Integrin 5 and Delta/Notch Signaling Have ... - Cell Press

Developmental Cell, Vol. 8, 575–586, April, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.devcel.2005.01.016

Integrin␣5 and Delta/Notch Signaling Have Complementary Spatiotemporal Requirements during Zebrafish Somitogenesis Dörthe Ju¨lich,1 Robert Geisler,2 Tu¨bingen 2000 Screen Consortium,3,4 and Scott A. Holley1,* 1 Department of Molecular, Cellular, and Developmental Biology Yale University New Haven, Connecticut 06520 2 Max Planck-Institut fu¨r Entwicklungsbiologie Tu¨bingen, D-72076 Germany

Summary Somitogenesis is the process by which the segmented precursors of the skeletal muscle and vertebral column are generated during vertebrate embryogenesis. While somitogenesis appears to be a serially homologous, reiterative process, we find that there are differences between the genetic control of early/ anterior and late/posterior somitogenesis. We demonstrate that point mutations can cause segmentation defects in either the anterior, middle, or posterior somites in the zebrafish. We find that mutations in zebrafish integrina5 disrupt anterior somite formation, giving a phenotype complementary to the posterior defects seen in the notch pathway mutants after eight/deltaD and deadly seven/notch1a. Double mutants between the notch pathway and integrina5 display somite defects along the entire body axis, with a complete loss of the mesenchymal-to-epithelial transition and Fibronectin matrix assembly in the posterior. Our data suggest that notch- and integrina5dependent cell polarization and Fibronectin matrix assembly occur concomitantly and interdependently during border morphogenesis. Introduction Somites form sequentially from anterior to posterior in bilateral pairs as the embryo extends posteriorly, giving rise to a species-specific number of segments. Studies in mouse, chick, Xenopus, and zebrafish support a model in which somite formation is governed by a molecular oscillator, called the “somite clock,” which *Correspondence: [email protected] 3 Members of the Tu¨bingen 2000 Screen Consortium affiliated with the Max-Planck-Institut fu¨r Entwicklungsbiologie, Spemannstrasse 35, 72076, Tu¨bingen, Germany: F. Van Bebber, E. Busch-Nentwich, R. Dahm, O. Frank, H.-G. Fronhöfer, H. Geiger, D. Gilmour, S. Holley, J. Hooge, D. Ju¨lich, H. Knaut, F. Maderspacher, H.-M. Maischein, C. Neumann, C. Nu¨sslein-Volhard, H. Roehl, U. Schönberger, C. Seiler, S. Sidi, M. Sonawane, A. Wehner. 4 Members of the Tu¨bingen 2000 Screen Consortium affiliated with Artemis Pharmaceuticals GmbH, 72076, Tu¨bingen, Germany: P. Erker, H. Habeck, U. Hagner, C.E. Hennen Kaps, A. Kirchner, T. Koblizek, U. Langheinrich, C. Loeschke, C. Metzger, R. Nordin, J. Odenthal, M. Pezzuti, K. Schlombs, J. deSantana-Stamm, T. Trowe, G. Vacun, B. Walderich, A. Walker, C. Weiler.

causes oscillations in gene expression within the presomitic mesoderm (PSM). Propagation of these oscillations requires notch pathway signaling (mouse, zebrafish) and wnt (mouse) and are further modulated by fgf signaling (mouse, chick, zebrafish) and the tbx gene fused somites (fss) (zebrafish). Stabilization of the oscillations in the anterior PSM leads to the establishment of segment polarity and the onset of morphological segmentation, which requires Mesps, Notch/Delta, and Eph/Ephrins, as blocks of cells pinch off from the anterior of the mesenchymal PSM to form an epithelial somite (in zebrafish, mouse, and chick) (Giudicelli and Lewis, 2004). Numerous genetic studies demonstrate that perturbation of notch signaling in mice, humans, and zebrafish (Bessho et al., 2001; Bulman et al., 2000; Conlon et al., 1995; Dunwoodie et al., 2002; Evrard et al., 1998; Holley et al., 2000, 2002; Hrabé Angelis et al., 1997; Kusumi et al., 1998; Oka et al., 1995; Sieger et al., 2003; van Eeden et al., 1996; Wong et al., 1997; Zhang and Gridley, 1998), wnt3a in mice (Aulehla et al., 2003; Takada et al., 1994), receptor tyrosine phosphatase j in zebrafish (Aerne and Ish-Horowicz, 2004), or mesp2 in mice (Saga et al., 1997) leads to a segmentation defect in the posterior, but not the anterior, somites, with the defects occurring posterior to the 5th–9th somites in zebrafish. It is believed that this polarity represents a gradual breakdown of a single patterning mechanism that normally governs the segmentation of the entire body axis (Jiang et al., 2000). This hypothesis is supported by the fact that most genes expressed in a segmental pattern within the somites are expressed in all somites. Additionally, fss mutant embryos show segmentation defects along the entire body axis, indicating that there are common features in the genetic control of anterior and posterior somitogenesis (van Eeden et al., 1996). There are, however, several notable observations that distinguish the specification, formation, and differentiation of the anterior somites. Differences in the specification of the anterior paraxial mesoderm have been uncovered by genetic experiments in mice and zebrafish. Mice mutant for either of the transcription factors mesogenin or tbx6 form only the anterior paraxial mesoderm (Chapman and Papaioannou, 1998; Yoon and Wold, 2000). Similarly, in zebrafish, one eyed pinhead;no tail double mutants lack all but the anterior w5 somites (Schier et al., 1997). One consistent difference in anterior somitogenesis observed in mice, zebrafish, and the cephalochordate amphioxus is the more rapid progression of the somite cycle (Hanneman and Westerfield, 1989; Schubert et al., 2001; Tam, 1981). In the zebrafish, the anterior 6 somites form every 20 min, while the 24 posterior somites form every 30 min. In amphioxus, this temporal difference is even more extreme in that the anterior w8 somites form every hour but each subsequent somite cycle is 18 hr. There is also precedent for mutations that more severely affect the differentiation of the anterior somites. In the mouse, PDGFRα is thought to mediate signaling between the

Developmental Cell 576

myotome and sclerotome, and mice mutant for this receptor show extensive fusion of the cervical vertebrae but milder defects in thoracic and lumbar vertebrae (Soriano, 1997; Tallquist et al., 2000). This is similar to congenital human defects known as Klippel-Feil syndrome in which the cervical vertebrae are fused but the rib cage is only moderately affected, if at all (Pourquié and Kusumi, 2001). In this study, we make several observations that further distinguish anterior somitogenesis and suggest that somites 7–9 represent a transition zone between anterior and posterior somitogenesis in zebrafish. We performed a genetic screen for mutants exhibiting morphological defects confined to the anterior somites and identified two alleles of a gene called before eight (bfe). We positionally cloned bfe and found that it encodes the zebrafish integrina5. Integrinα5 forms a heterodimer with Integrinβ1 to create the primary receptor for the extracellular matrix (ECM) protein Fibronectin (Fn) (Miranti and Brugge, 2002). Morpholino knockdown of the Integrinα5 ligand natter(nat)/fn1 (Trinh and Stainier, 2004) weakly phenocopies integrina5, while knockdown of a second ligand, fn3, affects all somites. We examined double mutants between integrina5 and the posterior-specific notch pathway mutants. integrina5;aei/deltaD double mutants exhibit somite defects along the entire anterior-posterior axis and display a loss of mesenchymal-to-epithelial transition (MET) and failure to assemble Fn matrix within the posterior somitic mesoderm. These effects on morphogenesis underscore the fact that the genetic differences between anterior and posterior segmentation described here largely pertain to the direct regulation of segment morphogenesis as opposed to differences in the creation of the segmental prepattern. Results Distinct Patterns of Gene Regulation in the Anterior Somites There are two classes of genes that display differences in their patterns of transcription when comparing the anterior 5–7 somites with their posterior counterparts. The first class of genes is segmentally expressed in all somites, but this expression arises simultaneously in the anterior 5–7 somites at the 6- to 10-somite stage, while subsequent stripes form successively with the creation of each new somite. The segmental expression of myoD, snail1, and engrailed show this temporal pattern of regulation (Figures 1A–1C) (Ekker et al., 1992; Hammerschmidt and Nu¨sslein-Volhard, 1993; Weinberg et al., 1996). The second class of genes are exclusively expressed in the anterior somites: sox11a (de Martino et al., 2000) (Figure 1D) and nanos2 (Figure 1E). Notably, the localized transcription of these two genes precedes or is concurrent with the establishment of hox expression in this region of the paraxial mesoderm (Prince et al., 1998). The Anterior Somites Are Resistant to Perturbation of notch Signaling Mutations in the notch pathway genes after eight (aei)/ deltaD (Figure 1G) and deadly seven (des)/notch1a and

