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2004 Wiley-Liss, Inc. Key words: Notch; endoderm; ... et al., 1998; Appel and Eisen, 1998;. Park and Appel, 2003), as well as the decision between hypochord ...

DEVELOPMENTAL DYNAMICS 229:756 –762, 2004

RESEARCH ARTICLE

Notch Signaling Can Regulate Endoderm Formation in Zebrafish Yutaka Kikuchi,1,2* Heather Verkade,2 Jeremy F. Reiter,2 Cheol-Hee Kim,3 Ajay B. Chitnis,3 Atsushi Kuroiwa,1 and Didier Y.R. Stainier2

Early in vertebrate development, the processes of gastrulation lead to the formation of the three germ layers: ectoderm, mesoderm, and endoderm. The mechanisms leading to the segregation of the endoderm and mesoderm are not well understood. In mid-blastula stage zebrafish embryos, single marginal cells can give rise to both endoderm and mesoderm (reviewed by Warga and Stainier [2002] The guts of endoderm formation. In: Solnica-Krezel L, editor. Pattern formation in zebrafish. Berlin: Springer-Verlag. p 28 – 47). By the late blastula stage, however, single marginal cells generally give rise to either endoderm or mesoderm. To investigate this segregation of the blastoderm into cells with either endodermal or mesodermal fates, we analyzed the role of Notch signaling in this process. We show that deltaC, deltaD, and notch1 are expressed in the marginal domain of blastula stage embryos and that this expression is dependent on Nodal signaling. Activation of Notch signaling from an early stage leads to a reduction of endodermal cells, as assessed by sox17 and foxA2 expression. We further find that this reduction in endoderm formation by the activation of Notch signaling is preceded by a reduction in the expression of bonnie and clyde (bon) and faust/gata5, two genes necessary for endoderm formation (Reiter et al. [1999] Genes Dev 13:2983–2995; Reiter et al. [2001] Development 128:125–135; Kikuchi et al. [2001] Genes Dev 14:1279 –1289). However, activation of Notch signaling in bon mutant embryos leads to a further reduction in endodermal cells, also arguing for a bon-independent role for Notch signaling in endoderm formation. Altogether, these results suggest that Notch signaling plays a role in the formation of the endoderm, possibly in its segregation from the mesoderm. Developmental Dynamics 229: 756 –762, 2004. © 2004 Wiley-Liss, Inc. Key words: Notch; endoderm; sox17; foxA2; zebrafish Received 29 September 2003; Accepted 6 October 2003

INTRODUCTION During vertebrate development, the endodermal germ layer gives rise to the epithelial lining of the digestive tract as well as its associated organs. In zebrafish, the endoderm originates from the most marginal blastomeres of blastula stage embryos (Warga and Nu¨sslein-Volhard, 1999).

1

Fate mapping analyses show that at the mid-blastula stage single cells can give rise to both endoderm and mesoderm. By the late blastula stage, single cells generally give rise to either endoderm or mesoderm, indicating that these lineages have segregated. However, the mechanisms leading to the segregation of

the endoderm and mesoderm are unknown. Endodermal progenitors are found no more than four cells away from the blastoderm margin intermingled with cells that give rise to some mesodermal derivatives such as the somites and the heart, indicating that endodermal and meso-

Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan Department of Biochemistry and Biophysics, Programs in Developmental Biology, Genetics and Human Genetics, University of California at San Francisco, San Francisco, California 3 Laboratory of Molecular Genetics, NICHD/NIH, Bethesda, Maryland Grant sponsors: NIH; AHA; MEXT (The Ministry of Education, Culture, Sports, Science, and Technology); Grant number: KAKENHI 13138204. Dr. Kim’s present address is Department of Biology, Chungnam National University, Daejeon 305-764, Korea. *Correspondence to: Yutaka Kikuchi, Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan. E-mail: [email protected] 2

DOI 10.1002/dvdy.10483

© 2004 Wiley-Liss, Inc.

