View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Available online at www.sciencedirect.com R Developmental Biology 254 (2003) 172–187 www.elsevier.com/locate/ydbio wnt1 and wnt10b function redundantly at the zebrafish midbrain–hindbrain boundary Arne C. Lekven,a,* Gerri R. Buckles,a Nicholas Kostakis,b and Randall T. Moonb a Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843-3258, USA b Howard Hughes Medical Institute, Department of Pharmacology and Center for Developmental Biology, Box 357750, University of Washington School of Medicine, Seattle, WA 98195, USA Received for publication 28 May 2002, revised 8 October 2002, accepted 1 November 2002 Abstract Wnt signals have been shown to be involved in multiple steps of vertebrate neural patterning, yet the relative contributions of individual Wnts to the process of brain regionalization is poorly understood. Wnt1 has been shown in the mouse to be required for the formation of the midbrain and the anterior hindbrain, but this function of wnt1 has not been explored in other model systems. Further, wnt1 is part of a Wnt cluster conserved in all vertebrates comprising wnt1 and wnt10b, yet the function of wnt10b during embryogenesis has not been explored. Here, we report that in zebrafish wnt10b is expressed in a pattern overlapping extensively with that of wnt1. We have generated a deficiency allele for these closely linked loci and performed morpholino antisense oligo knockdown to show that wnt1 and wnt10b provide partially redundant functions in the formation of the midbrain–hindbrain boundary (MHB). When both loci are deleted, the expression of pax2.1, en2, and her5 is lost in the ventral portion of the MHB beginning at the 8-somite stage. However, wnt1 and wnt10b are not required for the maintenance of fgf8, en3, wnt8b, or wnt3a expression. Embryos homozygous for the wnt1–wnt10b deficiency display a mild MHB phenotype, but are sensitized to reductions in either Pax2.1 or Fgf8; that is, in combination with mutant alleles of either of these loci, the morphological MHB is lost. Thus, wnt1 and wnt10b are required to maintain threshold levels of Pax2.1 and Fgf8 at the MHB. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Zebrafish; wnt1; wnt10b; Midbrain–hindbrain boundary; Acerebellar; No isthmus Introduction Subsequent steps of neural A/P patterning appear to involve the refinement of initial A/P domains into smaller Patterning of the vertebrate nervous system requires the subdivisions. These steps of vertebrate neural patterning input from members of the Wnt family of secreted glyco- and morphogenesis are dependent on at least three Wnts. proteins at many points. For example, during neural induc- For example, in the mouse, the wnt3a locus is required for tion, a Wnt signal from the paraxial mesoderm is required to the formation of the hippocampus (Lee et al., 2000). Studies send a “posteriorizing” signal that establishes anterior–pos- of the regulation of the mouse emx2 promoter suggest that terior (A/P) polarity and induces the expression of posterior inputs of wnt3a in addition to wnt8b (Richardson et al., neural markers (i.e., posterior to forebrain; Bang et al., 1999) are involved in dorsal telencephalon formation (Theil 1999; McGrew et al., 1995). In zebrafish, the expression of et al., 2002). Further, in the zebrafish, the wnt8b locus wnt8 in the paraxial mesoderm has been shown to be the (Kelly et al., 1995) has been shown to be involved in earliest part of this “posteriorizing” signal (Erter et al., establishing subdivisions within the anterior neural plate 2001; Lekven et al., 2001). (Houart et al., 2002; Kim et al., 2002). In this case, regional inhibition of wnt8b activity permits the expression of telen- cephalic fates, but the mechanism that establishes telence- * Corresponding author. Fax: ϩ1-979-845-2891. phalic identity downstream of wnt8b inhibition is not E-mail address: [email protected] (A.C. Lekven). known. 0012-1606/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0012-1606(02)00044-1 A.C. Lekven et al. / Developmental Biology 254 (2003) 172–187 173 Genetic studies of wnt1 in the mouse have shown that the et al., 1992a; Postlethwait et al., 1998). (Note: zebrafish formation of a portion of the midbrain and anterior hind- wnt10b has been referred to as wnt10br; because this locus brain, a region that encompasses a structure called the isth- represents the ortholog of other vertebrate wnt10b loci, we mus or midbrain–hindbrain boundary (MHB), is dependent will refer to this locus as zebrafish wnt10b.) The linkage of on this locus (McMahon and Bradley, 1990; Thomas and wnt1 and wnt10b paralogs has been conserved in the verte- Capecchi, 1990; Thomas et al., 1991). This structure, a brate lineage, and this relationship may reflect the existence constriction in the neural tube