activity is required in the anterior PSM in the absence of des/notch1 and aei/deltaD. Thus, in the anterior PSM, fss functions downstream of des/notch1 and aei/deltaD. Ectopic expression of her1 in the anterior PSM is also observed in deltaCmo embryos, her1mo embryos and bea embryos (Fig. 3G,H,J; Fig. 4B). We have found that this anterior expression is lost in fss;deltaCmo embryos and fss;bea embryos but not fss;her1mo embryos (Fig. 5E-G). Therefore, while fss functions downstream of both deltaC and bea, her1 is the only gene found so far that functions downstream of fss in the anterior PSM.
The second set of epistasis experiments makes use of a unique feature of the deltaC expression pattern in her1mo embryos: the strong domain of deltaC expression in the anterior PSM is refined, resulting in stripes of deltaC expression that persist in the somitic mesoderm (Fig. 4D; Fig. 5I). These stripes resemble the stripes of deltaC expression seen in wild-type embryos (Fig. 3K, Fig. 4C, Fig. 5H). We have used this refinement of deltaC expression in her1mo embryos as an assay to test for additional functions for fss and the notch pathway in the anteriormost PSM, downstream of her1.
her1 is epistatic to fss with regard to deltaC expression in the anterior PSM [i.e. deltaC, like her1, is expressed in the anterior PSM of her1 mo;fss embryos (Fig. 5J) but not fss embryos (Jiang et al., 2000)]. However, the refining of the deltaC expression domain observed in her1mo embryos is lost (compare Fig. 4D and Fig. 5I with Fig. 5J). Thus, while her1 acts downstream of fss with regard to the maintenance of deltaC expression in the anterior PSM, fss functions downstream of her1 with regard to the later refining of deltaC expression in the anteriormost PSM. Analysis of double mutants between her1mo and either aei/deltaD, des/Notch1, deltaCmo or bea, indicate that each of these latter genes functions downstream of her1 in the anteriormost PSM to create the refining pattern of deltaC (Fig. 5K-N). In these double mutant embryos, this refining pattern is converted into a weak salt and pepper pattern, and the stripes of deltaC expression in the somitic mesoderm are eliminated (Fig. 4E; Fig. 5J-M).
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DISCUSSION |
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The loss-of-function phenotype of these genes now can be explained within the context of this model. After the expression of each of these genes is initiated at the posterior of the tailbud, the resulting proteins would initiate the oscillations. If Her1 is absent, then her1 expression is never downregulated in the PSM (Fig. 4B). If aei/deltaD or des/notch1 function is lost, then Her1 derived from the initial burst of her1 expression in the tailbud will repress the transcription of its own mRNA, and the loss of notch signaling would then lead to a failure to re-initiate her1 transcription (Fig. 5B). The phenotypes seen in the anterior PSM in the notch pathway mutants (the salt and pepper pattern) are likely to be the result of an anterior-specific activity.
This model is in agreement with misexpression studies in Xenopus, suggesting that periodic changes in notch signaling activity occur in the PSM (Jen et al., 1999). Our model also can explain the observation that the anterior progression of a wave of chick hairy expression is unperturbed when the PSM is physically separated into anterior and posterior halves (Palmeirim et al., 1997
). The gradient of the repressive activity of chick hairy could provide an instructive memory within the cells of the PSM, and the remaining cell-cell contacts would provide the required Notch-Delta signaling interactions needed to re-initiate chick hairy expression. This type of regulation would not require the oscillating signal to always be propagated from the posterior by an intercellular relay.
notch-dependent or notch-independent oscillations?
In wild-type embryos, neighboring cells oscillate together (they turn on her1 expression together and turn off her1 expression together). This coordination creates the stripes of her1 expression. It has been proposed that the function of the notch pathway during somitogenesis is to synchronize, not to generate, the oscillations of gene expression (Jiang et al., 2000). According to this model, perturbation of notch pathway signaling will cause the cells to lose coordination, and the cells will continue to oscillate independently of their neighbors. These de-synchronized oscillations would not create stripes of gene expression. Instead, a salt and pepper pattern is created in which there is random heterogeneity in levels of gene expression among neighboring cells. The important difference between the de-synchronization model and the model presented in this paper is that the former proposes that the notch pathway does not create the oscillations in gene expression and that in the absence of notch signaling, the oscillations in gene expression persist. The model presented here proposes that the notch pathway generates the oscillations in gene expression and that in the absence of notch pathway signaling, oscillations in gene expression no longer occur.