antisense inhibition of Su(H) affect the posterior somites while the anterior somites are normal, and double mutant analysis suggests that the resistance of the anterior somites to genetic perturbation is not due to redundancy (Holley et al., 2000, 2002; Sieger et al., 2003; van Eeden et al., 1996, 1998). Overexpression of an activated form of Notch (NICD) or a dominant-negative form of the notch pathway gene Su(H) (X-Su(H)DBM) can perturb Xenopus and zebrafish somite formation (Jen et al., 1997; Takke and Campos-Ortega, 1999; Wettstein et al., 1997). We repeated these experiments in zebrafish and found that injection of low amounts of either NICD or X-Su(H)DBM mRNA preferentially affects the posterior somites (Figures 1H and 1J), while injection of higher levels of either gene blocks the formation of all somites (Figures 1I and 1K), demonstrating that the anterior somites are also resistant to “dominant” perturbation of notch signaling. Transient Segmentation Defects around the 7th–9th Somites In addition to the posterior-specific mutants, we observe transient segmentation defects centered around somites 7–9 in fused somites (fss) heterozygous embryos and in some trans-heterozygous combinations such as fsste314a/+;aeitg249/+;destx201/+ (Figure 1L). This perturbation may affect only one or two borders of the 7th, 8th, or 9th somites or may extend from the 6th to the 12th somite. We have not been able to detect transient perturbations in the somite clock in these mutant embryos, suggesting that the perturbation may specifically affect segment border formation (data not shown). This is consistent with the requirement of fss in the anterior PSM (Barrios et al., 2003; van Eeden et al., 1998). These transient segmentation defects suggest that a transition occurs around somites 7–9 and that this transition is particularly sensitive to a reduction in fss gene function. It further demonstrates that point mutations can affect the segmentation of a restricted part of the body axis without affecting the posterior somites. before eight Is Required for Anterior Somitogenesis We performed a morphological screen of 5- to 15-somitestage embryos from 3532 mutagenized families representing 3265 genomes for anterior-specific somite mutants and isolated two phenotypically indistinguishable alleles of a locus called before eight (bfe). bfe mutant embryos display a segmentation defect complementary to that of the notch pathway mutants: the anterior somites are irregular, but posterior segmentation is normal (Figure 2A). While some homozygous embryos can be identified at the 3-somite stage, the mutants cannot be reliably sorted until the 5- to 7-somite stage. In homozygous embryos, somite morphogenesis appears to arrest after the border cells align and the borders subsequently disappear. Examination of prepatterning within the morphologically unsegmented PSM revealed no perturbation in her1, her7, or deltaC expression (Figures 2B–2D), indicating that the somite clock functions in bfe, again contrasting its phenotype to that of the notch pathway mutants (Figures 2E–2G) (Gajewski et al., 2003; Holley et al., 2000, 2002; Jiang et al., 2000; Oates and Ho, 2002). Patterning defects can be seen

Integrinα5, Notch, and Somite Morphogenesis 577

Figure 1. Distinguishing Features of the Anterior Somites (A–E) Dorsal views of dissected trunks and tails. Anterior is pointing up. (A)–(D) show one bilateral half for the early (left) and late (right) expression of (A) myoD at the 3- and 7-somite stage, (B) snail1 at the 3- and 7-somite stage, (C) engrailed at the 3- and 10-somite stage, and (D) sox11a at the 7- and 16-somite stage, while (E) shows nanos2 expression in whole trunks at 6- and 16-somite stages. Somitic expression of nanos2 fades by the 16-somite stage. Arrowheads in (C) and (D) indicate the anterior segmental gene expression. (F–L) Lateral DIC images of the trunks of live w12- to 16-somite-stage embryos. Anterior is toward the left. (F) Wild-type. (G) aei/deltaD homozygote. (H–K) Injection of low levels of either (H) NICD or (J) X-Su(H)DBM mRNA preferentially affects the posterior somites, while injection of higher levels of either (I) NICD or (K) X-Su(H)DBM affects all somites. For each set of injections, presented are the number of experiments (exp.) and the percentage of embryos that form the specified number of somites (/s). Percentage is calculated relative to the total number of nonnecrotic embryos (n). 75 ng/␮l NICD: (seven exp.) 47.5/0s, 3.8/3s, 8.4/4s, 7.6/5s, 8.4/6s, 1.3/7s, 0.4/10s; 22.7/wt [n = 238]. 200 ng/␮l NICD: (4 exp.) 77/0s, 1.0/2s, 3/3s, 2/4s, 2/5s, 2/7s; 13/wt [n = 100]. 100 ng/␮l X-Su(H)DBM: (4 exp.) 3.7/0s, 0.7/2s, 3/3s, 16.3/4s, 18.5/5s, 20.7/6s, 15.6/7s, 8/8s, 4.5/9s, 0.7/10s; 8/wt [n = 135]. 250 ng/␮l X-Su(H)DBM: (6 exp.) 34.2/0s, 3/2s, 8.7/3s, 20/4s, 12/5s, 10/6s, 4.3/7s, 0.5/8s, 0.5/9s; 7/wt [n = 231]. (L) fsste314a/+;aeitg249/+;destx201/+ embryos show a transient segmentation defect centered on the 7- to 9-somite region (asterisks). 5 of 14 fss/fss × aei/+;des/+ crosses produced transient segmentation defects around the 7- to 9-somite region in 35 of 124, 30 of 102, 14 of 63, 54 of 98, and 4 of 60 embryos, respectively. In some genetic backgrounds, fss/+ embryos display a similar transient segmentation defect (observed in 11 crosses involving 2 independent fss alleles).

in the segmental expression of myoD (Figure 2I). However, the expression of tbx24, notch1a, notch5, notch6, mespa, mespb, papc, lfringe, and hoxy6 appears to be relatively normal in bfe embryos, suggesting that bfe plays a minor role in establishing or maintaining polarity within each somite (data not shown). bfe homozygotes die between days 3 and 14.

bfe Is Necessary for Normal Myotome Shape and Myotome Border Recovery in fss Homozygotes In the posterior of bfe mutant embryos, somites adopt a U-shape rather than the chevron shape seen in wildtype embryos (Figures 3A and 3B). U-shaped somites are usually indicative of perturbation of hedgehog signaling from the notochord to the somites, leading to

Figure 2. before eight Is Required for Anterior Somitogenesis (A) Lateral DIC image of a live w14-somite stage bfe mutant embryo. (B–G) Dorsal views of the tailbud with anterior pointing up. The oscillating expression of (B and E) her1, (C and F) her7, and (D and G) deltaC is normal in (B–D) 3-somite-stage bfe embryos, but not in (E–G) 8-somite-stage aei/deltaD mutant embryos. (H–J) Dorsal views of dissected trunks and tails showing myoD expression (red) and her1 expression (blue). (H) Wild-type. (I) bfe mutant embryos display defects in the anterior segmental expression of myoD, whereas (J) aei/deltaD embryos show aberrant myoD expression in the posterior.