at the junction between the of an ancient cluster of three wnt paralogs present before the midbrain and hindbrain, serves as an important signaling divergence of protostomes and deuterostomes; wnt1, wnt6, center for patterning the midbrain and anterior hindbrain and wnt10 (Nusse, 2001). The conservation of the spatial (Rhinn and Brand, 2001; Wassef and Joyner, 1997). In this arrangement of wnt1 and wnt10b raises the possibility that case, the function of wnt1 may, at least in part, be to regulate their expression may be regulated in a coordinated fashion, the expression of engrailed homologs, as expression of for example, similar to the vox and vent loci in Xenopus mouse en1 under the control of a wnt1 enhancer element can (Rastegar et al., 1999) and zebrafish (Melby et al., 2000). In rescue the wnt1 knockout phenotype (Danielian and Mc- support of this assertion, a fragment of the zebrafish wnt10b Mahon, 1996). Thus, Wnt signaling is required to pattern gene showed an expression pattern that may be similar to the early neurectoderm and also to establish a signaling that shown by wnt1 (Krauss et al., 1992a). If zebrafish center that will further pattern brain subdivisions. wnt10b and wnt1 are expressed in similar patterns and are at In zebrafish, mutagenesis screens have identified three least partially functionally redundant, this could explain the loci required for the formation of the MHB (Brand et al., lack of an identified wnt1 point-mutant recovered from two 1996). acerebellar (ace) mutants, which carry mutations in large-scale mutagenesis screens (Driever et al., 1996; Haff- the fgf8 gene (Reifers et al., 1998), fail to develop a MHB ter et al., 1996). and cerebellum and lose the expression of MHB markers We have analyzed the relationship of wnt1 and wnt10b during early somite stages (Reifers et al., 1998), no isthmus embryonic expression in zebrafish in addition to addressing (noi) mutants, which carry mutations in the pax2.1 gene their functions through a genetic loss-of-function approach (Brand et al., 1996; Lun and Brand, 1998), also fail to form (Lekven et al., 2000). We have found that zebrafish wnt10b the MHB, but also lack a portion of the midbrain, the optic is expressed in a pattern substantially overlapping that of tectum (Brand et al., 1996). In both of these mutants, the wnt1, including the time of onset of expression. We gener- expression of several markers for the prospective MHB is ated a zebrafish line carrying a deficiency allele for wnt1, initiated properly but quickly disappears, indicating that wnt10b, and neighboring loci and show that, in contrast to these genes are required for the early maintenance of gene mouse wnt1, zebrafish wnt1 and wnt10b are not required for expression at the MHB (Lun and Brand, 1998). spiel ohne the formation of the midbrain and cerebellum. However, gretzen (spg) mutants also fail to form the MHB, and similar these loci are required for the maintenance of the expression to ace mutants, have enlarged optic tecta (Brand et al., of several genes at the MHB, with the notable exceptions of 1996). The spg locus has recently been shown to encode fgf8, en3, wnt8b, and wnt3a. Through a combination of zebrafish Pou2 (Belting et al., 2001; Burgess et al., 2002), rescue experiments and morpholino antisense oligo knock- and analysis of early markers of the MHB domain show that down, we also show that wnt1 and wnt10b are at least spg is also required for the early maintenance of gene partially functionally redundant in their capacity to regulate expression (Reim and Brand, 2002). The function of spg gene expression at the MHB. Finally, we show through may be to mediate regional competence in the neurectoderm double mutant analyses that zebrafish lacking wnt1 and to Fgf8 signals (Reim and Brand, 2002). Thus, a complex wnt10b, though they retain expression of pax2.1 and fgf8, genetic network exists that includes at least Pax2.1, Fgf8, are sensitized to reductions in the levels of these genes. Pou2, and Wnt1, to perform several aspects of MHB for- mation: initiation of the MHB program, maintenance of gene expression, and morphogenesis (Rhinn and Brand, Materials and methods 2001). During two large-scale mutagenesis screens in the ze- Fish care and maintenance brafish, only the ace, noi, and spg loci were identified as being required for MHB formation (Driever et al., 1996; Routine fish care and maintenance were performed as Haffter et al., 1996); no wnt1 mutants were apparently described (Westerfield, 1995). Wild type fish used were of recovered. However, wnt1 has been isolated in zebrafish and the AB strain, or were a derivative of the AB strain we refer was shown to be expressed in a conserved pattern similar to to as KWT (Lekven et al., 2001). The alleles used in this other vertebrate wnt1 homologs (Molven et al., 1991).