casanova Plays an Early and Essential Role in Endoderm Formation in Zebrafish

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Developmental Biology 215, 343–357 (1999)Article ID dbio.1999.9441, available online at http://www.idealibrary.com on

casanova Plays an Early and Essential Rolein Endoderm Formation in Zebrafish

Jonathan Alexander,* Michael Rothenberg,* Gilbert L. Henry,†and Didier Y. R. Stainier*,1

*Programs in Developmental Biology and Human Genetics, Department of Biochemistry andBiophysics, University of California at San Francisco, San Francisco, California 94143-0448;and †Department of Molecular and Cellular Biology, Harvard University,7 Divinity Avenue, Cambridge, Massachusetts 02138

The cellular and molecular mechanisms that regulate endoderm development in vertebrates have only recently begun to beexplored. Here we show that the zebrafish locus casanova plays an early and essential role in this process. casanova mutantsack a gut tube and do not express any molecular markers of endoderm differentiation. The early endodermal expression ofenes such as axial, gata5, and fkd2 does not initiate in casanova mutants, indicating that the endoderm is defective fromhe onset of gastrulation. Mosaic analysis demonstrates that casanova functions cell autonomously within the endodermal

progenitors. We also report the isolation of a zebrafish homologue of Mixer, a gene important for early endoderm formationn Xenopus. casanova does not encode zebrafish Mixer, and mixer expression is normal in casanova mutants, indicatinghat casanova acts downstream of, or parallel to, mixer to promote endoderm formation. We further find that the forerunnerells, a specialized group of noninvoluting dorsal mesendodermal cells, do not form in casanova mutants. Studies ofasanova mutants do not support an important role for the forerunner cells in either dorsal axis or tail development, as haseen previously proposed. In addition, although different populations of mesodermal precursors are generated normally inasanova mutants, morphogenetic defects in the heart, vasculature, blood, and kidney are apparent, suggesting a possibleole for the endoderm in morphogenesis of these organs. © 1999 Academic Press

Key Words: gut; axial; gata5; mixer; forerunner cells; Kupffer’s vesicle; cardia bifida; endothelium; knypek; one-eyed
pinhead.
INTRODUCTION

The three fundamental germ layers of the vertebrateembryo—ectoderm, mesoderm, and endoderm—form dur-ing gastrulation. The induction and patterning of the ecto-derm and mesoderm have been studied extensively, result-ing in a detailed though still incomplete understanding ofhow these tissues arise (Kessler and Melton, 1994; Slack,1993). In contrast, development of the endoderm, whichforms the gut tube, its associated organs such as the liverand pancreas, and the lining of the respiratory tract, hasuntil recently been relatively unexplored.

Most of our knowledge about endoderm developmentcomes from studies of the amphibian Xenopus laevis. Theendoderm in Xenopus arises from the yolk-rich cells of the

1 To whom correspondence should be addressed. Fax: (415) 476-3892. E-mail: [email protected].

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vegetal hemisphere (Dale and Slack, 1987). These cellscommit to an endodermal fate by early in gastrulation, butprior to this stage they can be redirected to other fates byvarious experimental manipulations (Heasman et al., 1984;Henry et al., 1996; Wylie et al., 1987). Importantly, substan-tial endodermal differentiation occurs in isolated Xenopusvegetal pole explants (Gamer and Wright, 1995; Henry etal., 1996; Jones et al., 1993), suggesting that the endodermforms through a process that is largely cell and/or tissueautonomous.

Certain growth factors that induce mesoderm can alsoinduce endoderm. For example, the related transforminggrowth factor (TGF)-b superfamily members Activin andVg1 are capable of inducing the expression of severalendodermal markers in isolated Xenopus animal caps(Gamer and Wright, 1995; Henry et al., 1996; Jones et al.,
1993); experiments using an inhibitory Vg1 ligand confirman endogenous role for a Vg1-like activity in dorsal
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endoderm formation in Xenopus (Joseph and Melton, 1998).ibroblast growth factors and the secreted bone morphoge-etic protein antagonists chordin and noggin may alsounction in Xenopus endoderm induction (Henry et al.,996; Jones et al., 1993; Sasai et al., 1996). Together these

observations suggest a general model in which the highlevels of mesoderm inducers produced by vegetal cellscreate a local signaling environment that directs thesevegetal cells themselves to an endodermal fate.

A critical player in Xenopus endoderm induction is theT-box transcription factor VegT. Ectopic expression of VegTin animal caps causes expression of several endodermalmarkers (Horb and Thomsen, 1997). Conversely and signifi-cantly, depletion of the vegetally localized maternal depositof VegT using antisense oligonucleotides blocks endodermformation entirely (Zhang et al., 1998). These results fur-ther support the idea that Xenopus endoderm inductionoccurs at least tissue-autonomously. How the VegT-regulated zygotic genes interact with the signaling path-ways described above to induce endoderm formation is notknown.

Several recently identified zygotically expressed genesmay act within the presumptive endoderm in response toinducers such as Activin. Two Xenopus homologues of themouse Sox17 gene, Xsox17a and Xsox17b, are capable ofdirecting presumptive ectodermal tissue to an endodermalfate (Hudson et al., 1997). Expression of Xsox17 becomesestricted to the endoderm at the onset of gastrulation ands induced in animal caps by treatment with activin (Hud-on et al., 1997). Overexpression of Xsox17 results in highevels of endodermal marker expression in isolated Xenopusnimal caps, while overexpression of a fusion of Xsox17 andhe repressor domain of Drosophila Engrailed (EnR) inhibits

the expression of such markers in both vegetal pole ex-plants and activin-treated animal caps (Hudson et al., 1997).Mix homeobox genes also appear to play an important rolein endoderm formation. Several such genes have beenisolated in Xenopus, all of which show endodermal expres-sion and are induced in animal caps by Activin treatment(Ecochard et al., 1998; Henry and Melton, 1998; Rosa, 1989;Tada et al., 1998). Expression of at least some Mix genes isalso induced by VegT. Ectopic overexpression of Mix genesresults in different degrees of endodermal gene expressionin isolated Xenopus animal caps; two in particular, Mixerand milk, induce high levels of endodermal marker expres-sion (Ecochard et al., 1998; Henry and Melton, 1998).Experiments using Mixer–EnR and Xsox17–EnR fusionsstrongly suggest that Mixer likely promotes endodermdevelopment principally or perhaps entirely throughXsox17 (Henry and Melton, 1998). The maintenance ofXsox17 expression in the presumptive endoderm by Mixer(and perhaps other Mix proteins) therefore likely representsa critical early event in endoderm formation.