Figure 3. bfe Mutant Embryos Have U-Shaped Posterior Somites (A and B) Lateral DIC images of the trunk and tail of larvae 96 hr postfertilization (hpf). (A) Wild-type. (B) bfe homozygotes. (C and D) Lateral DIC images of the 10th–15th somites of 72 hpf embryos. The horizontal myoseptum (arrows) is present in both (C) wild-type and (D) bfe homozygotes. (E and F) Engrailed is expressed in the muscle pioneers of 24 hpf embryos in both (E) wild-type and (F) bfe mutant embryos. Both images are lateral views of the first 11–12 somites. (G and H) Projections of stacks of confocal fluorescence images of 36 hpf embryos stained for slow muscle myosin heavy chain. Images are of the 10–15 somite region. (G) Wild-type. (H) bfe homozygotes. (I–N) Dorsal views of the w7th–15th somites of embryos 36 hpf. (I and L) Widefield images of the slow muscle fibers. (J and M) DIC images. (K) An overlay of (I) and (J). (N) An overlay of (L) and (M). nt, neural tube. s, somites. (O–R) Lateral, darkfield images of muscle fiber birefringence of 72 hpf embryos. Shown are the w7th–15th somite region. Myotome borders are evident in (O) wild-type embryos, in (P) bfe homozygotes, and in (Q) fss mutant embryos (arrowheads), but they are largely absent in (R) fss;bfe double homozygotes . In all panels, anterior is toward the left.

mispecification of the adaxial cells and their derivatives: the horizontal myoseptum, muscle pioneers, and slow muscle fibers (Schauerte et al., 1998; van Eeden et al., 1996). In contrast to most U-type mutants, bfe embryos have a horizontal myoseptum (Figures 3C and 3D) and Engrailed-expressing muscle pioneers (Figures 3E and 3F). Similarly, bfe embryos display normally arranged slow muscle fibers within the U-shaped myotomes (Figures 3G and 3H). These slow muscle fibers are induced at the medial edge of the somite, and 3–6 hr after segmentation, they migrate through the somite to the lateral surface of the myotome (Figures 3I– 3K)(Blagden et al., 1997; Devoto et al., 1996). Examination of bfe embryos reveals that the lateral migration of the slow muscle fibers is normal (Figures 3L–3N). Finally, we investigated the ability of the adaxial cell derivatives to create irregular myotome borders in fss mutant embryos. Myotome borders are present in wildtype, bfe, and fss embryos but are largely absent in bfe;fss double mutant embryos (Figures 3O–3R). Thus,

while we find no obvious defect in the expression of direct targets of hedgehog signaling, such as patched (data not shown), nor in the specification or development of the horizontal myoseptum or the slow muscle fibers, mutations in bfe do affect the ability of the slow muscle fibers to make/shape myotome borders. In contrast to the anterior segmentation defect, this aspect of the bfe phenotype affects the entire anterior-posterior axis during a process that occurs hours after somite formation. bfe Encodes integrina5 bfe was mapped via meiotic recombination to a 144 kb region containing a predicted homolog of integrina5 (Figure 4A). We sequenced the coding region of integrina5 from wild-type embryos and both bfe alleles. In each mutant allele, premature stop codons were found within the 5th repeat of the β propeller, which forms the domain containing the binding sites for Fn in the α5/β1 heterodimer (Figures 4B and 4C) (Humphries et al., 2003).


Figure 4. before eight Is integrina5 (A) bfe was mapped via meiotic recombination to chromosome 23 between the simple sequence length polymorphisms (SSLPs) z4421 (126 recombinations in 1470 meioses) and z5141 (12 recombinations in 2536 meioses). z5141 and expressed sequence tags (ESTs) between the two SSLPs were used to isolate BACs (horizontal gray bars), and the end sequences of the BACs generated by the Zebrafish Genome Project were used to generate single nucleotide polymorphisms (SNPs), which then were used to refine the genetic interval to 144 kb (light gray box). This interval contained a zebrafish homolog of integrina5. (B and C) Sequence analysis identified independent premature stop codons within the fifth β propeller repeat of integrina5 in both (B) tbfe1 and tbfe2 and in gray boxes in (C). (C) The predicted amino acid sequence of Integrinα5 displays 57%, 53%, and 54% identity to the Xenopus, mouse, and human Integrinα5, respectively. The protein domains are color coded as in (B). (D) integrina5 mRNA is deposited maternally and can be seen in the two-cell blastula. (E–G) At the shield stage, (E) integrina5 is transcribed ubiquitously, while during both (F) early and (G) late somitogenesis, it is expressed in the posterior tip of the tail bud, t, and weakly in the PSM and the adaxial cells, a. 3s, 3-somite stage. 15s, 15-somite stage. In (E), the shield is to the right. (F) and (G) are dorsal views. (H and I) Lateral DIC views of live 10- to 15-somite-stage embryos. (H) Injection of 300 ␮M of an integrina5 morpholino phenocopies bfe (five experiments, 90% affected, n = 263). (I) Injection of the integrina5 morpholino into integrina5 mutant embryos produces a stronger defect in the anterior somites (four experiments, n = 324, 25.6% no anterior somite borders, 69.7% irregular anterior somite borders, and 4.6% wild-type). (J) Injection of 250 ng/␮l in vitro-synthesized integrina5 mRNA into bfe embryos rescues the segmentation phenotype (four experiments, n = 382, 99.7% were wild-type). Injection of 250 ng/␮l of an mRNA encoding for an Integrinα5-GFP fusion protein also rescues integrina5 mutants (green in [J]). Coinjection of an mRNA encoding for mRFP with a nuclear localization signal (red in [J]) allows simultaneous examination of the alignment and polarization of the somite border cells along with Integrinα5 clustering. The arrowhead indicates Integrin clustering along the basal surface of the somite border cells. Note that the cells along this border have adopted a columnar morphology, while the cells along the less mature border to the right have not completed epithelialization. Arrows indicate Integrinα5-GFP clustering along the nascent somite border. Pictured is a confocal image (dorsal view) of the anterior PSM and the most recently formed somites of a w10-somite-stage embryo. Injection experiments in (I) and (J) used embryos derived from bfe/+ × bfe/+ matings. In (F)–(J), anterior is toward the left.

