1 Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0101, Japan
2 Center for Developmental Biology, RIKEN, Japan
*Author for correspondence (e-mail: yotayota{at}bs.aist-nara.ac.jp)
Accepted 3 May 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Segmentation, Boundary formation, Notch, Lunatic fringe, Somites, Induction, Chick
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The somite in vertebrates is a transient structure that is reiterated along the body axis by segmentation processes and makes a major contribution to the formation of the axial structures including vertebral bones and skeletal muscles (Christ and Ordahl, 1995; Pourquié, 2001
; Stern and Vasiliauskas, 2000
; Stockdale et al., 2000
). We reasoned that the somitic segmentation serves as a useful model because it offers the following advantages: (1) the segmentation repeatedly takes place one pair at a time with regularity in time and distance in an anterior to posterior order, and (2) overt proliferation or movement of cells does not occur when a fissure forms (Primmett et al., 1989
; Stern et al., 1988
), allowing an evaluation of consequences of various embryonic manipulations such as we describe in this paper.
In the anterior end of the unsegmented paraxial mesoderm (presomitic mesoderm: PSM), an expression boundary of genes including MesP2 that coincides with the next border being formed is established prior to a morphological change. The segmental patterns of these genes are thought to be regulated by a "segmentation clock", first demonstrated by wavy and cyclic expression of c-hairy1 (Maroto and Pourquié, 2001; Palmeirim et al., 1997
; Pourquié, 2001
). Thus, the segmentation clock operates in the continuous young PSM to establish the segmental patterns of gene expression in the anterior PSM, which eventually implements a morphological fissure formation. Both clock and segmentation genes are tightly related to Notch signaling, as revealed mainly by recent knockout and mutant studies: an animal where Notch signaling is (at least in part) deficient displays perturbed patterns of cyclic and segmental expression of genes in PSM, and also shows its consequent malformation of segmented structures later in development (Bessho et al., 2001
; Conlon et al., 1995
; Evrard et al., 1998
; Holley et al., 2000
; Hrabe de Angelis et al., 1997
; Jiang et al., 2000
; Kusumi et al., 1998
; Oka et al., 1995
; Saga et al., 1997
; Saga and Takeda, 2001
; Shen et al., 1997
; Wong et al., 1997
; Zhang and Gridley, 1998
). In general, studies using mutants or knockout animals unveil the first stage where the gene of concern is essential during development. However, if a given gene plays a role in the fissure formation as well as at earlier steps of segmentation, it would be difficult to distinguish between them. This may be the reason why the molecular mechanisms underlying the fissure formation have been poorly addressed.
In this paper we first describe a novel inductive event taking place when a segmentation fissure forms, in which posterior border cells located immediately posterior to the next forming boundary instruct the anterior ones. We next address molecular mechanisms underlying these events by focusing on Notch signals where Lunatic fringe (Lfng) is involved. Lfng is a modulator of the Notch receptor (Bruckner et al., 2000; Moloney et al., 2000
) with glycosyltransferase activity, and is expressed in a region coinciding with the segmentation border in PSM. By combining DNA in ovo electroporation with embryological manipulations to make an ectopic boundary of a transgene activity in PSM, we found that Notch signals play major roles in the formation of a fissure. We present a model in which specific localization of Lfng determines the site of Notch action relevant to the morphological segmentation. We also discuss a mode of action for Notch in vertebrate somitogenesis using an analogy of that known for Drosophila.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histological analyses
Embryos were fixed in Carnoys solution (60% ethanol, 30% chloroform, 10% acetic acid) dehydrated in ethanol and embedded in paraffin wax. For staining with anti-quail QCPN monoclonal antibody, 7 µm histological sections were incubated with hybridoma supernatant (DHSB) for 2 hours. After washing in phosphate-buffered saline (PBS), they were reincubated for 90 minutes with horseradish peroxidase-conjugated anti-mouse immunoglobulin (Dako) diluted 1:100 with 2% skim milk in PBS. The reaction was developed in 80 µg/ml diaminobenzidine (DAB), 0.004% H2O2/PBS. Mayers Hematoxylin solution (Wako) was used for a background nuclear staining of paraffin sections of embryonic day 4 (E4) embryos. For phalloidin staining, embryos were fixed in 4% paraformaldehyde in PBS and immersed in a graded series of sucrose solutions up to 30%, then embedded in Tissue-Tek (Sakura). Ten µm cryostat sections were incubated for 30 minutes with 5 Units/ml of Alexa FlourTM 568 or 647 phalloidin (Molecular Probes) dissolved in PBS.