Mutational analyses have identified few genes essentialfor vertebrate endoderm formation. Tetraploid embryo-ES
cell aggregation experiments in mouse demonstrate anessential role for the transcription factor HNF3b in fore-
Copyright © 1999 by Academic Press. All right

nd midgut development (Dufort et al., 1998). Zebrafishygotic one-eyed pinhead (oep) mutants lack endoderm asell as prechordal plate and ventral neuroectoderm (Schier

t al., 1997). oep encodes a member of the EGF–CFC proteinamily that appears to act as an essential cofactor inignaling by nodal-related growth factors (Gritsman et al.,999). Also, zebrafish embryos mutant for both squint andyclops, two genes that encode nodal-related growth fac-ors, form essentially no mesendoderm (Feldman et al.,998). Zebrafish embryos lacking both maternal and zygoticne-eyed pinhead protein display an identical phenotype

Gritsman et al., 1999). In these cases involution does notccur, however, leaving it unclear whether these factorsirectly induce mesendodermal fates or promote the cellovements necessary for mesendoderm formation during

astrulation.In this report we demonstrate an essential role for the

ebrafish locus casanova (cas) in endoderm development.as mutants appear to lack endoderm entirely from thenset of gastrulation. cas functions cell autonomously

within the endodermal progenitors and acts either down-stream of, or parallel to, a zebrafish Mixer homologue. casmutants also appear to lack forerunner cells and displaymorphogenetic defects in several mesodermal derivatives.

MATERIALS AND METHODS

StrainsAdult zebrafish and embryos were maintained and staged as

described (Westerfield, 1995). The casta56 and knypekm119 (kny)mutations were identified in screens for ENU-induced embryonic-lethal mutations (Chen et al., 1996; Solnica-Krezel et al., 1996).

Phenotypic AnalysisIn situ hybridizations were performed as described (Alexander et

l., 1998). For sectioning, embryos were embedded in JB4 (Poly-ciences) and counterstained with neutral red. Labeling of forerun-er cells with syto-11 was performed as described (Cooper and’Amico, 1996). Photographs were taken on either a Leica MZ12

tereomicroscope or a Zeiss Axioplan using Kodak Ektachrome60T or Fujichrome 1600 ASA film and processed using AdobehotoShop 4.0.

Cell TransplantationCell transplantations were performed essentially as described

(Ho and Kimmel, 1993). Cells were transplanted from labeled donorto unlabeled host embryos at mid- to late-blastula stages. Hostembryos were fixed at approximately 80% epiboly (midgastrulastage), and donor embryos were raised to determine their genotype.Host embryos were examined for expression of the axial gene, andiotin-labeled donor cells were subsequently detected using theBC peroxidase kit (Vector Laboratories, Inc.).

Isolation of mixer
A fragment of mixer was isolated from midgastrula-stage cDNA
using degenerate PCR primers 59-CCCGAGTGCAGGTGTG-

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345Zebrafish Endoderm Formation Requires casanova

GTTYCARAA-39 and 59-GGTGTTCATGTCGGGTGTG-TNDTYTTRTT-39 and standard touchdown PCR protocols.

Gene-specific primers were then used to screen a gastrula-stagecDNA library by PCR for a full-length mixer clone. The GenBankaccession number for mixer is AF121771.

Linkage Analysis

We identified a single-strand conformational polymorphism inthe mixer 39 untranslated region (UTR) in a line containing thecasta56 allele. casta56 does not segregate with mixer (data not shown)and therefore cas does not encode Mixer.

RESULTS

cas Mutants Lack a Gut Tube

The cas locus is defined by a single recessive allele,asta56, identified in a large-scale screen for mutations that

affect zebrafish embryonic development (Chen et al., 1996).cas mutants are first identifiable at approximately 24 hpostfertilization (hpf) by the presence of cardia bifida—bilateral hearts resulting from a failure of cardiac fusion tooccur. cas mutants exhibit pericardial edema and collapsedbrain ventricles, common to all zebrafish cardiac mutants,as well as a thickened yolk extension (Figs. 1A and 1B). Thecas mutation was originally classified as affecting heartformation (Chen et al., 1996). Light microscopic examina-tion at 36 hpf and later stages, however, reveals that casmutants entirely lack a gut tube. The absence of a gut tubeis most easily seen just behind the yolk extension where theintestine exits the body at the anal opening, immediatelyanterior to the pronephric ducts (Fig. 1C). cas mutants haveneither an anal opening nor an intestine, nor is a well-formed pronephric duct visible (Fig. 1D). Small cysts formposterior to the yolk extension in cas mutants (Fig. 1D). Wehypothesize that these cysts result from the failure of thepronephric ducts to form normally in the absence of a guttube (see below).