Thus, both bfe alleles should be null alleles of integrina5. Recently, a missense mutation in the zebrafish integrina5 was isolated in a screen for craniofacial defects (Crump et al., 2004). We observe similar defects in the hyosymplectic, ceratohyal, and gill cartilages (data not shown). integrina5 mRNA is maternally deposited and later ubiquitously expressed at the beginning of gastrulation (Figures 4D and 4E). During both early and late somitogenesis, integrina5 is expressed in the posterior tip of

the tailbud and in the adaxial cells (Figures 4F and 4G). The Integrinα5 protein required for somite border formation likely derives from the ubiquitous maternal and early zygotic expression as well as the expression in the posterior of the tailbud, while the later defect in myotome morphology correlates with the elevated expression in the adaxial cells. Indeed, injection of an antisense morpholino against integrina5 phenocopies the integrina5 mutant (Figure 4H), and injection into the integrina5 mutant embryos produces a stronger defect


Figure 5. nat/fn1 and fn3 Are Necessary for Zebrafish Somitogenesis (A–G) integrinß1 mRNA expression during (A) early and (B) late somitogenesis. nat/fn mRNA expression during (C) early and (D) late somitogenesis. fn3 mRNA expression during (E) early and (F) late somitogenesis. (A, C, and E) 4-somite-stage embryos. (B, D, and F) 15-somitestage embryos. (G) Fn matrix localization in a 14-somite-stage embryo. (A–G) Dorsal views with anterior to the left. (H) Injection of 750 ␮M of the nat/fn1 morpholino phenocopies the integrina5 anterior somite phenotype (three experiments, 30% of 98 embryos displayed irregular anterior somites). (I) 750 ␮M nat/fn1 morpholino was injected into embryos derived from a cross of bfe heterozygotes (four experiments, n = 178, 27% strong anterior somite phenotype and an elongation defect). Injection of fn3mo1 up to 1 mM by itself produces no clear phenotype, but injection of 750 ␮M fn3mo2 produces irregular somites and a tail extension defect (two experiments, 82% affected, n = 55). (J and K) We used two different morpholinos to knockdown fn3 activity. Coinjection of 500 ␮M each of fn3mo1 and fn3mo2 produces the same phenotype but with less toxicity (three experiments, 97% affected, n = 127). The embryo in (J) is at the w7-somite stage, and the same embryo is pictured in (K) at the 20-somite stage. (L and M) Coinhibition of fn1 and fn3 produces defects ranging from an enhancement of the (L) somite defect (three experiments, 100% affected, n = 74) to a (M) dramatically shortened body axis (four experiments, 100% affected, n = 192), depending upon the combination and concentration of the injected morpholinos. (H)–(M) are lateral views of live 12- to 14-somite-stage embryos. Anterior is toward the left.

in the anterior somitic mesoderm (Figure 4I). The enhancement of the zygotic phenotype is likely due to the morpholino inhibition of translation of the maternal mRNA, suggesting that the wild-type maternal contribution buffers some of the effects of the loss of zygotic activity in integrina5 homozygotes. Injection of integrina5 or integrina5-GFP mRNA rescues the integrina5 mutant phenotype (Figure 4J). Visualization of Integrinα5-GFP along with either a nuclear RFP or DIC in live embryos via timelapse microscopy reveals Integrinα5GFP around the cell cortex and Integrinα5-GFP clustering along the future basal side of the somite border cells. This clustering along nascent somite borders is concomitant with alignment and polarization of the border cells (Figure 4J, arrows, and Movie S1; see the Supplemental Data available with this article online). Knockdown of nat/fn1 or fn3 Leads to Defects in Somitogenesis Integrinα5 heterodimerizes with Integrinβ1 to form one of eight known receptors for the ECM protein Fn. Throughout somitogenesis, integrinb1 is expressed in the adaxial cells, posterior tailbud, and PSM and is weakly expressed in the somites (Figures 5A and 5B) (Thisse et al., 2001). In zebrafish, there are two fibronectin genes, nat/fn1 and fn3, that are expressed in the paraxial mesoderm during segmentation. During both early/anterior and late/posterior somitogenesis, nat/fn1 is expressed in the notochord and in the posterior tailbud and PSM (Figures 5C and 5D). fn3 is more broadly

and strongly expressed throughout the paraxial mesoderm during segmentation, but shows a difference in its early and late expression in that the PSM/tailbud expression is much weaker during early/anterior somitogenesis (Figures 5E and 5F). Despite the broad expression of integrina5, integrinb1, and the two fibronectins, assembly of the Fn matrix is localized to the somite borders and surfaces of the paraxial mesoderm (Figure 5G) (Crawford et al., 2003). Due to the possible extensive redundancy among the numerous Fn receptors, we undertook a functional analysis of the two ligands to determine if Integrin-Fn interactions are necessary for posterior segmentation. Perturbation of fn1 function in zebrafish in the mutant natter or via morpholino was recently shown to affect migration of the heart field (Trinh and Stainier, 2004). We find that inhibition of fn1 via injection of the nat/fn1 morpholino gives a weak anterior somite border defect, first visable around the 8- to 10-somite stage, similar to a phenotype observed in some integrina5 clutches (Figure 5H). The relative subtlety of the defect likely explains why this aspect of the nat/fn1 phenotype has not been reported previously. Further injection of the nat/ fn1 morpholino into integrina5 mutant embryos causes an enhancement of the integrina5 phenotype, though posterior somitogenesis still recovers (Figure 5I). We used two different morpholinos to knockdown fn3 and find that combining the two gives a stronger, more penetrant phenotype (Figures 5J and 5K). Knockdown of fn3 produces embryos that initially form somites (Figure


5J). However, by the 15- to 20-somite stage, the regular somite morphology is lost, and tail extension defects are apparent (Figure 5K). Coinjection of low concentrations of fn1 and fn3 morpholinos leads to an enhanced somite defect (Figure 5L). Increasing the dosage of the fn1 and fn3 morpholinos leads to more profound defects in axis formation/elongation (Figure 5M). These phenotypes suggest the existence of a spatiotemporal division of labor among the fibronectins such that fn1 is more important for anterior somitogenesis than fn3. Moreover, these experiments imply that Integrin-Fn interactions are important for somitogenesis along the entire body axis, with other integrins playing a more prominent role in posterior somitogenesis in lieu of integrina5. integrina5;notch Pathway Double Mutants Display Synergistic Defects in Somite Border Formation integrina5 has an anterior segmentation phenotype complementary to the posterior segmentation defect observed in notch pathway mutants such as aei/deltaD (Figures 6B and 6C). We thus sought to characterize the phenotype of embryos double mutant for integrina5 and the notch pathway genes. integrina5;aei/deltaD double mutant embryos display segmentation defects along the entire anterior-posterior axis (Figure 6D). Moreover, the irregular borders seen in the posterior of aei/deltaD embryos (Figure 6C, arrow) are missing in the double mutants. Since the posterior somite borders form in integrina5 single mutants, the lack of even irregular borders in the double mutants is a synergistic effect of the loss of both integrina5 and aei/deltaD. This same effect was seen in a parallel analysis of integrina5;bea double mutants (Figures 6E and 6F) and integrina5;des/notch1a (data not shown). We further found that perturbation of integrina5 function eliminates the resistance of the anterior somites to ectopic, low-level expression of NICD and X-Su(H)DBM, with the injected mutant embryos displaying a defect stronger than that of integrina5 mutants (data not shown). The synergy in the posterior of integrina5;aei/deltaD embryos is apparent too early in development to be due to either a defect in somite border maintenance or to the adaxial cell/slow muscle fiber phenotype in the posterior somites of integrina5. We therefore examined the phenotype of the integrina5;aei/deltaD embryos in more detail in order to pinpoint the nexus of the synergy. titin expression marks the segment borders of wild-type embryos (Figure 6G) (Oates and Ho, 2002), the irregular posterior somite borders in aei/deltaD embryos (Figure 6H), and irregular anterior somite borders and the U-shaped posterior somites of integrina5 embryos (Figure 6I). There is a marked increase in perturbation of the titin staining in the integrina5;aei/deltaD double mutant embryos as compared to either single mutant (Figure 6J). Likewise, examination of myotome organization reveals irregular myotome borders in the posterior of aei/deltaD embryos (Figure 6L), U-shaped myotomes in integrina5 embryos (Figure 6M), and complete disorganization in integrina5;aei/deltaD double mutant embryos (Figure 6N). We next investigated cellular morphology within the region that should encompass the nascent somites in 10- to 16-somite-stage wild-type, mutant, and double mutant embryos. Examination of f-actin reveals the co-