The phenotype of the her1mo embryos supports the model in which her1 and the notch pathway create the oscillations in gene expression and is inconsistent with the de-synchronization model. her1 is expressed throughout the PSM in her1mo embryos and there is no significant variation in this expression between sibling embryos, i.e. there is no evidence of coordinated oscillations. Moreover, her1mo embryos show no variation in the levels of her1 expression among neighboring cells, i.e. there is no evidence of de-synchronized oscillations (Fig. 4B; see http://www.eb.tuebingen.mpg.de/papers/holley_dev_2002.html). The expression of deltaC in her1mo embryos is more similar to the expression pattern seen in the notch pathway mutants: deltaC is expressed weakly in the posterior PSM and in a strong domain always found in the anterior PSM. However, this anterior expression domain of deltaC in her1mo embryos is uniform and not in a salt and pepper pattern, i.e. there is no evidence of asynchronous oscillations (Fig. 4D, Fig. 5I). These phenotypes indicate that the oscillations in her1 and deltaC expression do not occur if her1 function is absent.
The de-synchronization model originally suggested that the salt and pepper patterns of her1 and deltaC expression seen in aei/deltaD embryos are indicative of continued but de-synchronized cellular oscillations in gene expression (Jiang et al., 2000). However, this model does not account for the absence of stripes of gene expression within the posterior PSM of aei/deltaD embryos because the salt and pepper pattern is restricted to the anterior PSM. aei/deltaD embryos do not have cells within the posterior PSM that express her1 at levels equivalent to the high levels of expression seen within the posterior stripes in wild-type sibling embryos (compare Fig. 5A with 5B). Therefore, there is nothing to indicate that these cells in the posterior and intermediate PSM are oscillating in the absence of aei/deltaD function (Holley et al., 2000
). Furthermore, the de-synchronization model does not explain why there is an abrupt or coordinated onset of the salt and pepper pattern within the middle of the domain in which the oscillations normally are observed. If the oscillations in her1 and deltaC expression persisted in aei/deltaD embryos, then virtually all of these embryos should exhibit a strong salt and pepper pattern gradually arising within the more posterior PSM, as observed for her1 expression in bea embryos (Fig. 3J).
In fss embryos, the oscillations in gene expression occur, but the anteriormost stripe is always missing, indicating that fss is not required to generate the oscillating pattern but is required to maintain this pattern in the anterior PSM (Fig. 5C) (van Eeden et al., 1998; Holley et al., 2000
; Jiang et al., 2000
). Here, the de-synchronization model would make a simple prediction: removal of notch pathway activity in the fss background via fss;aei/deltaD and fss;des/notch1 double mutant combinations should create a de-synchronized version of the oscillating pattern observed in fss embryos, i.e. a salt and pepper pattern instead of stripes. However, only weak posterior expression is observed in these embryos and there is no variation in levels of expression among neighboring cells (Fig. 5D; see http://www.eb.tuebingen.mpg.de/papers/holley_dev_2002.html) (van Eeden et al., 1998
; Holley et al., 2000
). The cells turn on her1 expression posteriorly and together, gradually lose their expression as they mature and become relatively more anterior. Thus, the loss of Notch pathway function results in an elimination, not de-synchronization, of oscillations in gene expression.
These analyses indicate that all evidence of the oscillations in her1 and deltaC expression is absent in backgrounds in which either her1 or aei/deltaD function is missing. This suggests that the generation of the oscillations and the coordination of the oscillations between cells are one and the same, and that the two processes cannot be separated. Nevertheless, one cannot exclude the possibility that the oscillations persist in these mutants in some way that is not observed and that the oscillations in gene expression are a subset of a more general, unseen oscillation.