RNA probes and whole-mount in situ hybridization
Chicken Lfng and Notch1 were provided by Drs C. Tabin (Laufer et al., 1997) and Y. Wakamatsu (Wakamatsu et al., 1999
), respectively. Delta1 was a 946 bp fragment obtained by the RT-PCR technique using the primers, 5'-TACTGCACTCACCACAAGCC-3' and 5'-TGATGGAGATGTCCTTCTCG-3'. Preparation of Dig-labeled RNA probes and whole-mount in situ hybridization followed by histological sectioning were performed as previously described (Takahashi et al., 1996
).
In ovo DNA microelectroporation
The entire cDNA for chicken Lfng tagged with FLAG (a gift from Dr C. Tabin) (Laufer et al., 1997), and mouse Notch
E-6myc and Notch LNG-6myc (gifts from Dr R. Kopan) (Schroeter et al., 1998
) were subcloned into the pCAGGS expression vector (Niwa et al., 1991
). They were co-electroporated with pCAGGS-GFP (green fluorescence protein) into the presumptive somitic mesoderm of HH stage 7-8 chicken embryos. Microelectroporation was carried out essentially according to the method previously reported (Momose et al., 1999
; Yasuda et al., 2000
) with slight modifications as follows: DNA solution was prepared at 5 µg/µl, colored with 2% Methylgreen (Nakalai) and placed onto the anterior primitive streak using a glass capillary. A plus electrode (platinum) was positioned under the embryo, and a minus electrode (sharpened tungsten) was put near the DNA solution. An electric pulse of 6V, 25 mseconds was charged three times.
Western blotting
COS cells transfected using Lipofectamine (Gibco BRL) were subjected to western blotting analyses as described previously (Kopan et al., 1996). Anti-FLAG M2 monoclonal antibody (Sigma) and anti-Myc (9E10) monoclonal antibody (Santa Cruz Biotechnology) were diluted 1:8000 and 1:1000, respectively, with 5% skim milk in PBST (0.1% Tween 20 in PBS).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Lfng mediated the boundary-forming activity
We next investigated the molecular mechanisms underlying the inductive events that we found by focusing on the roles of Lfng. Lfng mRNA in the anterior PSM displays a sharp anterior boundary of expression coinciding with 1 (Aulehla and Johnson, 1999; McGrew and Pourquié, 1998
), which was confirmed in histological sagittal sections (Fig. 4A,A'). A clear edge to the signal at 1 contrasts with its variable position in posterior PSM. Lfng is known to modify Notch receptor with the glycosyltransferase activity (Bruckner et al., 2000
; Ju et al., 2000
; Moloney et al., 2000
; Panin et al., 1997
). A growing number of reports, mainly using studies of mutants, have shown essential roles of Notch signaling during somite segmentation (Bessho et al., 2001
; Conlon et al., 1995
; Evrard et al., 1998
; Holley et al., 2000
; Hrabe de Angelis et al., 1997
; Jiang et al., 2000
; Kusumi et al., 1998
; Oka et al., 1995
; Saga et al., 1997
; Saga and Takeda, 2001
; Shen et al., 1997
; Wong et al., 1997
; Zhang and Gridley, 1998
), although the widely distributed patterns of the transcripts of Notch and Notch-related molecules including ligands in PSM (Saga and Takeda, 2001
) (also Fig. 4) have made it difficult to precisely locate the site of Notch actions. We therefore reasoned that Lfng at 1 would have the role in boundary forming activity which we found in the present study.
|
|
|
Given these results, we conclude that during normal segmentation Lfng activity demarcating the prospective line 1 influences Notch receptor so that Notch signals become active at this site to mediate a morphological segmentation. Thus, establishment of the Lfng boundary in the anterior PSM appears to determine the site of Notch action within the area where mRNAs of Notch and its ligand are widely distributed (Fig. 4, see also Fig. 10).