lumnar border cells, along the regular somite borders in wild-type embryos (Figure 6S), along the irregular borders in aei/deltaD embryos (Figure 6T), and along the regular posterior somite borders in integrina5 embryos (Figure 6U). However, the cells in the posterior paraxial mesoderm of integrina5;aei/deltaD double mutant embryos fail to adopt the columnar cell shape and make segment borders, suggesting a defect in the MET that occurs during morphological segmentation (Figure 6V). Cell polarity can also be visualized by β-catenin localization (Figures 6W–6Z, green), which lines the cell cortex, and by the localization of the nuclei (Figures 6W–6Z, red). The columnar cell shape and basal localization of the nuclei can be seen along the regular somite borders in wild-type embryos (Figure 6W), along irregular borders in aei/deltaD embryos (Figure 6X), and along regular borders in integrina5 embryos (Figure 6Y), but cannot be seen in integrina5;aei/deltaD double mutant embryos (Figure 6Z). These findings suggest that the posterior paraxial mesoderm cells of the double mutant are unable to undergo MET. The cells of the PSM in fss mutant embryos also fail to undergo MET, and this has been shown to be due largely to loss of ephA4 expression (Barrios et al., 2003). In contrast, the onset of ephA4 expression in the anterior PSM can be seen in integrina5;aei/deltaD double mutant embryos (Figure 6R, arrowhead). Therefore, the lack of MET is not due to loss of either ephA4 expression or the expression of ephrinB2b, ephrinA1, or ephrinB2a (data not shown). Cell-ECM interactions are important for establishing and maintaining cell polarity. Indeed, Integrins were so named for their ability to integrate the ECM with the actin cytoskeleton (Miranti and Brugge, 2002). In the zebrafish, Fn protein is detected on the surfaces of the paraxial mesoderm and along the somite borders, but not within the PSM (Crawford et al., 2003). Costaining for Fn matrix (Figures 6A#–6D#, green) with the nuclei (Figures 6A#–6D#, red) shows that in wild-type embryos the Fn matrix is assembled concomitantly with the basal alignment of the nuclei along the nascent somite borders (Figure 6A#). Some organization can be seen along the irregular borders in aei/deltaD embryos (Figure 6B#) and in integrina5 embryos (Figure 6C#), but Fn matrix assembly is virtually absent in integrina5;aei/deltaD double mutant embryos (Figure 6D#), again illustrating the synergy between notch signaling and integrina5 during somite morphogenesis. A parallel analysis of integrina5;des/notch1a and integrina5;bea double mutants yielded comparable results (data not shown). In summary, catechization of the double mutants indicates that integrina5 and notch signaling function in parallel to regulate segment border morphogenesis, MET, and assembly of the Fn matrix during posterior somitogenesis. Discussion integrina5 Is Required for Anterior Somitogenesis We performed a genetic screen for anterior-specific somite mutants and found two alleles of integrina5. This screen was as large as the 1996 Tu¨bingen genetic screen that yielded 18 posterior-specific somite mutants representing four genes: aei/deltaD, des/notch1a,


Figure 6. Concomitant Perturbation of integrina5 and notch Signaling Eliminates All Somite Formation (A) A 17-somite-stage wild-type embryo. (B) A w13-somite-stage integrina5 homozygote. (C) A w15-somite-stage aei/deltaD homozygote. The arrow indicates the most obvious posterior border. (D) integrina5;aei/deltaD double mutant. Pictured is a w13-somite-stage embryo. (E) bea homozygote. Arrows indicate partial borders in the posterior paraxial mesoderm. (F) bea;integrina5 double mutants. In each experiment, embryos were derived from parents double heterozygous for either integrina5tbfe1 or integrina5tbfe2 and either aei/deltaDtg249, beatit446 (shown), des/notch1atx201, or fss/tbx24ti1 (data not shown). (G–J) Lateral views of titin expression, which marks each somite border in embryos 30 hpf. (G) Wild-type. (H) aei/deltaD homozygotes. (I) integrina5 embryos. (J) aei/deltaD;integrina5 double mutants. (K–N) Myotome organization in the posterior trunk is revealed by slow muscle myosin localization in embryos 36 hpf. (K) Wild-type. (L) aei/ deltaD. (M) integrina5. (N) aei/deltaD;integrina5. Projections of stacks of confocal images of lateral views of the 10th–15th somites. (O–D#) Dorsal views of one bilateral half of the most recently formed somites of 10- to 16-somite-stage embryos. (O–R) Induction of ephA4 transcription (arrowheads) can be seen in the anterior PSM in (O) wild-type, (P) aei/deltaD, (Q) integrina5, and (R) aei/deltaD;integrina5. (S–V) Examination of cell morphology within the somitic mesoderm via phalloidin staining. (S) Wild-type. (T) aei/deltaD. (U) integrina5. (V) aei/deltaD;integrina5. (W–Z) Examination of β-catenin localization (green) and nuclei (prodidium iodide, red) show epithelial borders with basal localization of nuclei along somite borders. (W) Wild-type. (X) aei/deltaD. (Y) integrina5. (Z) aei/deltaD;integrina5. The arrows in (X) point to polarized cells along an irregular border. (A#–D#) The basal side is the actual somite border and can be visualized via Fn localization (green) and nuclear orientation (propidium iodide, red). (A#) Wild-type. Examination of a number of wild-type embryos suggests that Fn matrix assembly begins w5–10 min after the somite border cells begin to align. (B#) aei/deltaD. (C#) integrina5. (D#) aei/deltaD;integrina5. (S)–(D#) are confocal images. In all panels, anterior is toward the left.

white tail/mindbomb, and bea. While the results of the 1996 screen underscore the importance of notch signaling in posterior somitogenesis, the results presented here imply a central function of integrina5 in anterior somitogenesis. The integrina5 phenotype varies signifi-

cantly from clutch to clutch due to genetic background and possibly due to disparities in the degree of maternal rescue. The weak phenotype consists of regular, but somewhat indistinct, borders affecting the first 2–6 somites, while reduction of the maternal contribution in


strong zygotic mutants eliminates almost all evidence of border formation in the anterior somites. In all cases, border formation is initiated in the anterior, but it is not completed or maintained, and the embryos recover with normal posterior somitogenesis. In many clutches, it is difficult to recognize the irregular anterior somite borders in an older embryo, and we believe that our discovery of these mutations is due to screening the embryos between 5 and 15 somites, when the phenotype is most obvious. integrina5 Is Required for the Morphogenic Activity of the Slow Muscle Fibers integrina5 mutant embryos have U-shaped posterior somites, and our characterization suggests that this is due to a subtle defect in the slow muscle fibers. Given that integrina5 is highly expressed in the adaxial cells and that Fn matrix is present in the myotome borders, we interpret the U-shaped phenotype as a result of reduced adhesion between the slow muscle fibers and the myotome borders. This is supported by the occasional disruption of the myotome border in the posterior of integrina5 homozygotes (Figure 6M). The elevated levels of integrina5 expression in the adaxial cells may also allow the slow muscle fibers to assemble myotome borders de novo in fss mutant embryos, leading to the adaxial cell-dependent “rescue” of borders in this mutant. Comparison of Zebrafish integrina5 to Other Somite Mutants Generally speaking, the zebrafish integrina5 morphological phenotype is most similar to that of PDGFRα mutant mice and the congenital human disease KlippelFeil syndrome. While the etiology of Klippel-Feil syndrome is unknown, the defect in PDGFRα mutant mice is thought to result from aberrant signaling between the myotome and sclerotome rather than in segmentation itself (Pourquié and Kusumi, 2001; Soriano, 1997; Tallquist et al., 2000). Injection of morpholinos against her1 have been reported to specifically affect the anterior somites, but it is not clear if this is due to the actual requirement for her1 or if it is due to a reduced efficacy of the morpholino over time, as was shown to occur with morpholino-mediated inhibition of foxc1a function during somitogenesis (Henry et al., 2002; Oates and Ho, 2002; Topczewska et al., 2001). In vertebrates, there are 18 α and 8 β Integrin subunits that are known to produce at least 24 different heterodimers. Integrinα5β1 is thought to be the primary receptor for Fn, though α3β1, α4β1, α8β1, αvβ1, αvβ3, αvβ6, and αIIbβ3 can also bind Fn (Yang et al., 1999). Gene knockout experiments in the mouse identified requirements only for fn and integrina5 in somitogenesis. The fn knockout mouse phenotype is more severe than the mouse integrina5 mutant phenotype in that it completely lacks both a notochord and somites. This fn phenotype is reminiscent of the fn1;fn3 double knockdown phenotype in zebrafish (Figure 5M) (GeorgesLabouesse et al., 1996). The strongest integrina5 knockout phenotype consists of irregular anterior somites and a truncated posterior body. This general requirement for integrina5 in posterior development in mouse