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dermal progenitors are intermingled to some degree along the margin of the blastoderm. Genetic analyses in zebrafish have identified three genes (bonnie and clyde [bon], faust [fau], and casanova [cas]) that play essential roles in endoderm formation. bon and fau mutants contain ⬃10% and ⬃60% of the normal number of endodermal cells, respectively (Kikuchi et al., 2000; Reiter et al., 2001), while cas mutants lack all endodermal cells (Alexander et al., 1999). bon, fau, and cas encode a Mixtype homeodomain transcription factor (Kikuchi et al., 2000), the zinc finger transcription factor Gata5 (Reiter et al., 1999), and a high-mobility group (HMG) domain transcription factor (Dickmeis et al., 2001; Kikuchi et al., 2001; Sakaguchi et al., 2001), respectively. More recently, another Mix-type homeodomain transcription factor, Mezzo, was identified to be involved in endoderm formation, in a manner partially redundant with Bon (Poulain and Lepage, 2002). Gene expression analyses and misexpression experiments in wildtype and mutant embryos have revealed that these four transcription factors function downstream of Nodal signaling and upstream of sox17, an HMG transcription factor gene (Alexander and Stainier, 1999) and foxA2, a winged helix/forkhead transcription factor gene (also known as axial; Stra ¨ hle et al., 1993), both of which are expressed in endodermal cells (reviewed in Stainier, 2002). Numerous studies have revealed that Notch signaling plays important roles in cell fate specification during development (reviewed by Artavanis-Tsakonas et al., 1999; Mumm and Kopan, 2000). A mechanism of lateral inhibition involving Notch signaling was proposed to explain the acquisition of different cell fates by neighboring cells. In zebrafish, lateral inhibition has been demonstrated to regulate primary neuron and oligodendrocyte development (Haddon et al., 1998; Appel and Eisen, 1998; Park and Appel, 2003), as well as the decision between hypochord and notochord cell fate (Latimer et al., 2002). bon, mezzo, and fau/gata5 are expressed in all cells around the circumference of the margin, while

cas, sox17, and foxA2 are expressed in the scattered endodermal cells around the marginal domain of the late-blastula and early gastrula stage embryos. The salt-and-pepper pattern of cas, sox17, and foxA2 expression suggests that endodermal cells may be selected by lateral inhibition. In this study, we test whether Notch signaling regulates endoderm formation in the marginal domain. Activation of the Notch signaling pathway by overexpression of Notch intracellular domain (NICD) in zebrafish embryos leads to a reduction in the number of cells expressing sox17 and foxA2. We further show that expression of bon, which functions upstream of sox17 and foxA2, is significantly reduced by the activation of Notch signaling. Our data suggest that Notch signaling may influence the decision to become endoderm.

RESULTS AND DISCUSSION Zebrafish endodermal cells can be identified during gastrulation by their distinctive morphology and expression of sox17 and foxA2. During early gastrulation, these cells are distributed around the margin in a saltand-pepper pattern (Fig. 1A-D). The formation of the zebrafish endoderm depends upon bon, fau/gata5, and mezzo, three transcription factor genes expressed throughout the marginal domain. That only a subset of marginal cells expressing these three transcription factor genes become endodermal suggests that some other mechanism is further restricting the endoderm-forming capacity of marginal cells. The salt-andpepper distribution of endodermal progenitors further suggests that a lateral specification mechanism may participate in this restriction, prompting us to investigate the role of Notch signaling in this process. deltaC (Smithers et al., 2000), deltaD (Haddon et al., 1998), and notch1 (Bierkamp and Campos-Ortega, 1993) are also expressed in this marginal domain during both late blastula and early gastrula stages (Fig. 1E,G,I). Because the marginal expression of bon, fau/gata5, and cas is regulated by Nodal signaling