The anterior presomitic mesoderm
A salt and pepper expression pattern could be created by a number of patterning processes gone awry and is not indicative inherently of oscillations. In fact, non-oscillating genes such as deltaD and mesp-b also can exhibit a patchy salt and pepper pattern in the anterior PSM of the notch pathway mutants (not shown) (Durbin et al., 2000; Sawada et al., 2000
). More importantly, we know that the anterior PSM is distinct from the posterior PSM, and the analysis of fss;aei/deltaD and fss;des/Notch1 embryos indicates that the strong anterior salt and pepper expression domain of her1 and deltaC in aei/deltaD and des/notch1 embryos is dependent entirely upon fss (van Eeden et al., 1998
; Holley et al., 2000
) and, therefore, is dependent upon an activity specific to the anterior PSM (Holley et al., 2000
). This explains why the salt and pepper pattern is found only in the anterior PSM of aei/deltaD embryos, and also led us to propose previously that this anterior expression was induced de novo by a separate, anterior wave-front activity. The wave-front would move from anterior to posterior along the body axis as the embryo extends posteriorly. This wave-front activity requires the function of the fss gene that normally functions in the anterior PSM to maintain or stabilize the oscillating pattern emanating from the posterior tailbud. In the absence of oscillations, this wave-front activity can induce or facilitate the expression of the oscillating genes in the anterior PSM, leading to the abrupt onset of the salt and pepper pattern in the anterior PSM of the aei/deltaD embryos (Holley et al., 2000
). Recent studies performed in the chick suggest that the wave-front could correlate with a drop in the level of FGF signaling, which is highest in the posterior PSM (Dubrulle et al., 2001
).
The analysis of deltaC expression in her1mo embryos uncovers an additional Notch-dependent patterning activity in the anterior PSM. This activity can create a segmental pattern of gene expression in the absence of any evidence of oscillations in her1 and deltaC expression: a smooth domain of deltaC expression is refined anteriorly to create stripes of expression that persist in the somitic mesoderm. This refinement requires the activity of fss, aei/deltaD, des/notch1, deltaC and bea, indicating that each of these genes has an additional function in the anterior-most PSM, downstream of her1. This is consistent with the fact that aei/deltaD, deltaC and des/notch1 are each transcribed within the PSM and later in the somitic mesoderm. In fact, this refining pattern is likely to be revealed only within the her1mo embryos because her1 is the only one of these cloned genes whose expression is restricted to the PSM (Bierkamp and Campos-Ortega, 1993; Dornseifer et al., 1997
; Jiang et al., 2000
; Müller et al., 1996
). Ultimately, this indicates that the phenotypes observed in aei/deltaD and des/notch1 embryos are composites of defects that occur both upstream and downstream of her1 (oscillator) function. It has been shown that notch pathway signaling is involved in establishing the anteroposterior pattern within each somite (Conlon et al., 1995
; Oka et al., 1995
; Evrard et al., 1998
; Hrabé Angelis et al., 1997
; Kusumi et al., 1998
; Wong et al., 1997
; Zhang and Gridley, 1998
; Takahashi et al., 2000
). The late activity of the notch pathway described here probably represents this same anteroposterior patterning function. What is remarkable is that this late function can create a segmental pattern in the absence of prior oscillations in her1 and deltaC expression.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Aoyama, H. and Asamoto, K. (1988). Determination of somite cells: independence of cell differentiation and morphogenesis. Development 104, 15-28.[Abstract]
Aulehla, A. and Johnson, R. L. (1999). Dynamic expression of lunatic fringe suggests a link between notch signaling and an automomous cellular oscillator driving somite segmentation. Dev. Biol. 207, 49-61.[Medline]
Bierkamp, C. and Campos-Ortega, J. A. (1993). A zebrafish homologue of the Drosophila neurogenic gene Notch and its pattern of transcription during early embryogenesis. Mech. Dev. 43, 87-100.[Medline]
Conlon, R. A., Reaume, A. G. and Rossant, J. (1995). Notch1 is required for the coordinate segmentation of somites. Development 121, 1533-1545.
Cooke, J. (1998). A gene that resuscitates a theory-somitogenesis and a molecular oscillator. Trends Genet. 14, 85-88.[Medline]
Cooke, J. and Zeeman, E. C. (1975). A clock and wavefront model for control of the number of repeated structures during animal morphognesis. J. Theor. Biol. 58, 455-476.
del Barco Barrantes, I., Elia, A., Wunnch, K., Hrabde De Angelis, M., Mak, T., Rossant, J., Conlon, R., Gossler, A. and Luis de la Pompa, J. (1999). Interaction between Notch signaling and Lunatic Fringe during somite boundary formation in the mouse. Curr. Biol. 9, 470-480.[Medline]
Dornseifer, P., Takke, C. and Campos-Ortega, J. A. (1997). Overexpression of a zebrafish homologue of the Drosophila neurogenic gene Delta perturbs differentiation of primary neurons and somite development. Mech. Dev. 63, 159-171.[Medline]
Dubrulle, J., McGrew, M. J. and Pourquié, O. (2001). FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106, 219-232.[Medline]
Durbin, L., Sordino, P., Barrios, A., Gering, M., Thisse, C., Thisse, B., Brennen, C., Green, A., Wilson, S. and Holder, N. (2000). Anteroposterior patterning is required within segments for somite boundary formation in developing zebrafish. Development 127, 1703-1713.
