Regulation of eye development by frizzled signaling in Xenopus

Eye development in both invertebrates and vertebrates is regulated by a network of highly conserved transcription factors. However, it is not known what controls the expression of these factors to regulate early eye formation, and whether transmembrance signaling events are involved. Her we establish a role for signaling via a member of the frizzled family of receptors in regulating early eye development. We show that overexpression of Xenopus frizzled 3 (Xfz3), a receptor expressed during normal eye development, functions cell autonomously to promote ectopic eye formation, and can also perturb endogenous eye development. Ectopic eye obtained with Xfz3 overexperession have a laminar organization similar to endogenous eyes and contain differentiated retinal cell types. Ectopic eye formation is preceded by ectopic expression of transcription factors involved in early eye development, including Pax6, Rx and Otx2. Conversely, targeted overexpression of a dominant-negative form of Xfz3 (Nxfz3), consisting of the soluble extracellular domain of the receptor, results in suppression of endogenous Pax6, Otx2 and Rx expression and suppression of endogenous eye development. This effect can be rescued by co-expression of Xfz3. Finally, overexpression of Kermit, a protein that interacts with the C-terminal intracellular domain of Xfz3, also blocks endogenous eye development, suggesting that signaling through Xfz3 or a related receptor is required for normal eye development. In summary we show that frizzled signaling is both necessary and sufficient to regulate eye development in Xenopus.

REGlJLATION OF EYE DEVELOPMENT BY FRIZZLED SIGNALING IN XENOPUS by Jennifer Terry Rasmussen A thesis submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Master of Science In Anatomy Department of Neurobiology and Anatomy The University of Utah August 2001 Copyright © Jennifer Terry Rasmussen'2001 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a thesis submitted by Jennifer Terry Rasmussen This thesis has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. Chair: Monica L. Vetter o Joseph Yost THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the thesis of Jennifer Terry Rasmussen in its final form and have found that (1) its format, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School. Date Chair, Supervisory Committee Approved for the Major Department Approved for the Graduate Council ~...:...\ S. C01-- · David S. Chapman Dean of The Graduate School ABSTRACT Eye development in both invertebrates and vertebrates is regulated by a network of highly conserved transcription factors. However, it is not known what controls the expression of these factors to regulate early eye formation, and whether transmembrane signaling events are involved. Here we establish a role for signaling via a member of the frizzled family of receptors in regulating early eye development. We show that overexpression of Xenopus frizzled 3 (Xfz3), a receptor expressed during normal eye development, functions cell autonomously to promote ectopic eye formation, and can also perturb endogenous eye development. Ectopic eyes obtained with Xfz3 overexpression have a laminar organization similar to endogenous eyes and contain differentiated retinal cell types. Ectopic eye formation is preceded by ectopic expression of transcription factors involved in early eye development, including Pax6, Rx and Otx2. Conversely, targeted overexpression of a dominant-negative form ofXfz3 (Nxfz3), consisting of the soluble extracellular domain of the receptor, results in suppression of endogenous Pax6, Otx2 and Rx expression and suppression of endogenous eye development. This effect can be rescued by co-expression of Xfz3. Finally, overexpression of Kermit, a protein that interacts with the C-terminal intracellular domain ofXfz3, also blocks endogenous eye development, suggesting that signaling through Xfz3 or a related receptor is required for normal eye development. In summary we 'show that frizzled signaling is both necessary and sufficient to regulate eye development in Xenopus. v TABLE OF CONTENTS ABSTRACT ................................................... i v ACKNOWLEDGMENTS • • • • • • • • • • • • • • . • • • . • • • • • • • • • • • • • • • • • • • • • V1.1. Chapter 1. INTRODUCTION ............................................ 1 2. REGULATION OF FRIZZLED SIGNALING IN EYE DEVELOPMENT Introduction ............................................. 5 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 7 Results ................................................. 9 Discussion .............................................. 18 3. CONCLUSIONS ............................................. 25 REFERENCES ................................................. 