Histological sections of 48-hpf embryos confirm the ab-sence of a gut tube in cas mutants. While the sonichedgehog (shh)-expressing gut tube is clearly visible be-tween the notochord and the yolk in wild-type embryos(Fig. 1E), in cas mutants the notochord is positioned almostdirectly atop the yolk (Fig. 1F). Floor-plate cells in casmutants express shh normally (Figs. 1E and 1F), but noother shh-expressing cells are seen, suggesting that theendoderm is absent in cas mutants.

cas Mutants Do Not Express Molecular Markersof Endoderm Differentiation

In order to test further whether any endoderm is presentin cas mutants we examined the expression of variousendoderm markers. Several genes that encode transcriptionfactors related to Drosophila Forkhead are expressed in the
endoderm during zebrafish development (Odenthal andNusslein-Volhard, 1998). axial, a zebrafish homologue of

ouse HNF3b (Strahle et al., 1993), is expressed in theanterior endoderm and the ventral neuroectoderm duringsomitogenesis (Fig. 2A). No endodermal axial expression isdetectable in cas mutants, although neural expression ofxial appears normal (Fig. 2B). Two other forkhead-relatedenes, fkd7 and fkd2, are expressed in the endoderm as wells in subpopulations of the neural crest and axial mesodermFig. 2C) (Odenthal and Nusslein-Volhard, 1998). Again cas

utants specifically lack endodermal expression of fkd7nd fkd2 (Fig. 2D and data not shown). Additionally, fkd7xpression reveals that the hypochord forms but is short-ned posteriorly in cas mutants (Fig. 2D).The gata genes encode zinc finger-containing transcrip-

ion factors, several of which are expressed in the develop-ng gut and heart (Laverriere et al., 1994). We examined thexpression of gata4 in wild-type and cas mutant embryosuring late somitogenesis. At these stages wild-type em-ryos express gata4 in the myocardium as well as a regionf the endoderm from which the liver will later developFig. 2E). Cardiac expression of gata4 is evident in cas

utants, demonstrating cardia bifida, but no endodermalata4 expression is seen (Fig. 2F). Examination of gata6xpression in wild-type and cas mutant embryos yieldedimilar results (data not shown). Thus, we see no molecularvidence for the presence of endoderm in cas mutantsuring somitogenesis stages.

The Endoderm Is Defective in cas Mutantsfrom the Onset of Gastrulation

Endodermal expression of axial initiates soon after theonset of gastrulation (Fig. 3A) and is maintained throughoutgastrulation (Fig. 3C). These axial-expressing cells are iden-tifiable as endodermal precursors by their close appositionto the yolk and their large flattened morphology (Warga andNusslein-Volhard, 1999). cas mutants specifically lackendodermal axial expression, while axial expression in theprechordal plate and notochord appears normal (Figs. 3B and3D). These data indicate that the endoderm in cas mutantsis defective when the hypoblast first forms.

The zebrafish gata5 homologue has recently been iso-lated and shown to be expressed in endodermal precursorsas the hypoblast forms in the early gastrula (Rodaway et al.,1999). We therefore examined gata5 expression in wild-typeand cas mutant embryos. By midgastrulation gata5 expres-sion appears in endodermal precursors distributed through-out the forming hypoblast (Fig. 3E). This endodermal gata5expression is strikingly absent in cas mutants (Fig. 3F).Endodermal expression of fkd2 (Odenthal and Nusslein-Volhard, 1998) also initiates during gastrulation (Fig. 3G),but again is specifically absent in cas mutants (Fig. 3H).These data reinforce the conclusion that the endoderm in
cas mutants is defective, and perhaps entirely absent, fromthe onset of gastrulation.

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cas Functions Cell Autonomously in the EndodermThe above results demonstrate that the endoderm is abnor-

mal in cas mutants from a very early stage of development.his defect could result from a failure by cas mutants to

FIG. 1. cas mutants lack a gut tube. Nomarski optical images (A–48 hpf (E, F). Compared to a wild-type sibling (A), the cas mutanasterisks), and a thicker yolk extension (arrow). In wild-type emrrowhead) are visible, immediately anterior to the pronephric duvident in cas mutants (D); arrowhead indicates the cyst present in

notochord (arrowhead) and the yolk in wild-type embryos. In cas mand the notochord (arrowhead) rests nearly upon the yolk. Expressiembryos. (A–D) Lateral views, anterior to the left and dorsal to th

enerate endoderm-inducing signals. Alternatively, the pre-umptive endodermal progenitors in cas mutants may fail to

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eceive or to respond to such signals. In order to test directlyhere cas functions in endoderm development we used cell

ransplantation to create genetic mosaics (Ho and Kimmel,993). We then assessed the behavior of wild-type cells trans-

d histological sections (E, F) of embryos at 24 (A, B), 36 (C, D), andshows pericardial edema (arrowhead), collapsed brain ventricles

s (C) the still-forming intestine (arrow) and anal opening (black(white arrowhead). Neither the intestine nor the anal opening ismutants. (E) The shh-expressing gut tube (arrow) sits between thents (F) no gut tube or shh-expressing endodermal cells are evidentshh in the floor plate is evident in both wild-type and cas mutant

; (E, F) transverse sections through the upper trunk and yolk ball.

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lanted into cas mutant hosts and vice versa by analyzing thexpression of axial. Wild-type cells transplanted into cas


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mutant hosts can form endoderm, as assayed by their expres-sion of axial, their endodermal morphology (Warga andNusslein-Volhard, 1999), and their lateral location in theembryo (Fig. 4). In contrast, cas mutant cells were neverobserved to form endoderm (Fig. 4 and data not shown). These

FIG. 2. cas mutants do not express molecular markers of endoderwere examined at the 25-somite stage (21.5 hpf) for expression ofentral neuroectoderm and anterior endoderm (arrowhead); endoderata4 are expressed in the endoderm of wild-type (arrowheads in C,late and hypochord (C), which is posteriorly shortened in cas mutut tube that will form the liver. Myocardial gata4 expression (arroA–D) Lateral views, anterior to the left, dorsal to the top; (E, F) do

experiments demonstrate that cas functions cell-auto-nomously within the endoderm to allow its proper development. (


A Zebrafish Mixer Homologue Is ExpressedNormally in the Prospective Mesendodermin cas Mutants

The homeobox gene Mixer has recently been shown to

fferentiation. Wild-type (A, C, E) and cas mutant (B, D, F) embryos(A, B), fkd7 (C, D), and gata4 (E, F). (A) axial is expressed in the

axial expression is absent from cas mutants (B). Similarly, fkd7 andt not cas mutant (D, F) embryos. fkd7 is also expressed in the floor(arrow in D). Endodermal gata4 expression defines a region of theE) illustrates the cardia bifida present in cas mutants (arrows in F).views, anterior to the left.