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Inductive events occur near the prospective line 1
PSM is known to possess an intrinsic ability to establish a segmented pattern from specific gene expression since, deprived of its surrounding tissues, including the ectoderm, it still displays a normal pattern of reiterated Delta1 and Delta-like1 (Dll1) expression (Correia and Conlon, 2000; Palmeirim et al., 1998
). As for the mechanisms by which the morphological fissure forms, however, up to now it was known only that the PSM requires the ectoderm (Correia and Conlon, 2000
; Palmeirim et al., 1998
; Schmidt et al., 2001
). In this report we have for the first time demonstrated that an inductive event takes place instructed by the cells located posterior to 1 (posterior border cells). This instructive phenomenon contrasts with a permissive effect of the surface ectoderm which supports but apparently does not instruct the fissure formation (Correia and Conlon, 2000
; Palmeirim et al., 1998
; Schmidt et al., 2001
). In our experimental system in which a small group of cells (50
100 cells) were dissected from a quail donor embryo, it was virtually impossible to identify whether they were only posterior border cells, or whether they included cells straddling the prospective line. Nevertheless, the simplest and most reasonable interpretation of the results we obtained is that during normal segmentation the posterior border cells play primary roles in the instruction to make a fissure. This is further supported by another line of evidence shown in this study: an interface of Lfng activity, mRNA of which is normally expressed with a sharp anterior boundary coinciding with 1, mimics the actions of the posterior border cells. We designate this boundary-forming activity as a "segmenter" (Fig. 10A).
Segmenter activity is mediated by Lfng and Notch signals
Lfng has been shown to modify Notch by its glycosyltransferase activity in the same cells that express Notch (Bruckner et al., 2000; Moloney et al., 2000
; Panin et al., 1997
). Thus, the segmenter appears to be mediated by Notch actions. Given these facts, we present a model as shown in Fig. 10A that shows how the segmenter activity is generated. Within anterior PSM where Notch1 and Delta1 mRNAs are widely distributed, a specific localization of Lfng activity restricts the site of Notch action to posterior to 1. The Notch-activated cells then signal to the cells anterior to them which become separated and eventually epithelialized. The molecular nature of this signal(s) remains to be determined, but could be direct cell-cell interactions and/or some secretory factors. Candidates for the former signals involve Eph/ephirin-directed repulsion and also cadherin-mediated segregation between two types of cells (Durbin et al., 1998
; Durbin et al., 2000
; Holder and Klein, 1999
; Inoue et al., 2001
; Nose et al., 1988
; Schmidt et al., 2001
; Takeichi, 1995
; Wilkinson, 2001
). Notch-Delta signals themselves might directly contribute to this segregation as shown in more detail below. This does not exclude the possibility that the posterior border cells simply produce some extracellular matrix so that a mechanical gap forms between the anterior cells. Nevertheless, since in several specimen we have observed that segmenter- or Notch
E/Lfng-producing cells caused the anterior cells to rearrange their AP polarity, although with a relatively low efficiency, it is reasonable to propose that the posterior border cells possess an instructive capability over the anterior ones. Although Lfng knockout mice show a severe phenotype in segmentation where the proper spacing and segmented border of somites are lost (Evrard et al., 1998
; Zhang and Gridley, 1998
), it has been unclear whether the deficiency was attributable to Lfng that cycles in the posterior PSM or to Lfng restricted to 1 (Aulehla and Johnson, 1999
; Forsberg et al., 1998
; McGrew et al., 1998
). In this study we were able to distinguish between them by determining the role of Lfng at the prospective line 1.
The Notch-mediated segmenter appears to act only anteriorly. This was shown by the fact that NotchE/Lfng-expressing cells located in the anterior half of the presumptive somite did not make a fissure even though the interface of the transgene activity was midway along the AP axis of the somite (Fig. 7A). It was also supported by the finding that in the embryos that made supernumerary somites by receiving 1-derived cells, the grafted cells were located either posterior to- or on both sides of the ectopic fissure, but never confined only to the anterior region (Fig. 3). Although the precise mechanisms of this unidirectional action are unknown, the presence of a global gradient of morphogen-like signaling cannot account for it. It is possible that AP polarity is present in each single cell of the PSM (=recipient cells in our experiments) so that the cells can detect where the segmenter signal comes from. This polarization may include planar cell polarity as seen in epithelial cells in the Drosophila wing (Usui et al., 1999
). The directional signaling in fissure formation shown in the present study is also consistent with the finding that zebrafish double mutant for knypek and trilobite have a single somite unit consisting of only two rows of cells (Henry et al., 2000
), indicating that two types of cells suffice in boundary formation. Thus, these findings argue against the model in which the existence of a third cell state alternating with the anterior (A) and posterior (P) ones was proposed to explain why a fissure forms at alternate confrontations between the A and P (Meinhardt, 1986
).