(reviewed by Stainier, 2002), we investigated whether Nodal signaling also regulates deltaC, deltaD, and notch1 expression. We found that embryos lacking both the maternal and zygotic complement of the Nodal cofactor Oep (MZoep mutants; Gritsman et al., 1999) display no deltaC, deltaD, and notch1 expression, indicating that the expression of these genes is also regulated by Nodal signaling (Fig. 1F,H,J). The expression patterns of deltaC, deltaD, and notch1 during late blastula and early gastrula stages raise the possibility that Notch signaling is involved in the formation of the endoderm and mesoderm. Expression of the Notch intracellular domain activates the Notch signaling pathway in a ligand-independent manner (Fortini et al., 1993; Lieber et al., 1993; Struhl et al., 1993). To investigate the role of Notch signaling in endoderm formation, we injected mRNA encoding the intracellular domain of zebrafish Notch1 (NICD; Takke et al., 1999). Injection of 100 pg of NICD mRNA led to a substantial decrease in the number of sox17- and foxA2-expressing endodermal cells (Fig. 2B,E; Table 1). We also found a reduced number of cas-expressing endodermal cells during early gastrulation stages and reduced endodermal tissues at pharyngula stages after NICD injections (data not shown). Unfortunately, as specific pan-mesodermal markers remain to be identified in zebrafish, we have been unable to analyze directly the role of Notch signaling in mesoderm formation. Instead, we generated embryos consisting primarily of endodermal cells by injecting 500 pg of a constitutively active form of the type-I TGF␤ receptor Taram-a, designated Taram-a* (Peyrieras et al., 1998). We found that, while 18% (39 of 214) of the embryos injected with Taram-a* alone expressed the somitic marker myoD, coinjection with 100 pg of NICD increased that proportion to 28% (66 of 235), suggesting that activation of the Notch signaling pathway can convert endodermal cells to mesodermal cells. Altogether, these results indicate that activation of the Notch signaling pathway during early embryonic stages can sup-

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Fig. 1. Nodal-dependent deltaC, deltaD, and notch1 gene expression in the zebrafish margin. A and C are lateral views, and B and D–J animal pole views, of shield stage (6 hours postfertilization) embryos. All images are oriented with dorsal to the right. A,B: sox17 is expressed dorsally in the forerunner cells (arrows) and in the endodermal cells (arrowheads). C,D: foxA2 is expressed in the axial mesoderm (arrows) and in the endodermal cells (arrowheads). E,G,I: deltaC, deltaD, and notch1 are expressed in the marginal domain. F,H,J: Marginal deltaC, deltaD, and notch1 expression is absent in MZoep mutants. Note the low level of notch1 expression in MZoep mutants, which is likely to be due to maternal message. wt, wild-type.

Fig. 2. Notch signaling suppresses endodermal sox17 and foxA2 expression. Dorsal views (anterior to the top) of 80% epiboly stage embryos (8.3 hours postfertilization). A–F: Embryos were injected with 100 pg of lacZ (A,D), notch1 intracellular domain (NICD, B,E), or her1 (C,F) mRNA at the one- to four-cell stage. B,E: NICD mRNA overexpression reduced the number of sox17- and foxA2-expressing endodermal cells. C,F: her1 mRNA overexpression also reduced the number of sox17-expressing endodermal cells but did not appear to affect endodermal foxA2 expression.

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TABLE 1. Effects of Notch Signaling on Endodermal sox17 and foxA2 Expressiona NICD

Number of embryos injected Number of embryos showing decreased expression of marker gene Number of embryos showing increased expression of marker gene

XSu(H)/ANK

her1

sox17

foxA2

sox17

foxA2

sox17

foxA2

105 104 (99%)

64 62 (97%)

100 92 (92%)

67 65 (97%)

77 77 (100%)

62 3 (5%)

0

0

0

0

0

0

a

Embryos at 80% epiboly (8.3 hours postfertilization) were scored as demonstrating decreased sox17 expression if they exhibited less than half as many sox17-expressing endodermal cells as control siblings, and scored as demonstrating increased sox17 expression if they exhibited a greater than 10% increase in endodermal cells.

press endoderm formation, and at some frequency activate somitic mesoderm formation. To further analyze the potential role of Notch signaling on endoderm formation, we tested the effects of two different Notch effectors in these processes. XSu(H)1/ANK encodes a constitutively active form of Xenopus Suppressor of Hairless [Su(H)], a downstream effector of Notch signaling (Wettstein et al., 1997). Overexpression of XSu(H)1/ ANK, like that of NICD, led to a substantial decrease in the number of cas-, sox17-, and foxA2-expressing endodermal cells (Table 1; data not shown). These data show that Notch signaling can affect endoderm formation by acting through Su(H). Zebrafish hairy/Enhancer of split homologue her1 is expressed in the entire marginal domain at late blastula and early gastrula stage embryos (Mu¨ller et al., 1996). Activation of Notch signaling causes ectopic her1 expression in the presomitic mesoderm, indicating that, at least during somite formation, her1 is downstream of Notch (Takke and Campos-Ortega, 1999). These data together suggest that her1 may also be involved in endoderm formation in blastula and gastrula stage embryos. Overexpression of her1 mRNA (100 pg) reduced the number of casand sox17-expressing endoderm cells (Fig. 2C; Table 1; data not shown), whereas the number of foxA2-expressing endodermal cells appeared unaffected in her1-injected embryos (Fig. 2F; Table 1). These data indicate that Her1 may play a role downstream of Notch signaling in the formation of the endoderm.

