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INTRODUCTIONStudies of axis formation in Xenopus laevis have led to modelsin which two signaling events that are initiated by localizedmaternal determinants cause mesendoderm induction in themarginal zone, and organizer formation on its dorsal side atblastula-to-gastrula stages. Mesendoderm induction divides theembryo into three germ layers along the vegetal-to-animal axis,while signals from the organizer induce neural tissue at thedorsal side of the ectoderm. The neural ectoderm is patternedalong its anteroposterior (AP) axis, coincident with, andsubsequent to, its initial specification; this patterning isinitiated by posteriorizing signals derived from prospective ordefinitive mesendodermal tissues (Nieuwkoop, 1950;Toivonen, 1968). Three candidate posteriorizing signals havebeen suggested: retinoic acid (RA) (Blumberg et al., 1997;Conlon, 1995; Durston et al., 1989; Sive et al., 1990); fibroblastgrowth factors (Fgfs) (Cox and Hemmati-Brivanlou, 1995;Kengaku and Okamoto, 1993; Kengaku and Okamoto, 1995;Koshida et al., 1998; Lamb and Harland, 1995) and Wnts(Fekany-Lee et al., 2000; Kazanskaya et al., 2000; Kelly et al.,1995; Kiecker and Niehrs, 2001; McGrew et al., 1995;Yamaguchi, 2001). Although it is likely that factors in all threefamilies participate in this process, the precise role of each inthe temporal and spatial aspects of neural patterning as well asthe molecular consequences of their action have not been fullyclarified. In zebrafish, a region called the yolk syncytial layer (YSL),which is located beneath the blastoderm, may have a role inboth mesendoderm and organizer induction (Mizuno et al.,1996). As a consequence of inductive signals that emanatefrom the YSL, the blastoderm margin forms the mesendoderm,one side of which develops into the organizer (which, in turn,induces neural specification within the dorsal ectoderm). As inXenopus, there is evidence for signals emanating from theprospective mesendodermal layer that transform early neuralectoderm from an anterior to a posterior fate (Koshida et al.,1998). One feature that adds complexity to the mechanisms of APpatterning is the repeated use of the same type of signal indifferent stages and regions of the embryo, with context-dependent consequences. Fgfs and Wnts are both expressed inundifferentiated mesendoderm from the blastula stageonwards, and in the area of the presumptive midbrain-4335Development 129, 4335-4346 (2002)Printed in Great Britain © The Company of Biologists Limited 2002DEV2859Early neural patterning in vertebrates involves signals thatinhibit anterior (A) and promote posterior (P) positionalvalues within the nascent neural plate. In this study, wehave investigated the contributions of, and interactionsbetween, retinoic acid (RA), Fgf and Wnt signals in thepromotion of posterior fates in the ectoderm. We analyzeexpression and function of cyp26/P450RAI, a gene thatencodes retinoic acid 4-hydroxylase, as a tool forinvestigating these events. Cyp26 is first expressed in thepresumptive anterior neural ectoderm and the blastodermmargin at the late blastula. When the posterior neural genehoxb1b is expressed during gastrulation, it shows astrikingly complementary pattern to cyp26. Using these twogenes, as well as otx2 and meis3 as anterior and posteriormarkers, we show that Fgf and Wnt signals suppressexpression of anterior genes, including cyp26.Overexpression of cyp26 suppresses posterior genes,suggesting that the anterior expression of cyp26 isimportant for restricting the expression of posterior genes.Consistent with this, knock-down of cyp26 by morpholinooligonucleotides leads to the anterior expansion ofposterior genes. We further show that Fgf- and Wnt-dependent activation of posterior genes is mediated by RA,whereas suppression of anterior genes does not depend onRA signaling. Fgf and Wnt signals suppress cyp26expression, while Cyp26 suppresses the RA signal. Thus,cyp26 has an important role in linking the Fgf, Wnt andRA signals to regulate AP patterning of the neuralectoderm in the late blastula to gastrula embryo inzebrafish. Key words: Fgf, Wnt, Retinoic acid, Posteriorization, Cyp26,Raldh2, ZebrafishSUMMARYDistinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neuralectodermTetsuhiro Kudoh1,2,*, Stephen W. Wilson1and Igor B. Dawid21Department of Anatomy and Developmental Biology, University College London, Gower St, London WC1E 6BT, UK2Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health,Bethesda, MD, USA*Author for correspondence (e-mail: [email protected])Accepted 25 June 20024336hindbrain region, beginning at the late gastrula stage (Furthaueret al., 1997; Kelly et al., 1995; Phillips et al., 2001). Signalsmediated by members of these two major classes of secretedfactors are involved in early AP patterning in the neuralectoderm, as well as subsequent regional patterning processeswithin the developing brain (Houart et al., 2002; Kim et al.,2000; Reifers et al., 1998). To avoid having to consider a largerange of these complexities, we have focused on the earliestmanifestation of AP specification that is evident from the lateblastula through gastrula stages.We have searched for genes that may have an early role inaxis formation as part of a random in situ screen for regionallyexpressed genes in zebrafish embryos (Kudoh et al., 2001). Inthis screen, we noted the anterior neural ectodermal expressionof the gene cyp26/P450RAI, which encodes all trans retinoicacid 4-hydroxylase, an enzyme that degrades and inactivatesRA. Zebrafish cyp26 has originally been cloned fromregenerating fin tissue as an RA-responsive gene (White et al.,1996). We find that cyp26 is specifically expressed in thepresumptive anterior neural ectoderm from a surprisingly earlystage (from the late blastula onwards). At the early gastrulastage, the earliest known marker of posterior neural ectoderm,hoxb1b (Alexandre et al., 1996), is expressed in acomplementary pattern to cyp26. We focused on the regulationof this earliest subdivision along the AP axis in the neuralectoderm, using cyp26 and hoxb1b as our primary tools ofanalysis. We show that posteriorization of the neural ectodermhas two separable steps: suppression of anterior geneexpression and activation of posterior gene expression.Suppression of anterior gene expression in the posterior regionis the first step of AP differentiation and is caused byFgfs/Wnts in an RA-independent pathway.


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