In our experiments, making an interface of pCAGGS/Lfng-electroporated cells at level 1.5 resulted in an ectopic fissure. Level 1.5 of the normal embryo is the site where Lfng mRNA is also present (Fig. 4A and Fig. 10A). Since the presence of transcripts of a given gene does not necessarily reflect protein activity, we interpret our results as being brought about by the creation of a boundary between Lfng on/off, if not, high/low regions. Thus, during normal segmentation Lfng activity affecting Notch appears highly confined to 1 (posterior border cells). It is also conceivable that since Lfng mRNA is expressed in waves that stops at 1 during each cycle, the level of Lfng protein would be highest at this point, and this accumulation might be a requisite for fissure formation. The precise localization of Lfng protein/activity in normal PSM needs to be determined in order to distinguish between these possibilities.
We have shown that only a sharp interface between Lfng on and off (or high and low) regions resulted in an ectopic fissure, whereas widely distributed Lfng in PSM did not significantly affect the morphological segmentation. This could be due to a mosaic pattern of transgene activity (50%) in PSM, and therefore, widely electroporated Lfng was not sufficient to interfere with the endogenous boundary of Lfng at 1. It is apparent that Notch signaling needs to be precisely regulated in PSM to manifest segmentation: expression of some Notch-related genes oscillates in a coordinated manner during the segmentation cycle, and perturbation of these stereotyped patterns leads to defects in segmentation (Barrantes et al., 1999
; Holley et al., 2000
; Holley et al., 2002
; Jiang et al., 2000
; Jouve et al., 2000
; Takke and Campos-Ortega, 1999
).
How does Notch/Lfng make a morphological boundary?
The mechanisms by which Notch signaling establishes a boundary between cells and tissues have been extensively studied in Drosophila, where two modes of actions have been proposed, the lateral inhibition type and the wing disk type. At present both models fit the interpretation of our results (Fig. 10B). In the lateral inhibition type, confrontation between Notch-active and -inactive cells produces two types of cells, which exclude each other on either side of the interface (Artavanis-Tsakonas et al., 1999). One can depict a possible mechanism for vertebrate segmentation by analogy with this model (Fig. 10B): in the posterior border cells (green) Lfng modifies Notch so that it efficiently binds to Delta1 and transmits signals intracellularly and Notch-activated cells produce the segmenter that acts on the anterior cells. In this model, the posterior and anterior cells are negative for actions of Delta1 and Notch1, respectively, generating a sharp interface for Notch activity at 1. A good candidate for an effecter working downstream of Notch signaling is Hes7 since Hes7 knockout mice are affected in somite segmentation. In addition, the expression of Hes7 mRNA is almost identical to Lfng with a sharp anterior boundary at 1 (Bessho et al., 2001
).
In the wing disk of Drosophila, Fringe which is expressed in the dorsal region modifies Notch so that it can efficiently transmit a signal with Delta, expressed in the ventral region (negative for Fringe), whereas Fringe-modified Notch cannot interact with another ligand, Serrate, present in the dorsal half. These interactions between Notch, Fringe, Delta and Serrate result in a sharp peak of Notch activity that straddles the boundary between the dorsal and ventral regions (Panin et al., 1997). A similar relationship could occur at the next forming boundary of vertebrate segmentation (Fig. 10B): Lfng-modified Notch in the posterior border cells (green) transmits a signal with Delta1, presumed to be active in the anterior border cells (yellow), and another ligand Delta3, known to be localized in the posterior border cells in mice and essential for segmentation (Dunwoodie et al., 1997
; Kusumi et al., 1998
) might activate Notch signal in the anterior border cells, leading to a sharp peak of Notch activation straddling 1. In this case Lfng might confer differential susceptibility on Notch receptor to Delta1 and Delta3 as has been shown for Delta1 and Jagged1 (Hicks et al., 2000
). Hes1 and Hes5, presumed downstream effectors of Notch pathway, are indeed expressed in the anterior border cells (Ishibashi et al., 1995
; Ohtsuka et al., 1999
). It remains uncertain, however, whether they really act as an effecter since no defects have been reported even in double knockout mice for the Hes1 and Hes5 (Ohtsuka et al., 1999
). It is of interest to learn whether other Hes or Hes-related members are localized in this region.