precluded a direct analysis of posterior segment border formation in these mutants (Yang et al., 1999). integrina5 and notch Signaling during Zebrafish Somite Border Morphogenesis Somite border morphogenesis begins with alignment of the border cells and continues for the next hour as these cells develop a columnar morphology with an apical-basal polarity and assemble a basal Fn matrix. The analysis of integrina5;aei/deltaD double mutants unveiled redundancy between integrin and notch pathway function in promoting somite border morphogenesis and Fn matrix assembly. This is a role for Integrin-Fn not revealed in either integrina5 mutants or fibronectin morphants, probably due to redundancy. The synergy observed in the integrina5;notch double mutants is not due to a failure to maintain irregular somite borders, as our cellular analysis specifically focused on the region that should encompass the nascent somites, and no evidence of somite border morphogenesis was detected. The synergy may be indicative of a regulatory relationship between Notch and Integrinα5 whereby Notch affects Integrinα5 activity directly or indirectly via Eph/Ephrin signaling, which is involved in governing somite border morphogenesis (Barrios et al., 2003; Durbin et al., 1998). Integrinα5-GFP begins to cluster along the future basal surface of the somite border cells as soon as cells begin to align at the onset of border formation, while Fn matrix assembly is detected w5–10 min later. These two observations suggest that the initial Integrinα5 clustering is not due to Fn-mediated “outside in” signaling, but is due to cytoplasmic, cellautonomous “inside out” regulation. The cumulative picture arising from the examination of integrina5;aei/ deltaD double mutants, Integrin clustering, and Fn matrix assembly suggests that morphological somite border formation encompasses concomitant and interdependent Notch/Integrin-mediated MET and Fn matrix assembly (Figure 7A). In addition to how Notch might affect the activity of Integrinα5, the question of why isn’t Notch necessary for anterior somite border formation remains. Irrespective of whether Notch generates the oscillations in the PSM or synchronizes them (Holley et al., 2000, 2002; Jiang et al., 2000), it is not clear why aei/deltaD or des/ notch1a would not be required for establishing segment polarity and regulating segment border formation since there is ample evidence for this later, postoscillator function of the notch pathway in somitogenesis (Sato et al., 2002; Takahashi et al., 2000, 2003). The fact that the anterior somites are resistant to dominant perturbation of notch signaling indicates that anterior somitogenesis is more robust than posterior somitogenesis. Rather than the robustness being due to redundancy whereby the generation of a segmentation signal, x, is divided equally between redundant genes, the ectopic NICD or X-Su(H)DBM experiments suggest that the robustness is due to the generation of a 2x segmentation signal whose greater amplitude would resist interference from dominant perturbation of notch signaling. Alternatively, there could be a qualitative difference in the control of anterior somite morphogenesis with notch being utilized in a reduced capacity, thereby curtailing the effects of perturbing notch signaling. The sensitivity of the 7th–9th somites to perturbation in fss/+ and fss/+;aei/+;des/+ embryos implies an important


Figure 7. A Model for Notch and Integrinα5 Function during Somite Border Morphogenesis (A) Notch may affect Integrin activity somewhat directly (orange arrow) by stimulating the separation of the cytoplasmic tails of the α5β1 heterodimer and thereby switching the extracellular domain of the α5β1 to the high-affinity, ligand binding conformation (insideout signaling). For example, expression of an activated form of Notch4 in cultured endothelial cells somehow increases the percentage of active, high-affinity Integrinβ1 conformation on the cell surface (Leong et al., 2002). Alternatively, notch signaling could affect α5β1 function more indirectly (red arrow) by causing a change in cytoskeletal organization or through Eph/Ephrin signaling (purple arrows). The Integrinα5β1 heterodimer functions in Fn matrix assembly during epithelialization. Binding of Fn to α5β1 affects cytoskeletal organization via “outside-in” signaling (Miranti and Brugge, 2002). One protein that may mediate outside-in signaling, Focal Adhesion Kinase, has been shown to localize basally in zebrafish somite boundary cells (Henry et al., 2001). The cytoskeleton-α5β1 interactions are in turn necessary for Fn matrix assembly (Wierzbicka-Patynowski and Schwarzbauer, 2003). The changes in the cytoskeleton, Integrinα5β1 activity, and Fn matrix would be self-reinforcing by leading to further Integrin clustering and thus further cytoskeletal polarization and Fn matrix assembly as morphological somite formation proceeds. (B) Integrinα5 and notch signaling have complementary spatiotemporal requirements during zebrafish somitogenesis. There is a greater requirement for integrina5 during early/anterior somitogenesis than in late/posterior somitogenesis (blue), while there is a complementary requirement for notch signaling (yellow). The transition (green) is coincident with changes in the way in which the paraxial mesoderm is specified, in the rate of segmentation, and in the regulation of somitic gene expression.

change in regulation or mechanism as opposed to a change in which there is greater redundancy, since there is no apparent reason that such a latter transition would be sensitive to fss heterozygosity. Why Are Anterior Somites Different from Posterior Somites? We found that point mutations can cause segmentation defects in either the anterior somites, the posterior somites, or at the transition from early to late somitogenesis (Figure 7B). At present, the genesis of the differ-

ences between anterior and posterior somitogenesis remains unknown. One possible explanation was that somites 7–9 represented the transition from the mesoderm generated during gastrulation to tailbud-derived tissue. However, fate mapping data indicate that this transition occurs around somites 11–13 (data not shown) (Agathon et al., 2003; Mu¨ller et al., 1996). Nevertheless, other observations do suggest that the anterior somitic mesoderm is specified in a manner that is different than the posterior somites (Chapman and Papaioannou, 1998; Schier et al., 1997; Yoon and Wold, 2000). It is plausible that variations in segmentation could be related to differences in the ontogeny of distinct anterior-posterior regions of the paraxial mesoderm. Our genetic data largely support a model in which the regulation of anterior somite morphogenesis is different than that in the posterior. In this regard, it is notable that when the initial w6 somite borders form, there is still extensive dorsal convergence occurring, as the somites are broad along the medial-lateral axis but converge such that they shrink medial-laterally and expand dorsal-ventrally. This may necessitate differences in the control of segment morphogenesis. Amphioxus offers a more extreme parallel in that the anterior, Engrailedexpressing somites form by pinching off from the roof of the archenteron (enterocoely), while the posterior somites bud off from the tailbud and later hollow out (schizocoely) (Holland et al., 1997). While anterior somitogenesis in higher vertebrates may not exactly resemble that in amphioxus, this anterior-posterior distinction may have a common origin. Experimental Procedures Zebrafish Care and Rearing Zebrafish were raised as described in Nu¨sslein-Volhard and Dahm, 2002. The two bfe alleles, tbfe1 and tbfe2, were originally designated thl030 and tig453, respectively. Positional Cloning of bfe Mapcrosses between bfe and WIK and a bulk-segregant genome scan were performed by using standard protocols (Nu¨sslein-Volhard and Dahm, 2002). Single embryo mapping placed bfe between SSLPs z4421 and z5141 on LG23. BAC libraries (RZPD) were screened via PCR by using the SSLPs as well as closely linked ESTs. and putatively identified BACs were retested individually via PCR. BAC end sequences generated by the Zebrafish Genome Project were used to screen for SNPs via resequencing the bfe mapcrosses. The SNPs were examined on recombinants identified with z4421 and z5141 to map bfe to the genetic interval containing a predicted homolog of integrina5. The full coding sequence was obtained from both bfe alleles by sequencing three independent RT-PCR products from each allele. Injection Experiments Full-length integrina5 lacking the endogenous 5# and 3# UTRs was generated via PCR and were cloned into pCS2+; subsequently, eGFP was spliced in frame at the C terminus of Integrinα5. mRNA for the integrins X-Su(H)DBM (Wettstein et al., 1997) and NICD (Takke and Campos-Ortega, 1999) were generated with the SP6 Message Machine Kit (Ambion). The integrina5 morpholino (GeneTools) (5#-taaccgatgtatcaaaatccactgc-3#) was designed to bind the 5# UTR just upstream of the start codon. The nat/fn1 morpholino sequence is as previously described (Trinh and Stainier, 2004). The fn3 morpholinos sequences are as follows: fn3mo1 (5#-tactg actcacgggtcattttcacc-3#) and fn3mo2 (5#-gcttctggctttgactgtattt cgg-3#).