29 ACKNOWLEDGMENTS We would like to thank Terry Van Raay for initiating the collaboration between our labs, Kathy Moore for advice on sectioning and immunohistochemistry, and Sheryl Scott, Villu Maricq, Joe Yost and Kathy Moore for critical reading of the manuscript. We are grateful to Grant Mastick for the anti-Pax6 antibody. Regulation of eye development by frizzled signaling in Xenopus was originally published by the National Academy of Sciences and is reprinted here with pelTIlission. Jennifer T. Rasmussen, Matthew A. Deardorff, Change Tan, Mahendra S. Rao, Peter S. Klein, and Monica L. Vetter. Regulation of eye development by frizzled signaling in Xenopus. (2001) PNAS 98: 3861-3866. Copyright © 2001 by The National Academy of Sciences of the United States of America, all rights reserved. The author also wishes to thank Thomas Rasmussen, Miriya Rasmussen and Taylor Rasmussen. Without their understanding and support, this work would not have been possible. CHAPTER 1 INTRODUCTION The eye begins as an evagination of the forebrain during neurulation of the embryo. As the tissue moves out, it folds in on itself, forming the optic cup. The region of the optic cup nearest to the brain forms the retinal pigment epithelium, or the non­neuronal portion of the retina, and the inner portion of the optic cup, nearest to the lens, forms the neural retina. The fully differentiated neural retina consists of seven distinct cell types arranged in characteristic layers. Formation of mature retinal cell types depends upon the activity of numerous eye specific transcription factors such as Pax6 and Rx. For example, when the Drosophila Pax6 homologue, eyeless, is overexpressed in the developing embryo, an ectopic eye can be produced in all imaginal tissues (Halder et al., 1995). Likewise, vertebrate Pax6 overexpression in Xenopus will pronl0te ectopic eyes in developing neuronal tissue (Chow et aI., 1999). Similarly, Xrx overexpression in Xenopus can produce ectopic eye tissue and also profoundly affects morphology of the endogenous eye (Mathers et aI., 1997). Although much is known about eye specific transcription factors and their role in eye development, how they are initially activated is still poorly understood. Communication between differentiating tissues is essential for correct formation of embryonic organ systems, including the eye. This communication normally occurs via cell-cell signaling. 2 Cell signaling plays a major role in many developmental processes. For example, Notch signaling helps to pattern the nervous system and also plays an active role in differentiation of glial cells (Frisen and Lendahl, 2001). The Notch signaling pathway patterns distinct regions within the developing embryo, while signaling by the vertebrate wingless homologue Wnt and its receptor, Frizzled, promotes more global effects. WntlFrizzled signaling has been shown to specify axis formation, morphogenetic movements and cell type specification (Sumanas et aI., 2000) (Djiane et aI., 2000). The diversity of WntlFrizzled signaling effects in vertebrates may be attributed to the size of the WntlFrizzled family. In Xenopus alone, there are 15 Wnt ligands and 8 Frizzled receptors, with many more being described every month. Although cell signaling has not yet been implicated in early eye development, a few known cell signaling molecules, such as Frizzled receptors, are expressed in the eye field at the appropriate time to be involved in eye formation. The Frizzled receptors are seven pass transmembrane proteins that contain an extracellular, cysteine rich ligand binding domain as well as a small, intracellular domain. The W nt molecules are putative ligands for the Frizzled receptors and when Frizzled receptors bind ligand, two downstream pathways can be activated, the canonical and the planar cell polarity pathway (PCP). Signaling via the canonical pathway has been extensively studied and involves ~-catenin, Dishevelled, and GSK3~. Within a cell the levels of ~-catenin are very tightly regulated. In the absence of Wnt signaling, GSK3~, a serine/threonine kinase, promotes the phosphorylation and subsequent degradation of~­catenin. In the presence of Wnt signaling, the small intracellular protein, Dishevelled, becomes activated. The nature of this activation is not known. Activated Dishevelled 3 inhibits GSK3~ kinase function resulting in a build up of ~-catenin in the cytoplasm. ~­catenin then translocates to the nucleus where it can initiate transcription of downstream target genes such as siamois and nodal related (Xnr) (Cadigan and Nusse, 1997). The second downstream pathway is called the noncanonical or planar cell polarity (PCP) pathway. Work in Drosophila has shown that this pathway is necessary for correct orientation of the ommatidia and wing bristles during development, hence the name planar polarity (Mlodzik, 1999). In vertebrates, the PCP pathway is important for convergent extension movements (Heisenberg et al., 2000). Activation of the PCP pathway is also linked to intracellular calcium release in response to activation of the phosphatidylinositol pathway in a G-protein dependent manner (Miller et aI., 1999). Not as much is known about the PCP pathway as the ~-catenin pathway; however, it is known that like the ~-catenin pathway, the PCP pathway is mediated by the Dishevelled protein (Mlodzik, 1999). Both the ~-catenin and the PCP pathways are known to function during early development of Xenopus, but whether or not they have a