The posterior and anterior border cells subsequently follow distinct morphogenetic pathways: the former remain mesenchymal during a period when two somites form, whereas the latter become epithelialized immediately after a gap forms (Fig. 1) (Duband et al., 1987). This distinction between the posterior and anterior border cells would be attributed, according to the lateral inhibition type model, to the presence or absence of Notch activity, and in the wing disk type model other molecules would work in concert with Notch signaling to differentiate between these cells. Whatever the mode of Notch action is, it is still unknown how the border cells manifest dynamic changes in morphology. Studies that link Notch signaling to cytoskeletal dynamism will be required.
Recently, another role for Notch has been reported in zebrafish, in which Notch signals are essential for coordination of oscillating expression in the posterior PSM during each cycle of segmentation (Holley et al., 2002; Jiang et al., 2000
), but at present this role seems distinct from those determined in this study. Thus, the role of Notch signaling and its contribution to the boundary formation may differ in each morphogenetic process.
Relationships between the segmenter and other factors important for morphological boundary
It has previously been shown that the paraxis gene (Tcf15 Mouse Genome Informatics), which encodes a bHLH molecule, plays essential roles in epithelialization during somite segmentation. In paraxis knockout mice, a gap forms between segments but a subsequent epithelialization does not proceed, resulting in malformation of somite-derived organs (Burgess et al., 1996). Since in normal segmentation processes the gap formation is immediately followed by the epithelialization of the anterior border cells, the segmenter seems to act close to the functional pathway of Paraxis. It remains to be clarified whether the ectoderm-derived signals, known to support the morphological segmentation, act during the gap formation or the epithelialization process. It is also important to consider the dorsoventral and mediolateral axes to further understand the mechanisms underlying generation and action of the segmenter, and this is currently underway.
Lastly, Fringe-mediated formation of a tissue boundary has previously been shown in vertebrates: in the limb bud Radical fringe determines the position of the apical ectodermal ridge (AER), and Lfng is involved in barrier formation in zona limitans intrathalamica (zli) of the diencephalon (Irvine, 1999; Laufer et al., 1997
; Rodriguez-Esteban et al., 1997
; Zeltser et al., 2001
). It is of interest to know whether these morphogenetic movements share mechanisms similar to those shown in this study. Unlike the cases for AER or zli, somite segmentation involves an overt morphological separation between cells followed by dynamic changes in cell shape. In combination with the advantage that somite segmentation proceeds in a geometrically simple reiterated structure, our finding of the segmenter opens a way to unravel complex events occurring during border formation at the molecular and cellular level, and to depict a general picture of morphogenesis in vertebrates.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J. (1999). Notch signaling: cell fate control and signal integration in development. Science 284, 770-776.
Aulehla, A. and Johnson, R. L. (1999). Dynamic expression of lunatic fringe suggests a link between notch signaling and an autonomous cellular oscillator driving somite segmentation. Dev. Biol. 207, 49-61.[Medline]
Barrantes, I. B., Elia, A. J., Wunsch, K., de Angelis, M. H., Mak, T. W., Rossant, J., Conlon, R. A., Gossler, A. and de la Pompa, J. L. (1999). Interaction between Notch signalling and Lunatic fringe during somite boundary formation in the mouse. Curr. Biol. 9, 470-480.[Medline]
Bessho, Y., Sakata, R., Komatsu, S., Shiota, K., Yamada, S. and Kageyama, R. (2001). Dynamic expression and essential functions of Hes7 in somite segmentation. Genes Dev. 15, 2642-2647.
Briscoe, J., Pierani, A., Jessell, T. M. and Ericson, J. (2000). A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101, 435-445.[Medline]
Bruckner, K., Perez, L., Clausen, H. and Cohen, S. (2000). Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406, 411-415.[Medline]
Buchberger, A., Seidl, K., Klein, C., Eberhardt, H. and Arnold, H. H. (1998). cMeso-1, a novel bHLH transcription factor, is involved in somite formation in chicken embryos. Dev. Biol. 199, 201-215.[Medline]
Burgess, R., Rawls, A., Brown, D., Bradley, A. and Olson, E. N. (1996). Requirement of the paraxis gene for somite formation and musculoskeletal patterning. Nature 384, 570-573.[Medline]
Catala, M., Teillet, M. A., De Robertis, E. M. and Le Douarin, M. L. (1996). A spinal cord fate map in the avian embryo: while regressing, Hensens node lays down the notochord and floor plate thus joining the spinal cord lateral walls. Development 122, 2599-2610.