In Situ Hybridization and Immunohistochemistry The zebrafish integrinß1 EST (http://www.zfin.org, Genbank: CF417014) was used to generate a riboprobe for expression analysis (Thisse et al., 2001). The anti-Engrailed monoclonal 4D9 (Developmental Studies Hybridoma Bank) was used 1:50 on PFA-fixed embryos and was detected by using the Elite ABC Peroxidase Kit (Vector). The rabbit anti-β-catenin antibody was used at a dilution of 1:100 on PFA-fixed embryos and was visualized with a goat anti-rabbit Alexa647 antibody (Molecular Probes) used at a 1:200 dilution. The mouse S58 anti-slow myosin antibody (Developmental Studies Hybridoma Bank) was used at a dilution of 1:10 on embryos fixed in Carnoy’s fixative and was visualized with a goat anti-mouse FITC antibody (Sigma) (1:16). The rabbit anti-Fn antibody (Sigma) (1:200) was visualized with the goat anti-rabbit Alexa647 (Molecular Probes) (1:200). Alexa488 phalloidin (Molecular Probes) was used at 400 nM. Propidium iodide (Molecular Probes) was used according to the manufacturer’s instructions.

Supplemental Data Supplemental Data including a timelapse movie of the Integrinα5GFP;nls-RFP-expressing embryo shown in Figure 4J (Quicktime) are available at http://www.developmentalcell.com/cgi/content/ full/8/4/575/DC1/.

Acknowledgments We thank John Carlson, Paul Mould, and Frank Ruddle for comments on the manuscript and Phil Ingham for sharing unpublished data. We thank Josh Schroeder for technical assistance; Stephen Devoto, José Campos-Ortega, Chris Kintner, Andy Oates, Richard Poole, Ralf Rupp, and Roger Tsien for plasmids; and Herbert Steinbeisser for the rabbit anti-β-catenin antiserum. We thank Marcus Dekens and Holger Knaut for the nanos2 partial cDNA and Claudia Nicolaije for nanos2 in situ images. We thank Haig Keshishian for instruction on the BioRad 1024 confocal microscope. We are grateful to Christiane Nu¨sslein-Volhard for her support. Received: October 28, 2004 Revised: January 5, 2005 Accepted: January 13, 2005 Published: April 5, 2005

Chapman, D.L., and Papaioannou, V.E. (1998). Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6. Nature 391, 695–697. Conlon, R.A., Reaume, A.G., and Rossant, J. (1995). Notch1 is required for the coordinate segmentation of somites. Development 121, 1533–1545. Crawford, B.D., Henry, C.A., Clason, T.A., Becker, A.L., and Hille, M.B. (2003). Activity and distribution of paxillin, focal adhesion kinase, and cadherin indicate cooperative roles during zebrafish morphogenesis. Mol. Biol. Cell 14, 3065–3081. Crump, J.G., Swartz, M.E., and Kimmel, C.B. (2004). An integrindependent role of pouch endoderm in hyoid cartilage development. PLoS Biol. 2, E244. de Martino, S., Yan, Y.L., Jowett, T., Postlethwait, J.H., Varga, Z.M., Ashworth, A., and Austin, C.A. (2000). Expression of sox11 gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development. Dev. Dyn. 217, 279–292. Devoto, S., Melançon, E., Eisen, J.S., and Westerfield, M. (1996). Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation. Development 122, 3373–3380. Dunwoodie, S.L., Clements, M., Sparrow, D.B., Sa, X., Conlon, R.A., and Beddington, R.S. (2002). Axial skeletal defects caused by mutation in the spondylocostal dysplasia/pudgy gene Dll3 are associated with disruption of the segmentation clock within the presomitic mesoderm. Development 129, 1795–1806. 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. Ekker, M., Wegner, J., Akimenko, M.A., and Westerfield, M. (1992). Coordinate embryonic expression of three zebrafish engrailed genes. Development 116, 1001–1010. 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. Gajewski, M., Sieger, D., Alt, B., Leve, C., Hans, S., Wolff, C., Rohr, K.B., and Tautz, D. (2003). Anterior and posterior waves of cyclic her1 gene expression are differentially regulated in the presomitic mesoderm of zebrafish. Development 130, 4269–4278. Georges-Labouesse, E.N., George, E.L., Rayburn, H., and Hynes, R.O. (1996). Mesodermal development in mouse embryos mutant for fibronectin. Dev. Dyn. 207, 145–156.

References

Giudicelli, F., and Lewis, J. (2004). The vertebrate segmentation clock. Curr. Opin. Genet. Dev. 14, 407–414.

Aerne, B., and Ish-Horowicz, D. (2004). receptor tyrosine phosphatase psi is required for Delta/Notch signalling and cyclic gene expression in the presomitic mesoderm. Development 131, 3391– 3399.

Hammerschmidt, M., and Nu¨sslein-Volhard, C. (1993). The expression of a zebrafish gene homologous to Drosophila snail suggests a conserved function in invertebrate and vertebrate gastrulation. Development 119, 1107–1118.

Agathon, A., Thisse, C., and Thisse, B. (2003). The molecular nature of the zebrafish tail organizer. Nature 424, 448–452.

Hanneman, E., and Westerfield, M. (1989). Early expression of acetyl-choline-sterase activity in functionally distinct neurons of the zebrafish. J. Comp. Neurol. 284, 350–361.

Aulehla, A., Wehrle, C., Brand-Saberi, B., Kemler, R., Gossler, A., Kanzler, B., and Herrmann, B.G. (2003). Wnt3a plays a major role in the segmentation clock controling somitogenesis. Dev. Cell 4, 395–406. Barrios, A., Poole, R.J., Durbin, L., Brennan, C., Holder, N., and Wilson, S.W. (2003). Eph/Ephrin signaling regulates the mesenchymal-to-epithelial transition of the paraxial mesoderm during somite morphogenesis. Curr. Biol. 13, 1571–1582. 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. Blagden, C.S., Currie, P.D., Ingham, P.W., and Hughes, S.M. (1997). Notochord induction of zebrafish slow muscle mediated by Sonic hedgehog. Genes Dev. 11, 2163–2175. Bulman, M.P., Kusumi, K., Frayling, T.M., McKeown, C., Garret, C., Lander, E.S., Krumlauf, R., Hattersley, A.T., Ellard, S., and Turnpenny, P.D. (2000). Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis. Nat. Genet. 24, 438–441.