Other her genes such as her4 (Takke et al., 1999) are also expressed in the marginal domain at late blastula and early gastrula stages (J.F.R. and D.Y.R.S., unpublished observations) and may function downstream of Notch signaling to regulate foxA2 expression. Alternatively, NICD might also affect these processes by regulating the activity of other transcription factors such as LEF-1 (Ross and Kadesch, 2001), although it is important to note that overexpressing NICD and XSu(H)1/ANK leads to similar phenotypes. bon and fau/gata5 regulate the endodermal expression of sox17 and foxA2, but are expressed throughout the marginal domain (Reiter et al., 1999; Kikuchi et al., 2000). We next tested whether Notch signaling can regulate bon and fau/gata5 expression in early gastrula stage embryos. Overexpression of NICD dramatically reduced bon expression and slightly reduced fau/gata5 expression at 50% epiboly (Fig. 3B,E). By contrast, her1 overexpression did not affect bon or fau/gata5 expression at 50% epiboly (Fig. 3C,F). These data suggest that NICD inhibits endodermal expression of cas, sox17, and foxA2 by down-regulating bon expression. her1, whose expression can be controlled by Notch signaling, does not appear to regulate bon and fau/gata5 expression, but can affect cas and sox17 expression. We next tested whether Notch signaling required bon function to regulate endoderm formation. We used homozygous bon mutant fish to generate bon-/- embryos and injected them with 100 pg of NICD. We observed that overexpres-

sion of NICD in bon mutant embryos led to a further reduction in the number of sox17-expressing endodermal cells (Fig. 4E,F), further arguing for a bon-independent role of Notch signaling in endoderm formation. Our data show that the activation of Notch signaling throughout the early zebrafish embryo can suppress endoderm formation. We also tested the effects of blocking Notch signaling on endoderm formation. Overexpression of a dominant negative form of Xenopus Delta-1 (DN X-Delta-1; Chitnis et al., 1995; Appel and Eisen, 1998), injection of morpholino antisense oligonucleotides (MOs) specific to either deltaC or deltaD, and coinjection of deltaC and deltaD MOs (Holley et al., 2002) did not appear to increase the number of endodermal cells (data not shown). Presenilin (␥-secretase) is required for the processing of Notch, and its inhibition blocks Notch signaling (reviewed by Kopan and Goate, 2000; Fortini, 2001). Overexpression of a dominant negative form of presenilin (DN presenilin) (Leimer et al., 1999) or treating early embryos with the ␥-secretase inhibitor DAPT (N-[N(3,5-difluorophenacetyl-L-alanyl)]-Sphenylglycine t-butyl ester; Dovey et al., 2001; Geling et al., 2002) did not appear to increase the number of endodermal cells either (data not shown). In summary, activating Notch signaling during early zebrafish development led to a reduction in the number of endodermal cells. However, our attempts to block Notch signaling did not result in an increase in the number of endodermal cells. It is possible that other mechanisms

760 KIKUCHI ET AL.

Fig. 3.

Fig. 4.

NOTCH SIGNALING AND ENDODERM FORMATION 761

are at play to regulate the number of endodermal cells and that blocking Notch signaling is not sufficient to overcome them. Whether Notch signaling is really implicated in the segregation of endoderm and mesoderm through a lateral inhibition process remains unclear and will require the development of techniques that allow the activation and blocking of Notch signaling in single cells at the time when the decision to become endoderm or mesoderm is being made, as well as the ability to monitor the fate of these single cells and their progeny. Alternatively, it is possible that Notch signaling regulates the temporal pattern of bon expression and, thereby, the number of endodermal cells generated. bon expression initiates at the mid-blastula stage and becomes undetectable by 60% epiboly. Our data show that premature activation of Notch signaling leads to a premature reduction of bon expression and a subsequent decrease in the number of endodermal cells; thus endogenous Notch signaling might turn off bon expression and thereby regulate endoderm formation. In addition and as shown by the further reduction of endodermal cells in bon mutant embryos in-