Evrard, Y. A., Lun, Y., Aulehla, A., Gan, L. and Johnson, R. L. (1998). lunatic fringe is an essential mediator of somite segmentation and patterning. Nature 394, 377-381.[Medline]
Fisher, A. L., Ohsako, S. and Caudy, M. (1996). The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4 amino acid transcription repression and protein-protein interaction domain. Mol. Cell. Biol. 16, 2670-2677.[Abstract]
Forsberg, H., Crozet, F. and Brown, N. A. (1998). Waves of mouse Lunatic fringe expression, in four-hour cycles at two-hour intervals, precede somite boundary formation. Curr. Biol. 8, 1027-1030.[Medline]
Geisler, R., Rauch, G.-J., Baier, H., van Bebber, F., Broß, L., Dekens, M., Finger, K., Fricke, C., Gates, M. A., Geiger, H. et al. (1999). A radiation map of the zebrafish genome. Nat. Genet. 23, 86-89.[Medline]
Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eeden, F. J. M., Jiang, Y.-J., Heisenberg, C.-P. et al. (1996). The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1-36.
Holley, S. A., Geisler, R. and Nüsslein Volhard, C. (2000). Control of her1 expression during zebrafish somitogenesis by a Delta-dependent oscillator and an independent wave-front activity. Genes Dev. 14, 1678-1690.
Hrabé Angelis, M., McIntyre, J. and Gossler, A. (1997). Maintenance of somite borders in mice requires the Delta homologue Dll1. Nature 386, 717-721.[Medline]
Jen, W.-C., Wettstein, D., Turner, D., Chitnis, A. and Kintner, C. (1997). The Notch ligand, X-Delta-2, mediates segmentation of the paraxial mesoderm in Xenopus embryos. Development 124, 1169-1178.
Jen, W.-C., Gawantka, V., Pollet, N., Niehrs, C. and Kintner, C. (1999). Periodic repression of Notch pathway genes governs the segmentation of Xenopus embryos. Genes Dev. 13, 1486-1499.
Jiang, Y.-J., Brand, M., Heisenberg, C.-P., Beuchle, D., Furutani-Seiki, M., Kelsh, R. N., Warga, R. M., Granato, M., Haffter, P., Hammerschmidt, M. et al. (1996). Mutations affecting neurogenesis and brain morphology in the zebrafish, Danio rerio. Development 123, 205-216.
Jiang, Y. J., Aerne, B. L., Smithers, L., Haddon, C., Ish Horowicz, D. and Lewis, J. (2000). Notch signaling and the synchronization of the somite segmentation clock. Nature 408, 475-479.[Medline]
Jouve, C., Palmeirim, I., Henrique, D., Beckers, J., Gossler, A., Ish Horowicz, D. and Pourquié, O. (2000). Notch signalling is required for cyclic expression of the hairy-like gene HES1 in the presomitic mesoderm. Development 127, 1421-1429.
Kanki, J. P. and Ho, R. K. (1997). The development of the posterior body in zebrafish. Development 124, 881-893.
Knapik, E., Goodman, A., Atkinson, O., Roberts, C., Shiozawa, M., Sim, C., Weksler-Zangen, S., Trolliet, M., Futrell, C., Innes, B. et al. (1996). A reference cross DNA panel for zebrafish (Danio rerio) anchored with simple sequence length polymorphisms. Development 123, 451-460.