Christ, B. and Ordahl, C. P. (1995). Early stages of chick somite development. Anat. Embryol. 191, 381-396.[Medline]
Conlon, R. A., Reaume, A. G. and Rossant, J. (1995). Notch1 is required for the coordinate segmentation of somites. Development 121, 1533-1545.
Correia, K. M. and Conlon, R. A. (2000). Surface ectoderm is necessary for the morphogenesis of somites. Mech. Dev. 91, 19-30.[Medline]
Dahmann, C. and Basler, K. (1999). Compartment boundaries: at the edge of development. Trends Genet. 15, 320-326.[Medline]
Duband, J. L., Dufour, S., Hatta, K., Takeichi, M., Edelman, G. M. and Thiery, J. P. (1987). Adhesion molecules during somitogenesis in the avian embryo. J. Cell Biol. 104, 1361-1374.[Abstract]
Dunwoodie, S. L., Henrique, D., Harrison, S. M. and Beddington, R. S. (1997). Mouse Dll3: a novel divergent Delta gene which may complement the function of other Delta homologues during early pattern formation in the mouse embryo. Development 124, 3065-3076.
Durbin, L., Brennan, C., Shiomi, K., Cooke, J., Barrios, A., Shanmugalingam, S., Guthrie, B., Lindberg, R. and Holder, N. (1998). Eph signaling is required for segmentation and differentiation of the somites. Genes Dev. 12, 3096-3109.
Durbin, L., Sordino, P., Barrios, A., Gering, M., Thisse, C., Thisse, B., Brennan, 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]
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]
Hamburger, V. and Hamilton, H. (1951). A series of normal stages in the development of chick embryo. J. Morphol. 88, 49-92.
Henry, C. A., Hall, L. A., Burr Hille, M., Solnica-Krezel, L. and Cooper, M. S. (2000). Somites in zebrafish doubly mutant for knypek and trilobite form without internal mesenchymal cells or compaction. Curr. Biol. 10, 1063-1066.[Medline]
Hicks, C., Johnston, S. H., diSibio, G., Collazo, A., Vogt, T. F. and Weinmaster, G. (2000). Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2. Nat Cell Biol 2, 515-520.[Medline]
Holder, N. and Klein, R. (1999). Eph receptors and ephrins: effectors of morphogenesis. Development 126, 2033-2044.
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.
Holley, S. A., Julich, D., Rauch, G. J., Geisler, R. and Nüsslein-Volhard, C. (2002). her1 and the notch pathway function within the oscillator mechanism that regulates zebrafish somitogenesis. Development 129, 1175-1183.
Horikawa, K., Radice, G., Takeichi, M. and Chisaka, O. (1999). Adhesive subdivisions intrinsic to the epithelial somites. Dev. Biol. 215, 182-189.[Medline]
Hrabe de Angelis, M., McIntyre, J. n. and Gossler, A. (1997). Maintenance of somite borders in mice requires the Delta homologue DII1. Nature 386, 717-721.[Medline]
Inoue, T., Tanaka, T., Takeichi, M., Chisaka, O., Nakamura, S. and Osumi, N. (2001). Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development. Development 128, 561-569.
Irvine, K. D. (1999). Fringe, Notch, and making developmental boundaries. Curr. Opin. Genet. Dev. 9, 434-441.[Medline]
Irvine, K. D. and Rauskolb, C. (2001). Boundaries in development: formation and function. Annu. Rev. Cell. Dev. Biol. 17, 189-214.[Medline]
Ishibashi, M., Ang, S. L., Shiota, K., Nakanishi, S., Kageyama, R. and Guillemot, F. (1995). Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects. Genes Dev. 9, 3136-3148.[Abstract]
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1, 20-29.[Medline]
Jiang, Y. J., Aerne, B. L., Smithers, L., Haddon, C., Ish-Horowicz, D. and Lewis, J. (2000). Notch signalling 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.
Joyner, A. L., Liu, A. and Millet, S. (2000). Otx2, Gbx2 and Fgf8 interact to position and maintain a mid-hindbrain organizer. Curr. Opin. Cell. Biol. 12, 736-741.[Medline]
Ju, B. G., Jeong, S., Bae, E., Hyun, S., Carroll, S. B., Yim, J. and Kim, J. (2000). Fringe forms a complex with Notch. Nature 405, 191-195.[Medline]
Kopan, R., Schroeter, E. H., Weintraub, H. and Nye, J. S. (1996). Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain. Proc. Natl. Acad. Sci. USA 93, 1683-1688.