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play a key early role in Xenopus endoderm formationHenry and Melton, 1998). The early occurrence of the cas



Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.

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endoderm defect and the cell autonomy of cas function inhe endoderm led us to examine the relationship betweenas and Mixer. We used degenerate PCR to isolate a ze-rafish mix gene (Fig. 5A). BLAST searches (Altschul et al.,990) suggest that this gene is most closely related toenopus Mixer, and we therefore refer to it provisionally asixer. The zebrafish Mixer homeodomain shows 69%

dentity with that of chick CMIX (Peale et al., 1998; Stein etl., 1998) and resembles about equally those of Xenopusixer (58% identity) and Milk (56% identity) (Fig. 5B). We

sed a SSCP polymorphism in the mixer 39 UTR to analyzeinkage with cas. We found that they are not linked,emonstrating that cas does not encode zebrafish Mixer

data not shown).We next examined mixer expression. In wild-type em-

bryos we first detect mixer expression at the sphere stage ina small group of dorsal cells (Fig. 5C). By dome stage mixerexpression has spread circumferentially throughout themarginal zone and also appears in the dorsal YSL (Fig. 5D).At subsequent stages we do not detect mixer expression inhe YSL. Expression in the marginal zone persists throughhe onset of gastrulation (Figs. 5E and 5F). Soon afterwardixer expression is downregulated, and by 60% epiboly is

ndetectable (data not shown). mixer expression in casutants is indistinguishable from that seen in wild-type

mbryos at all stages (data not shown).We also compared mixer expression to two other genes

xpressed in the marginal zone of the pregastrula zebrafishmbryo, no tail (ntl) and gata5. ntl encodes the zebrafishomologue of mouse Brachyury (Schulte-Merker et al.,994) and is expressed in all cells that will involute to formhe hypoblast (i.e., both endoderm and mesoderm) (Fig. 5G)Schulte-Merker et al., 1992). Prior to the onset of gastrula-ion gata5 is expressed in a subset of the marginal zonerom which all of the endoderm as well as some mesoderm

FIG. 3. The endoderm is defective in cas mutants from the onsetwild-type (A, C, E, G) and cas (B, D, F, H) mutant embryos at shiethe onset of gastrulation (A) axial is expressed in the embryonicEndodermal axial expression is absent in cas mutants, while expresjust outside the shield in the cas mutant are likely notochormidgastrulation wild-type embryos express axial in the endodermaxpression of axial is seen in cas mutants (D). gata5 expression alsout not cas mutant (F) embryos. gata5 expression in the anterior lormal in cas mutants (F). fkd2 is expressed in endodermal precursas mutants specifically lack endodermal fkd2 expression (H). (A, Borsal views, anterior to the top.IG. 4. cas acts cell autonomously in the endodermal progenitorransplanted contains several axial-expressing endodermal precursoonor as they also contain the biotin dextran lineage tracer (browns judged by axial expression. Under higher magnification (C) the ps clearly seen (arrowheads indicate brown cells with purple cytoarrows indicate brown cells). In 53 wild-type to wild-type controormed axial-expressing endoderm (data not shown). Wild-type cell
n 5 of 77 cases (P 5 0.5–0.9). cas mutant cells were never observed to fosts (33 events; P , 0.1). (A, B) Right lateral views, anterior to the top

ill emerge (Fig. 5H) (Rodaway et al., 1999; Warga andusslein-Volhard, 1999). This pregastrula phase of gata5

xpression is normal in cas mutants (data not shown). Theixer expression domain appears quite similar to that of ntl

nd includes substantially more of the marginal zone thanhe gata5 expression domain (compare Figs. 5F–5H). Thus,xpression of zebrafish mixer, unlike that of its Xenopusomologue, is not restricted to the prospective endoderm.

cas Mutants Lack Forerunner Cells

The forerunner cells (FRs) first appear as a group of highlyendocytic cells located at the dorsal margin of the late-blastula zebrafish embryo (Cooper and D’Amico, 1996).From there they migrate along the YSL in front of theadvancing blastoderm margin and upon completion of epi-boly come to occupy a position deep within the tailbud atthe chordoneural hinge (Cooper and D’Amico, 1996; Melbyet al., 1996). Shortly thereafter the FR cluster expands toform Kupffer’s vesicle, a fluid-filled sac unique to theteleost tailbud (Cooper and D’Amico, 1996). Late in somi-togenesis Kupffer’s vesicle disappears and the progeny ofthe FRs contribute to the notochord, muscle, and mesen-chyme of the tail (Melby et al., 1996).