Henry, C.A., Crawford, B.D., Yan, Y.L., Postlethwait, J., Cooper, M.S., and Hille, M.B. (2001). Roles for zebrafish focal adhesion kinase in notochord and somite morphogenesis. Dev. Biol. 240, 474–487. Henry, C.A., Urban, M.K., Dill, K.K., Merlie, J.P., Page, M.F., Kimmel, C.B., and Amacher, S.L. (2002). Two linked hairy/Enhancer of splitrelated zebrafish genes, her1 and her7, function together to refine alternating somite boundaries. Development 129, 3693–3704. Holland, L.Z., Kene, M., Williams, N.A., and Holland, N.D. (1997). Sequence and embryonic expression of the amphioxus engrailed gene (AmphiEn): the metameric pattern of transcription resembles that of its segment-polarity homolog in Drosophila. Development 124, 1723–1732. Holley, S.A., Geisler, R., and Nu¨sslein-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., Ju¨lich, D., Rauch, G.J., Geisler, R., and Nu¨sslein-Vol-


hard, C. (2002). her1 and the notch pathway function within the oscillator mechanism that regulates zebrafish somitogenesis. Development 129, 1175–1183. Hrabé Angelis, M., McIntyre, J., and Gossler, A. (1997). Maintenance of somite borders in mice requires the Delta homologue Dll1. Nature 386, 717–721. Humphries, M.J., Symonds, E.J., and Mould, A.P. (2003). Mapping functional residues onto integrin crystal structures. Curr. Opin. Struct. Biol. 13, 236–243. 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. 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. 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. Leong, K.G., Hu, X., Li, L., Noseda, M., Larrivee, B., Hull, C., Hood, L., Wong, F., and Karsan, A. (2002). Activated Notch4 inhibits angiogenesis: role of beta 1-integrin activation. Mol. Cell. Biol. 22, 2830–2841. Miranti, C.K., and Brugge, J.S. (2002). Sensing the environment: a historical perspective on integrin signal transduction. Nat. Cell Biol. 4, E83–E90. Mu¨ller, M., Weiszäcker, E., and Campos-Ortega, J.A. (1996). Expression domains of a zebrafish homologue of the Drosophila pairrule gene hairy correspond to primordia of alternating somites. Development 122, 2071–2078. Nu¨sslein-Volhard, C., and Dahm, R. (2002). Zebrafish (Oxford, UK: Oxford University Press). Oates, A.C., and Ho, R.K. (2002). Hairy/E(spl)-related (Her) genes are central components of the segmentation oscillator and display redundancy with the Delta/Notch signaling pathway in the formatoin of anterior segmental boundaries in the zebrafish. Development 129, 2929–2946. 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. Pourquié, O., and Kusumi, K. (2001). When body segmentation goes wrong. Clin. Genet. 60, 409–416. Prince, V.E., Joly, L., Ekker, M., and Ho, R.K. (1998). Zebrafish hox genes: genomic organization and modified colinear expression patterns in the trunk. Development 125, 407–420. 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.

sor of Hairless in Notch mediated signalling during zebrafish somitogenesis. Mech. Dev. 120, 1083–1094. Soriano, P. (1997). The PDGF alpha receptor is required for neural crest cell development and for normal patterning of the somites. Development 124, 2691–2700. Takada, S., Stark, K.L., Shea, M.J., Vassileva, G., McMahon, J.A., and McMahon, A.P. (1994). Wnt-3a regulates somite and tailbud formation in the mouse embryo. Genes Dev. 8, 174–189. 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. Takahashi, Y., Inoue, T., Gossler, A., and Saga, Y. (2003). Feedback loops comprising Dll1, Dll3 and Mesp2, and differential involvement of Psen1 are essential for rostrocaudal patterning of somites. Development 130, 4259–4268. Takke, C., and Campos-Ortega, J.A. (1999). her1, a zebrafish pairrule gene, acts downstream of notch signaling to control somite development. Development 126, 3005–3014. Tallquist, M.D., Weismann, K.E., Hellstrom, M., and Soriano, P. (2000). Early myotome specification regulates PDGFA expression and axial skeleton development. Development 127, 5059–5070. Tam, P.P. (1981). The control of somitogenesis in mouse embryos. J. Embryol. Exp. Morphol. 65, 103–128. Thisse, B., Pflumio, S., Fu¨rthauer, M., Loppin, B., Heyer, V., Degrave, A., Woehl, R., Lux, A., Steffan, T., Charbonnier, X.Q., and Thisse, C. (2001). Expression of the zebrafish genome during embryogenesis (NIH R01 RR15402). ZFIN Direct Data Submission. Topczewska, J.M., Topczewski, J., Shostak, A., Kume, T., SolnicaKrezel, L., and Hogan, B.L. (2001). The winged helix transcription factor Foxc1a is essential for somitogenesis in zebrafish. Genes Dev. 15, 2483–2493. Trinh, L.A., and Stainier, D.Y. (2004). Fibronectin regulates epithelial organization during myocardial migration in zebrafish. Dev. Cell 6, 371–382. van Eeden, F.J.M., Granato, M., Schach, U., Brand, M., FurutaniSeiki, 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., and Nu¨sslein-Volhard, C. (1998). Zebrafish segmentation and pair-rule patterning. Dev. Genet. 23, 65–76. Weinberg, E.S., Allende, M.L., Kelly, C.S., Abdelhamid, A., Murakami, T., Anderman, P., Doerre, O.G., Grunwald, D.J., and Riggleman, B. (1996). Developmental regulation of zebrafish MyoD in wildtype, no tail and spadetail embryos. Development 122, 271–280. Wettstein, D.A., Turner, D.L., and Kintner, C. (1997). The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis. Development 124, 693–702. Wierzbicka-Patynowski, I., and Schwarzbauer, J.E. (2003). The ins and outs of fibronectin matrix assembly. J. Cell Sci. 116, 3269– 3276.

Sato, Y., Yasuda, K., and Takahashi, Y. (2002). Morphological boundary forms by a novel inductive event mediated by Lunatic fringe and Notch during somitic segmentation. Development 129, 3633–3644.

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.

Schauerte, H.E., van Eeden, F.J.M., Fricke, C., Odenthal, J., Strähle, U., and Haffter, P. (1998). Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish. Development 15, 2983–2993.

Yang, J.T., Bader, B.L., Kreidberg, J.A., Ullman-Cullere, M., Trevithick, J.E., and Hynes, R.O. (1999). Overlapping and independent functions of fibronectin receptor integrins in early mesodermal development. Dev. Biol. 215, 264–277.

Schier, A.F., Neuhauss, S.C.F., Helde, K.A., Talbot, W.S., and Driever, W. (1997). The one-eyed pinhead gene functions in mesoderm and endoderm formation in zebrafish and interacts with no tail. Development 124, 327–342. Schubert, M., Holland, L.Z., Stokes, M.D., and Holland, N.D. (2001). Three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) associated with the tail bud: the evolution of somitogenesis in chordates. Dev. Biol. 240, 262–273. Sieger, D., Tautz, D., and Gajewski, M. (2003). The role of Suppres-

Yoon, J.K., and Wold, B. (2000). The bHLH regulator pMesogenin1 is required for maturation and segmentation of paraxial mesoderm. Genes Dev. 14, 3204–3214. Zhang, N., and Gridley, T. (1998). Defects in somite formation in lunatic fringe deficient mice. Nature 394, 374–377. Accession Numbers The integrina5 sequence is deposited with GenBank accession number AY861664.