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]
Laufer, E., Dahn, R., Orozco, O. E., Yeo, C. Y., Pisenti, J., Henrique, D., Abbott, U. K., Fallon, J. F. and Tabin, C. (1997). Expression of Radical fringe in limb-bud ectoderm regulates apical ectodermal ridge formation. Nature 386, 366-373.[Medline]
Lawrence, P. A. and Struhl, G. (1996). Morphogens, compartments, and pattern: lessons from drosophila? Cell 85, 951-961.[Medline]
Lumsden, A. (1999). Closing in on rhombomere boundaries. Nat. Cell. Biol. 1, 83-85.
Maroto, M. and Pourquié, O. (2001). A molecular clock involved in somite segmentation. Curr. Top. Dev. Biol. 51, 221-248.[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 somite segmentation in avian embryos. Curr. Biol. 8, 979-982.[Medline]
McGrew, M. J. and Pourquié, O. (1998). Somitogenesis: segmenting a vertebrate. Curr. Opin. Genet. Dev. 8, 487-493.[Medline]
McNeill, H. (2000). Sticking together and sorting things out: adhesion as a force in development. Nat. Rev. Genet. 1, 100-108.[Medline]
Meinhardt, H. (1986). Hierarchical inductions of cell states: a model for segmentation in Drosophila. J. Cell Sci. Suppl. 4, 357-381.
Moloney, D. J., Panin, V. M., Johnston, H. S., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K. D., Haltiwanger, R. S. et al. (2000). Fringe is a glycosyltransferase that modifies Notch. Nature 406, 369-375.[Medline]
Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K. and Yasuda, K. (1999). Efficient targeting of gene expression in chick embryos by microelectroporation. Dev. Growth. Differ. 41, 335-344.[Medline]
Niwa, H., Yamamura, K. and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193-199.[Medline]
Nose, A., Nagafuchi, A. and Takeichi, M. (1988). Expressed recombinant cadherins mediate cell sorting in model systems. Cell 54, 993-1001.[Medline]
Ohtsuka, T., Ishibashi, M., Gradwohl, G., Nakanishi, S., Guillemot, F. and Kageyama, R. (1999). Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation. EMBO J. 18, 2196-2207.
Oka, C., Nakano, T., Wakeham, A., de la Pompa, J. L., Mori, C., Sakai, T., Okazaki, S., Kawaichi, M., Shiota, K., Mak, T. W. et al. (1995). Disruption of the mouse RBP-J kappa gene results in early embryonic death. Development 121, 3291-3301.
Palmeirim, I., Dubrulle, J., Henrique, D., Ish-Horowicz, D. and Pourquié, O. (1998). Uncoupling segmentation and somitogenesis in the chick presomitic mesoderm. Dev. Genet. 23, 77-85.[Medline]
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]
Panin, V. M., Papayannopoulos, V., Wilson, R. and Irvine, K. D. (1997). Fringe modulates Notch-ligand interactions. Nature 387, 908-912.[Medline]
Pourquié, O. (2001). Vertebrate somitogenesis. Annu. Rev. Cell. Dev. Biol. 17, 311-350.[Medline]
Primmett, D. R., Norris, W. E., Carlson, G. J., Keynes, R. J. and Stern, C. D. (1989). Periodic segmental anomalies induced by heat shock in the chick embryo are associated with the cell cycle. Development 105, 119-130.[Abstract]
Psychoyos, D. and Stern, C. D. (1996). Fates and migratory routes of primitive streak cells in the chick embryo. Development 122, 1523-1534.
Rodriguez-Esteban, C., Schwabe, J. W., De La Peña, J., Foys, B., Eshelman, B. and Belmonte, J. C. (1997). Radical fringe positions the apical ectodermal ridge at the dorsoventral boundary of the vertebrate limb. Nature 386, 360-366.[Medline]
Saga, Y., Hata, N., Koseki, H. and Taketo, M. M. (1997). Mesp2: a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Genes Dev. 11, 1827-1839.[Abstract]
Saga, Y. and Takeda, H. (2001). The making of the somite: molecular events in vertebrate segmentation. Nat. Rev. Genet. 2, 835-845.[Medline]
Sanson, B. (2001). Generating patterns from fields of cells: Examples from Drosophila segmentation. EMBO Rep. 2, 1083-1088.
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, and Mesp-b confers the anterior identity to the developing somites. Development 127, 1691-1702.