The FRs have been proposed to represent the endodermalaspect of the neurenteric canal, a transiently existent spacethat connects the ependymal canal (the lumen of the spinalcord) to the anus at the end of gastrulation in numerouschordates (Cooper and D’Amico, 1996; Gont et al., 1993).

his hypothesis implies that the FRs are endodermal inrigin. Given the absence of other endodermal derivativesn cas mutants, we examined Kupffer’s vesicle, which theRs normally form (Fig. 6A), in embryos derived from aas/1 heterozygote intercross. Light microscopic observa-ion of embryos at the six-somite stage revealed that

strulation. axial (A–D), gata5 (E, F), and fkd2 (G, H) expression in, B), 80% epiboly (C–F), and 90% epiboly (G, H) stages. Soon afterld and endodermal precursors located throughout the hypoblast.n the embryonic shield is normal (B); the few axial-expressing cellsecursors that have not yet completed dorsal convergence. Atursors and the prechordal plate and notochord (C); no endodermaltifies endodermal precursors within the hypoblast of wild-type (E)

l mesoderm precursors and YSL, which is out of focus (E), appearswild-type embryos, as well as in the YSL and axial mesoderm (G).

imal pole views; (C, D) left lateral views, anterior to the top; (E–H)

cas mutant host at 80% epiboly into which wild-type cells werethe lateral hypoblast (A). These cells all derive from the wild-type

n in B); no mutant host cell was ever observed to form endoderm,ce of biotin dextran in the axial-expressing endodermal precursors); several cells not expressing axial also contain biotin dextran

splantations, we observed four cases in which transplanted cellssplanted into cas mutant hosts formed axial-expressing endoderm

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FIG. 5. Sequence and expression of a zebrafish Mixer homologue. (A) Predicted amino acid sequence of zebrafish Mixer; the homeodomainand a C-terminal acidic domain are underlined. (B) Comparison of the homeodomains of zebrafish Mixer (Mixer), CMIX, Xenopus Mixer(XMixer), and Milk; dashes indicate conserved residues. Expression of mixer (C–F), ntl (G), and gata5 (H) in wild-type embryos at sphere (C),dome (D), and 50% epiboly (E–H) stages. mixer expression initiates in a group of cells at the dorsal margin (C), then spreads throughout themarginal zone (D) where it is maintained at the onset of gastrulation (E). mixer also appears to be expressed in the dorsal YSL at dome stage(arrowhead in D). ntl expression (G) at the onset of gastrulation encompasses essentially the same cells as does mixer expression (F), while
ata5 expression (H) is limited to a subset of these cells (compare F–H). (C, D) Dorsal views; (E–H) lateral views. (F–H) High-magnificationomarski optics images.
Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.

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Kupffer’s vesicle did not form in approximately one-quarter(17/77) of these embryos (Fig. 6B); when raised these em-bryos were all cas mutants. This observation demonstratesa defect in the FRs in cas mutants.

In order to assess the FRs earlier in development wexamined expression of the ntl gene in wild-type and casutant embryos. At 80% epiboly ntl is expressed through-

out the involuting cells of the germ ring and in the devel-oping notochord; ntl is also expressed in the FRs (Melby etl., 1996), visible as an area of ntl expression that extends

posteriorly from the notochord below the level of themargin (Fig. 6C). FR ntl expression was absent in one-quarter (16/64) of the embryos derived from a cas/1 hetero-zygote intercross (Fig. 6D), while ntl expression in the germring and notochord was normal. Thus cas mutants appear tolack forerunner cell expression of ntl.

Functional defects in the FRs could cause the lack of FRntl expression and the failure of Kupffer’s vesicle to form incas mutants. Alternatively, cas mutants may not form FRsat all. In order to test the latter hypothesis we treatedembryos from a cas/1 heterozygote intercross at the domestage with syto-11, a fluorescent dye that labels the highlyendocytic FRs (Cooper and D’Amico, 1996). This procedurepermits visualization under fluorescence microscopy of theforerunner cell cluster late in gastrulation (Fig. 6E). Ap-proximately one-quarter of the embryos (6/22) lacked afluorescent forerunner cell cluster (Fig. 6F); when raisedthese embryos proved to be cas mutants. We also usedNomarski optics to examine the dorsal margin of shield-stage embryos derived from a cas/1 heterozygote intercross(Fig. 6G); this technique allows direct visualization of theforerunner cell cluster (Melby et al., 1996). Again, in ap-proximately one-quarter of the embryos (10/36) no FRs wereseen (Fig. 6H), and when raised these embryos were indeedcas mutants. Considered together these data demonstratethat the FRs do not form in cas mutants.

Defective Morphogenesis of MesodermalDerivatives in cas Mutants

The cas mutation was originally identified because itcauses cardia bifida, as shown by expression of the cardiac-specific homeobox gene nkx2.5 (Figs. 7A and 7B) (Chen andFishman, 1996; Lee et al., 1996). In order to determinewhether other mesodermal derivatives develop abnormallyin cas mutants we examined the expression of variousmesodermal markers. The bilaterally positioned endothe-lial precursors express the receptor tyrosine kinase genetie2 (Lyons et al., 1998) and normally assemble smoothly inthe midline to form the trunk vasculature during somito-genesis (Fig. 7C) (Liao et al., 1997). These endothelialprecursors are disorganized in cas mutants (Fig. 7D); moreanterior populations of endothelium, including the endo-cardium, are similarly abnormal (data not shown). Thegata1 gene labels differentiating erythroblasts arranged in
bilateral stripes within the posterior lateral plate mesoderm(Detrich et al., 1995). During somitogenesis these cells

move toward each other and join in the midline (Fig. 7E).This medial movement is also perturbed in cas mutants(Fig. 7F). Last, the pax2.1 gene (Mikkola et al., 1992) isexpressed in the nephrogenic mesoderm, which shows anarrangement similar to that of the differentiating erythro-blasts (Fig. 7G). Again, as development proceeds the pro-nephric ducts move medially in wild-type embryos, but failto do so normally in cas mutants (Fig. 7H); the kidneys alsoappear positioned more laterally in cas mutants. Thus,precursors of at least four different mesodermal organs—heart, vasculature, blood, and kidney—exhibit morphoge-netic defects in cas mutants.