Knapik, E., Goodman, A., Ekker, M., Chevrette, M., Delgado, J., Neuhauss, S., Shimoda, N., Driever, W., Fishman, M. and Jacob, H. (1998). A microsatellite genetic linkage map for zebrafish (Danio rerio). Nat. Genet. 18, 338-343.[Medline]
Kusumi, K., Sun, E. S., Kerrebrock, A. W., Bronson, R. T., Chi, D. C., Bulotsky, M. S., Spencer, J. B., Birren, B. W., Frankel, W. N. and Lander, E. S. (1998). The mouse pudgy mutation disrupts Delta homologue Dll3 and initiation of early somite boundaries. Nat. Genet. 19, 274-278.[Medline]
McGrew, M. J., Dale, J. K., Fraboulet, S. and Pourquié, O. (1998). The Lunatic Fringe gene is a target of the molecular clock linked to segmentation in avian embryos. Curr. Biol. 8, 979-982.[Medline]
Meinhardt, H. (1982). Models of Biological Pattern Formation. London: Academic Press.
Meinhardt, H. (1986). Models of segmentation. In Somites in Developing Embryos (ed. R. Bellairs, D. A. Ede and J. W. Lash), pp. 179-189. New York: Plenum Press.
Müller, M., Weiszäcker, E. and Campos-Ortega, J. A. (1996). Expression domains of a zebrafish homologue of the Drosophila pair-rule gene hairy correspond to primordia of alternating somites. Development 122, 2071-2078.
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene knockdown in zebrafish. Nat. Genet. 26, 216-220.[Medline]
Oka, C., Nakano, T., Wakeham, A., de la Pompa, J. L., Mori, C., Sakai, T., Okazaki, S., Kawaichi, M., Shiota, K., Mak, T. W. and Honjo, T. (1995). Disruption of the mouse RBP-J Kappa results in early embryonic death. Development 121, 3291-3301.
Palmeirim, I., Henrique, D., Ish Horowicz, D. and Pourquié, O. (1997). Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis. Cell 91, 639-648.[Medline]
Paroush, Z., Finley, R. L., Kidd, T., Wainwright, S. M., Ingham, P. L., Brent, R. and Ish-Horowicz, D. (1994). Groucho is required for Drosophila neurogenesis, segmentation, and sex determination and interacts directly with Hairy-related bHLH proteins. Cell 79, 805-815.[Medline]
Postlethwait, J., Yan, Y., Gates, M., Horne, S., Amores, A., Brownlie, A., Donovan, A., Egan, E., Force, A., Gong, Z. et al. (1998). Vertebrate genome evolution and the zebrafish gene map. Nat. Genet. 18, 345-349.[Medline]
Sawada, A., Fritz, A., Jiang, Y., Yamamoto, A., Yamasu, K., Kuroiwa, A., Saga, Y. and Takeda, H. (2000). Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic mesoderm, Mesp-b confers the anterior identity to the developing somites. Development 127, 1691-1702.
Shimoda, N., Knapik, E., Ziniti, J., Sim, C., Yamada, E., Kaplan, S., Jackson, D., de Sauvage, F., Jacob, H. and Fishman, M. (1999). Zebrafish genetic map with 2000 microsatellite markers. Genomics 58, 219-232.[Medline]
Takahashi, Y., Koizumi, K., Takagi, A., Kitajima, S., Inoue, T., Koseki, H. and Saga, Y. (2000). Mesp2 initiates somite segmentation through the Notch signaling pathway. Nat. Genet. 25, 390-396.[Medline]
Takke, C. and Campos-Ortega, J. A. (1999). her1, a zebrafish pair-rule gene, acts downstream of notch signaling to control somite development. Development 126, 3005-3014.
van Eeden, F. J. M., Granato, M., Schach, U., Brand, M., Furutani-Seiki, M., Haffter, P., Hammerschmidt, M., Heisenberg, C.-P., Jiang, Y.-j., Kane, D. A. et al. (1996). Mutations affecting somite formation and patterning in the zebrafish Danio rerio. Development 123, 153-164.
van Eeden, F. J. M., Holley, S. A., Haffter, P., Campos-Ortega, J. and Nüsslein-Volhard, C. (1998). Zebrafish segmentation and pair-rule patterning. Dev. Genet. 23, 65-76.[Medline]
Wong, P. C., Zheng, H., Chen, H., Becher, M. W., Sirinathsinghji, D. J. S., Trumbauer, M. E., Chen, H. Y., Price, D. L., Van der Ploeg, L. H. T. and Sisodia, S. S. (1997). Presenilin 1 is required for Notch1 and Dll1 expression in the paraxial mesoderm. Nature 387, 288-292.[Medline]
Zhang, N. and Gridley, T. (1998). Defects in somite formation in lunatic fringe deficient mice. Nature 394, 374-377.[Medline]