Schmidt, C., Christ, B., Maden, M., Brand-Saberi, B. and Patel, K. (2001). Regulation of Epha4 expression in paraxial and lateral plate mesoderm by ectoderm-derived signals. Dev. Dyn. 220, 377-386.[Medline]
Schroeter, E. H., Kisslinger, J. A. and Kopan, R. (1998). Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393, 382-386.[Medline]
Shen, J., Bronson, R. T., Chen, D. F., Xia, W., Selkoe, D. J. and Tonegawa, S. (1997). Skeletal and CNS defects in Presenilin-1-deficient mice. Cell 89, 629-639.[Medline]
Simeone, A. (2000). Positioning the isthmic organizer where Otx2 and Gbx2meet. Trends Genet. 16, 237-240.[Medline]
Sparrow, D. B., Jen, W. C., Kotecha, S., Towers, N., Kintner, C. and Mohun, T. J. (1998). Thylacine 1 is expressed segmentally within the paraxial mesoderm of the Xenopus embryo and interacts with the Notch pathway. Development 125, 2041-2051.
Stern, C. D., Fraser, S. E., Keynes, R. J. and Primmett, D. R. (1988). A cell lineage analysis of segmentation in the chick embryo. Development 104, 231-244.[Medline]
Stern, C. D. and Vasiliauskas, D. (2000). Segmentation: a view from the border. Curr. Top. Dev. Biol. 47, 107-129.[Medline]
Stockdale, F. E., Nikovits, W., Jr and Christ, B. (2000). Molecular and cellular biology of avian somite development. Dev. Dyn. 219, 304-321.[Medline]
Takahashi, Y., Koizumi, K., Takagi, A., Kitajima, S., Inoue, T., Koseki, H. and Saga, Y. (2000). Mesp2 initiates somite segmentation through the Notch signalling pathway. Nat. Genet. 25, 390-396.[Medline]
Takahashi, Y., Tonegawa, A., Matsumoto, K., Ueno, N., Kuroiwa, A., Noda, M. and Nifuji, A. (1996). BMP-4 mediates interacting signals between the neural tube and skin along the dorsal midline. Genes Cells 1, 775-783.
Takeichi, M. (1995). Morphogenetic roles of classic cadherins. Curr. Opin. Cell Biol. 7, 619-627.[Medline]
Takke, C. and Campos-Ortega, J. A. (1999). her1, a zebrafish pair-rule like gene, acts downstream of notch signalling to control somite development. Development 126, 3005-3014.
Tonegawa, A., Funayama, N., Ueno, N. and Takahashi, Y. (1997). Mesodermal subdivision along the mediolateral axis in chicken controlled by different concentrations of BMP-4. Development 124, 1975-1984.
Usui, T., Shima, Y., Shimada, Y., Hirano, S., Burgess, R. W., Schwarz, T. L., Takeichi, M. and Uemura, T. (1999). Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity under the control of Frizzled. Cell 98, 585-595.[Medline]
Wakamatsu, Y., Maynard, T. M., Jones, S. U. and Weston, J. A. (1999). NUMB localizes in the basal cortex of mitotic avian neuroepithelial cells and modulates neuronal differentiation by binding to NOTCH-1. Neuron 23, 71-81.[Medline]
Wilkinson, D. G. (2001). Multiple roles of EPH receptors and ephrins in neural development. Nat. Rev. Neurosci. 2, 155-164.[Medline]
Wong, P. C., Zheng, H., Chen, H., Becher, M. W., Sirinathsinghji, D. J., Trumbauer, M. E., Chen, H. Y., Price, D. L., van der Ploeg, L. H. and Sisodia, S. S. (1997). Presenilin 1 is required for Notch1 and DII1 expression in the paraxial mesoderm. Nature 387, 288-292.[Medline]
Wurst, W. and Bally-Cuif, L. (2001). Neural plate patterning: upstream and downstream of the isthmic organizer. Nat. Rev. Neurosci. 2, 99-108.[Medline]
Yasuda, K., Momose, T. and Takahashi, Y. (2000). Applications of microelectroporation for studies of chick embryogenesis. Dev. Growth Differ. 42, 203-206.[Medline]
Zeltser, L. M., Larsen, C. W. and Lumsden, A. (2001). A new developmental compartment in the forebrain regulated by Lunatic fringe. Nat. Neurosci 4, 683-684.[Medline]
Zhang, N. and Gridley, T. (1998). Defects in somite formation in lunatic fringe-deficient mice. Nature 394, 374-377.[Medline]