We considered the possibility that these mesodermalefects could result from decreased dorsal convergenceuring and after gastrulation. However, several lines ofvidence argue against this. First, the notochord in casutants is not abnormally broad (Figs. 3G and 3H). Second,

he pax2.1-expressing spinal commissural interneurons(Mikkola et al., 1992) are not wider apart in cas mutantscompared to wild-type (Figs. 7G and 7H). Finally, weexamined both the endoderm and these same mesodermalderivatives in knypek (kny) mutant embryos, which exhibitramatically diminished convergence and extensionSolnica-Krezel et al., 1996). kny mutants form endodermhat appears essentially normal although more broadlypread across the embryo (Figs. 7K and 7JL. The trunkndothelium in kny mutants is also spread more broadlycross the midline (Fig. 7J). kny mutants do manifestorphogenetic abnormalities in the blood and kidney pre-

ursors by the end of gastrulation that appear to be similaro cas mutants, but in kny mutants the pax2.1-expressingpinal commissural interneurons are more widely spacedhan normal (data not shown). These data suggest that theonvergence and extension defect in kny mutants affects allhree germ layers. Importantly, however, kny mutants doot exhibit cardia bifida and their endothelium does notppear disorganized (Figs. 7J and 7I). Also, the morphoge-etic defects in the kidney and blood progenitors of casutants do not appear until after the 10-somite stage, more

han 3 h later than in kny mutants, further suggesting thathe underlying problem in the two mutants is different.onsidering the above results, we conclude that the casesodermal defects likely do not result from a general

efect in dorsal convergence, but rather from a more spe-ific failure of lateral mesodermal cells to undergo appro-riate medial migration.

DISCUSSION

An Essential Role for cas in Endoderm FormationThe genetic networks that control development of the

vertebrate endoderm have only recently begun to be ex-plored. Several genes that appear to play key roles in theearly events of endoderm formation have now been identi-
fied. Important functions for these genes—in particularXsox17a and Xsox17b and Mix homeobox genes such as

Taait

mutants appear to lack a FR cluster (F); several syto-11-labeled EVLcells are seen. Using Nomarski optics the FRs can be directly



Mixer and milk—are suggested by their endodermally re-stricted expression patterns, their ability to promoteendodermal gene expression when ectopically overex-pressed, and, conversely, their ability to inhibit endodermalgene expression in presumptive endodermal tissue whenfused to the Drosophila EnR domain (Ecochard et al., 1998;Henry and Melton, 1998; Hudson et al., 1997; Lemaire etal., 1998; Tada et al., 1998). It will be important to testwhether mutations in these genes confirm their presumedroles in this process. In addition, genetic analyses havedemonstrated required roles for mouse HNF3b and ze-brafish oep, cyclops, and squint in formation of part or all ofthe endoderm (Dufort et al., 1998; Feldman et al., 1998;Gritsman et al., 1999; Schier et al., 1997).

Our data demonstrate an early and essential requirementfor the zebrafish cas locus in formation of the endoderm.cas mutants exhibit no evidence of endodermal differentia-tion; they lack a gut tube and show no endodermal geneexpression during somitogenesis and pharyngula stages(Figs. 1 and 2). Most interestingly, cas mutants lackendodermal expression of axial, gata5, and fkd2 from theonset of gastrulation (Fig. 3). These data place cas upstreamof these early endodermal markers and suggest that theendoderm in cas mutants is not merely defective but in factmay not form. What becomes of the endodermal progeni-tors in cas mutants is not known. These cells may die,although we have not observed increased apoptosis in casmutants during gastrulation (M.R. and D.Y.R.S., unpub-lished data). Alternatively these cells may be respecified, forexample to mesodermal fates. Testing this possibility willrequire the isolation of markers specific for the involutedmesoderm.

Mosaic analysis demonstrates that cas functions cell-autonomously within the endodermal progenitors (Fig. 4),presumably either to receive or to respond to endoderm-inducing signals. Directed misexpression of a constitutivelyactive type I TGF-b receptor (TARAM-A*) in a singleblastomere of 16-cell-stage zebrafish embryos cell-autonomously directs the progeny of that blastomere to anendodermal fate (Peyrieras et al., 1998). Interestingly,

ARAM-A* misexpression also restores both endodermnd prechordal plate formation in oep mutants (Peyrieras etl., 1998). The fact that the prechordal plate forms normallyn cas mutants suggests that cas is required specifically inhe endoderm, either downstream of or parallel to, oep and

TARAM-A*, but experiments to test this hypothesis di-rectly are needed.

Many studies have focused upon the role(s) of theendoderm in the induction or patterning of the mesoderm

visualized at the shield stage as a cluster of cells (arrowheads) thatobscures part of the dorsal margin (arrows). No FRs are seen in a casmutant embryo (H), allowing the margin to be easily traced

FIG. 6. The forerunner cells do not form in cas mutants. Wild-type and cas mutant embryos were examined by light microscopy(A, B), in situ hybridization for expression of ntl (C, D), syto-11fluorescence (E, F), and Nomarski optics (G, H). Embryos are at thefollowing stages: (A, B) 6-somite, (C, D) 70% epiboly, (E, F) 90%epiboly, and (G, H) shield. Kupffer’s vesicle, formed by the forerun-ner cells (FRs), is easily seen in the tail of wild-type embryos(arrowhead in A) but not in cas mutants (B). ntl is normallyexpressed in the FRs (arrowhead in C) but this expression is lackingin cas mutants (D). The FR cluster (arrowhead in E) can bevisualized by labeling with the fluorescent dye syto-11; cells of theenveloping layer (EVL) also take up syto-11 and thus fluoresce. cas

(arrows). (A, B, E, F) Posterior views, dorsal to the right; (C, D, G, H)dorsal views, anterior to the top.