Fig. 3. Notch signaling inhibits bon expression. A–F: Embryos were injected with 100 pg of lacZ (A,D), NICD (B,E), or her1 (C,F) mRNA at the one- to four-cell stage, and examined for bon or fau/gata5 expression at 50% epiboly (5.3 hours postfertilization). A–F: Animal pole views, dorsal to the right. B,E: NICD overexpression dramatically reduced bon expression and slightly reduced fau/gata5 expression. C,F: Unlike NICD, her1 overexpression did not affect bon or fau/gata5 expression. Fig. 4. Notch signaling also functions independently of bon function in endoderm formation. Dorsal views (anterior to the top) of 80% epiboly stage embryos (8.3 hours postfertilization). Embryos are wild-type (wt, A,B,C) or from an incross of bon-/- fish (D,E,F). Embryos were left uninjected (A,D), or injected with 100 pg of NICD mRNA at the one- to four-cell stage (B,C,E,F). A,D: The loss of bon function reduces the number of sox17 expressing endodermal cells. B,C: NICD overexpression also reduces the number of sox17 expressing endodermal cells. E,F: NICD overexpression in bon mutants further reduces the number of sox17 expressing endodermal cells.

jected with NICD, Notch signaling may also regulate endoderm formation in a bon-independent manner.

and treated with 100 ␮M DAPT from the one-cell stage until 80% epiboly (8.3 hours postfertilization).

EXPERIMENTAL PROCEDURES

ACKNOWLEDGMENTS

Zebrafish Strains

We thank Alex Navarro and Steven Waldron for invaluable technical assistance; Le Trinh for bon homozygous fish; Jose ` Campos-Ortega, Sharon Amacher, and Julian Lewis for DNA templates; and Jonathan Alexander, Yee-Ming Chan, Xin Sun, Mike Verzi, and Pia Aanstad for discussions and comments on the manuscript. Y.K. was funded by Postdoctoral Fellowships from the American Heart Association and the Human Frontier Science Program (HFSP). H.V. was funded by a Postdoctoral Fellowship from the HFSP. J.F.R. was a member of the Medical Scientist Training Program of the NIH and an Achievement Rewards for College Scientists scholarship recipient. C.-H. Kim was supported by a Vascular System Research Center grant from Korea Science and Engineering Foundation. This work was supported in part by grants to D.Y.R.S. from the American Heart Association, the Packard Foundation and the Life and Health Insurance Medical Research Fund, and by a Grant-in-Aid for Scientific Research to Y.K. from The Ministry of Education, Culture, Sports, Science, and Technology (MEXT).

Adult zebrafish were maintained and staged as described (Westerfield, 1995). Adult MZoepm134 fish were a generous gift of Drs. Will Talbot (Stanford University) and Marnie Halpern (Carnegie Institution of Washington). The bonm425 (Kikuchi et al., 2000) mutant allele was used and bonm425 homozygous fish were created by injecting wild-type bon mRNA into homozygous mutant embryos at the one- to four-cell stage to rescue embryonic defects.

Whole-Mount In Situ Hybridization Whole-mount in situ hybridization was performed as described (Kikuchi et al., 1997; Alexander et al., 1998), and riboprobes prepared according to published instructions.

mRNA and Morpholino Injections Capped mRNA was synthesized by using the SP6 mMessage mMachine system (Ambion) from the following previously described templates: NICD (Takke et al., 1999), XSu(H)1/ ANK (Wettstein et al., 1997), her1 (Takke and Campos-Ortega, 1999), Taram-a* (Peyrieras et al., 1998), DN X-Delta-1 (Chitnis et al., 1995; Appel and Eisen, 1998), DN presenilin (Leimer et al., 1999). For overexpression experiments, mRNAs of NICD (100 pg), XSu(H)1/ANK (100 pg), her1 (100 pg), Taram-a* (500 pg), DN X-Delta-1 (300 pg), DN presenilin (400 pg), and lacZ (100 pg) were microinjected at the one- to four-cell stage. Injections of morpholino antisense oligonucleotides for deltaC and deltaD were carried out as described (Holley et al., 2002).

DAPT Treatments DAPT treatments were performed as described previously (Geling et al., 2002): wild-type embryos were dechorionated at the one-cell stage

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