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and ectoderm (see for example Nieuwkoop, 1969; Bouw-meester et al., 1996). Given the apparent complete lack ofndoderm in cas mutants, the mutant embryos’ relatively

normal appearance (Fig. 1) is quite striking. Endodermalcells may be transiently present and able to fulfill theirnormal signaling functions in cas mutants. Alternatively,the relatively normal appearance of cas mutants may resultfrom the fact that in nonamphibian embryos these signalingfunctions appear to be performed at least in part by ex-traembryonic tissues, for example, in zebrafish the YSL andin mouse the visceral endoderm (Beddington and Robert-son, 1998; Fekany et al., 1999; Koos and Ho, 1998; Ya-manaka et al., 1998). At the same time, the mesodermaldefects seen in cas mutants (Fig. 7) may suggest a role(s) forthe endoderm in later morphogenetic events (see below).Further analyses of cas mutants will provide a uniqueopportunity to address the various roles played by theendoderm during vertebrate development.

Mesendodermal Expression of a Zebrafish MixerHomologue

FIG. 7. Morphogenesis of several mesodermal derivatives is defecJ, K, L) embryos showing expression of nkx2.5 (A, E, I), tie2 (B, F, J–L) and 21.5 hpf (B–D, F–H). By 24 hpf the definitive heart tube has

utants (E). Pharyngeal endodermal expression of nkx2.5 is also mimoothly in the midline of wild-type (B) but not cas mutant (F) emt the midline (C), but are positioned more laterally in cas mutantsositioned more laterally in cas mutants (compare D to H); the arrowutants form endoderm normally, although it is more broadly d

ndothelium appears smoothly although again more broadly arranghich is a lateral view, anterior to the left.

The early cell-autonomous role of cas in endoderm de-elopment led us to examine the relationship between cas

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nd Mixer. We have isolated a zebrafish Mix gene that,ased upon its overall homology to Xenopus Mixer, we callixer. Whether this gene represents an authentic zebrafishixer equivalent awaits further studies. mixer is not linked

o cas, and mixer expression is normal in cas mutants,uggesting that cas acts either downstream of, or parallel to,ixer to regulate endoderm formation.The mixer expression pattern (Fig. 5) raises intriguing

uestions regarding how endoderm and mesoderm are seg-egated before and during gastrulation. Unlike in Xenopus,n which Mixer expression is restricted to the presumptivendoderm, the mixer expression domain at the onset ofastrulation appears to encompass most if not all of thearginal cells that express ntl; both presumptive endoderm

nd mesoderm therefore express mixer. These results sug-est that zebrafish embryos may utilize a mechanism toegregate mesoderm from endoderm that is different fromhat of Xenopus. In Xenopus vegetally localized maternalegT appears to play a critical role in this process (Zhang etl., 1998). A zebrafish homologue of VegT, encoded by thepadetail locus, is not maternally expressed (Griffin et al.,

in cas mutants. Wild-type (A, B, C, D), cas (E, F, G, H), and kny (I,a1 (C, G), pax2.1 (D, H), and axial (K, L). Embryos are at 24 (A, E,ed in wild-type embryos (A) while two “hearts” are evident in casin cas mutants (compare A to E). Endothelial precursors assemble

. In wild-type embryos the bilateral red blood cell precursors meethe bilateral pronephroi (arrowheads) and pronephric ducts are also

dicate the pax2.1-expressing spinal commissural interneurons. knyuted (K, L); they do not exhibit cardia bifida (I); and their trunkthe midline (J). All are dorsal views, anterior to the left, except (K),

tive), gatformssingbryos(G). T

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998), perhaps providing further evidence of a differenceetween zebrafish and Xenopus. Comparison of the expres-


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sion domains of Brachyury homologues reveals yet anotherdistinction between zebrafish and Xenopus; while expres-ion of the Xenopus Brachyury homologue Xbra is re-

stricted to the mesoderm (Smith et al., 1991), in thezebrafish all involuting cells (i.e., both endoderm and me-soderm) express ntl (Schulte-Merker et al., 1992). Interest-ingly, all ingressing cells in both mouse and chick gastrulaealso express Brachyury (Beddington et al., 1992; Kispert etal., 1995). This observation may suggest that these organ-isms use a mechanism to segregate endoderm from meso-derm that is more similar to that used in zebrafish than thatin Xenopus, although how this segregation is achievedremains unknown. Understanding how the pregastrula ex-pression of zebrafish gata5 is restricted to the portion of themarginal zone from which the endoderm will emerge mayprovide some insight into this question, as likely will themolecular identification of cas and subsequent analysis ofits expression.

Forerunner Cell Development Requires cas

Our data illuminate several aspects of FR cell biology.First, it has not been clear to which germ layer the FRsbelong. Kupffer’s vesicle has been proposed to represent theendodermal aspect of the neurenteric canal, implying thatthe FRs are endodermal (Cooper and D’Amico, 1996). Onthe other hand, the various fates to which the FRs’ progenyultimately contribute—notochord, muscle, and mesen-chyme of the tail—are generally considered mesodermal(Melby et al., 1996). We believe the fact that cas is requiredfor the formation of the FRs (Fig. 6), similar to the role of casin endoderm development, provides genetic evidence sup-porting the endodermal assignment of the FRs. However,we have not directly tested whether cas acts cell autono-mously in the FRs. The b-catenin signaling pathway thatetermines the embryonic dorsal axis also appears essentialor formation of the FRs (Fekany et al., 1999). We thereforeropose that the FRs represent a specialized dorsal subset ofhe endoderm.

A related point concerns the germ-layer assignment ofhe hypochord. Studies in amphibia have concluded thathis structure derives from the endoderm (Lofberg andollazo, 1997), and the same has therefore been assumed toe true in zebrafish (Appel et al., 1999). However, whileruncated posteriorly (Fig. 2), the hypochord clearly formsn cas mutants, which suggests that the hypochord may note endodermal. It has recently been proposed that in ze-rafish Notch–Delta signaling plays a role in the allocationf dorsal midline cells to the ectoderm (floor plate), meso-erm (notochord), and endoderm (hypochord) (Appel et al.,999). Considering that the hypochord is present in casutants, we would suggest an alternative interpretation of

hese results; that Notch–Delta signaling acts to subdividecommon progenitor population into three different deriva-

ives: the floor plate, the notochord, and the hypochord. We
ould propose that this progenitor population is most
ikely mesodermal: its formation clearly does not requireap


as, as floor plate, notochord, and hypochord are all presentn cas mutants, arguing against an endodermal assignment,nd studies in chick and zebrafish have demonstrated alose embryologic and molecular genetic relationship be-ween the notochord and the floor plate (Halpern et al.,997; Teillet et al., 1998).The functions of the FRs or their derivative, Kupffer’s

esicle, in zebrafish development remain mysterious. Theppearance of the FRs at approximately 30% epiboly pro-ides the earliest morphological landmark of the embryo’sorsal aspect (Cooper and D’Amico, 1996). LiCl treatmentesults in the appearance of ectopic FRs, while stronglyffected zebrafish bozozok mutants lack FRs, indicatinghat FR formation lies downstream of the same b-catenin

signaling pathway that specifies the dorsal axis (Cooper andD’Amico, 1996; Fekany et al., 1999). These observationshave led to the idea that the FRs may play a role in theinduction or maintenance of dorsal mesoderm (Cooper andD’Amico, 1996; Fekany et al., 1999). While our studies werenot exhaustive, we see no evidence for this hypothesis; casmutants express prechordal plate and notochord markersnormally and are not cyclopic (Figs. 1 and 3; J.A. andD.Y.R.S., unpublished data). It is also formally possible thatFR precursors are transiently present in cas mutants andprovide these functions. The fact that Kupffer’s vesicle doesnot form in ntl mutants has suggested a possible role forthis structure in tail development (Melby et al., 1996).Again, our results provide no clear evidence for such afunction; the tailbud extends in cas mutants and contains anormal number of somites (Fig. 1). As noted above, thehypochord in cas mutants does appear shortened posteri-orly (Fig. 2). The hypochord and floor plate connect in thetail of the late somitogenesis and pharyngula stage embryo,a position defined as the chordoneural hinge (Gont et al.,1993). As the FRs and their derivative, Kupffer’s vesicle, sitat this exact place during tail extension, the hypochorddefect in cas mutants may relate to the absence of the FRs.

ther than this possibility, however, we have not identifiedny specific essential function for the FRs in zebrafishevelopment.

A Possible Role for the Endoderm in MesodermalMorphogenesis

While the mesoderm appears to differentiate normally incas mutants, at least initially, several mesodermal organs—the heart, vasculature, blood, and kidneys—display mor-phogenetic defects (Fig. 7). These defects are unlikely toresult from reduced dorsal convergence, as kny mutants do

ot show similar abnormalities despite being strongly de-ective in convergence and extension (Fig. 7). Also, theormal width of the notochord and normal spacing of thepinal commissural interneurons in cas mutants arguegainst a general defect in dorsal convergence.These morphogenetic defects may be due to the cell-

utonomous action of cas in each of these mesodermal cellopulations or may instead result nonautonomously from


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the absence of the endoderm. For example, the endodermmay provide signals that guide mesodermal morphogenesis,may serve as a substrate for the migration of mesodermalcells, and/or may move adherent mesodermal cells in thecourse of its own morphogenesis. The cardia bifida ingata4-mutant mouse embryos provides one example of anendodermal defect that apparently underlies abnormal me-sodermal morphogenesis (Narita et al., 1997). Studies of oepmutants also support a role for the endoderm in the mor-phogenesis of the heart (Schier et al., 1997; Peyrieras et al.,998), vasculature (Fouquet et al., 1997), and kidneys andlood (J.A. and D.Y.R.S., unpublished data). Resolution ofhis issue in cas mutants awaits direct testing of the cellutonomy of these morphogenetic defects.It is also notable that together the two cas “hearts”

ppear to contain as much myocardial tissue as do wild-ype embryos (compare Figs. 7A and 7B) and that the casearts beat and express all myocardial markers thus farested (M.R., J.A., and D.Y.R.S., unpublished data; Yelon etl., 1999). Numerous studies have suggested importantoles for the endoderm in the induction, differentiation,nd/or maturation of the myocardium (see for exampleacobson and Sater, 1988; Gannon and Bader, 1995; Schul-heiss et al., 1995). Endodermal precursors may be tran-iently present in cas mutants and provide sufficient signalso induce and promote the differentiation of the myocar-ium. Alternatively, some other tissue may provide theseignals. Clearly further studies are needed to resolve theotential roles of the endoderm in zebrafish heart develop-ent.

Conclusion

The results presented in this report establish that the caslocus is required for endoderm and FR formation in ze-brafish. Our data also suggest that cas plays a direct orindirect role in the morphogenesis of numerous mesoder-mal derivatives. We have initiated efforts to isolate cas bypositional cloning. We expect that the molecular identifi-cation of cas, and the elucidation of its relationship to othergenes that act in the formation of the endoderm, willrepresent fundamental steps toward achieving a detailedunderstanding of vertebrate endoderm development.

ACKNOWLEDGMENTS

We are grateful to A. Navarro for technical assistance; L. Parkerfor help with cell transplantation; L. Zon for the gata4 and gata6cDNAs; A. Rodaway, R. Patient, and N. Holder for the gata5cDNA; D. Melton for communicating results prior to publication;T. Lepage, D. Kimelman, and B. Draper for the gastrula cDNAlibrary; L. Solnica-Krezel for kny fish; and J. DeYoung and T. Taylorfor sequencing. We thank members of the lab for their thoughtfulcomments on the manuscript. J.A. and M.R. are members of theMSTP at UCSF. Support for this work was provided in part by the
American Heart Association (J.A. and D.Y.R.S.) and the PackardFoundation (D.Y.R.S.).

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Received for publication June 16, 1999
Revised July 24, 1999
Accepted August 6, 1999


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casanova Plays an Early and Essential Role in Endoderm Formation in Zebrafish

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