role in eye formation is unclear. Early evidence implicating Frizzled receptors in eye development arose during a screen to isolate novel Frizzled receptors in the neural tube. From this experiment, zebrafish Frizzled 9 was cloned and overexpressed in Xenopus embryos to assay its effect on neural development. This overexpression resulted in production of an ectopic eye at low frequencies (unpublished data). However, upon further inspection, it was found that zebrafish Frizzled 9 was not normally expressed in the eye field and therefore not likely to have a role in eye development. It is known that Frizzled receptors can promiscuously bind to the Wnt ligands and can also activate similar downstream 4 signaling pathways. Therefore it is possible that a Frizzled receptor, under experimental conditions, can mimic the effects of a related receptor even though it may not normally regulate those events within the embryo. Although Frizzled 9 was not likely to be involved in retinogenesis, these data suggested that a Frizzled molecule may regulate eye development. Several Frizzled receptors, Frizzled 2, Frizzled 3, and Frizzled 4, are all expressed in the eye field during development (Deardorff and Klein, 1999) (Shi et aI., 1998) (Shi and Boucaut, 2000). Of these Frizzled receptors, Frizzled 3 was chosen for further analysis because overexpression of Xenopus Frizzled 3 (Xfz3) resulted in the ectopic formation of retina like tissue (unpublished, Deardorff, MA; Klein, PS). The research presented here suggests a novel role for a cell signaling pathway in early eye development. Overexpression of Frizzled 3 can produce an ectopic eye that contains fully differentiated retinal cells organized into the characteristic laminar arrangement of the neural retina. Overexpression of Frizzled 3 can also perturb development of the endogenous eye, similar to the effects seen with overexpression of Pax6. Blocking Frizzled function, including Xfz3, with a dominant negative Frizzled molecule either reduces the size of the endogenous eye or completely eliminates the eye on the injected side. In summary, we show that the Frizzled receptor is both necessary and sufficient for development of the eye. CHAPTER 2 REGULATION OF EYE DEVELOPMENT BY FRIZZLED SIGNALING IN XENOPUS Introduction Vertebrate eye development begins with the formation of the eye field, a region within the anterior neural plate that is fated to give rise to eyes (Eagleson and Harris, 1990). The eye field is demarcated by the expression of several regulatory genes, such as the homeodomain transcription factors Pax6, Rx, and Six3 (Hill et aI., 1991; Oliver et aI., 1995; Mathers et aI., 1997). Pax6 in particular plays an essential role in defining the tissue that is fated to become eye, and appropriate expression is required for normal eye development. However, it is not yet known whether upstream signaling pathways control expression of these transcription factors and thus regulate eye development. Proteins belonging to the wnt family of secreted ligands serve important patterning functions during development (Cadigan and Nusse, 1997). Wnt proteins function by binding to seven-pass transmembrane receptors belonging to the frizzled family (Bhanot et at, 1996; Yang-Snyder and et aI., 1996; He et aI., 1997; Hsieh et aI., 1999). Multiple wnt ligands and frizzled receptors are expressed during vertebrate nervous system development (Grad! et aI., 1999). For example, wnt signaling regulates 6 formation of the neural crest (Dorsky et aI., 1998), and anterior-posterior patterning of the central nervous system (Niehrs, 1999). One mernber of the frizzled family, Xenopus frizzled 3 (Xfz3), shows an expression pattern consistent with a role in regulating eye development. Xfz3 is expressed throughout development, but beginning at stage 12 expression increases and becomes restricted to the developing nervous system with strong expression in the anterior neural plate in a region overlapping the early eye field (Shi et aI., 1998). Xfz3 continues to be expressed in the developing optic vesicle as development proceeds (Shi et aI., 1998). After neural tube closure Xfz3 is also expressed in the dorsal neural tube where it regulates neural crest formation (M.A. Deardorff, J.-P. Saint-Jeannet and P.S. Klein, submitted). Several wnt ligands are also expressed during eye development, although none have been specifically implicated at the early stages of this process. Wnts activate frizzled receptors by binding to the cysteine-rich extracellular domain of the receptor (Hsieh et aI., 1999). Frizzled activation can lead to signaling either through a canonical pathway involving ~-catenin, or noncanonical pathways that regulate planar cell polarity in Drosophila and possibly vertebrates (Vinson et aI., 1989; Mlodzik, 1999; Tada and Smith, 2000), as well as calcium mobilization and protein kinase C activation in Xenopus and zebrafish (Kiihl et aI., 2000). Additional novel proteins have been identified that may couple frizzled receptors to these downstream signaling pathways. In the case of Xfz3, a protein called Kermit has been identified that specifically interacts with the cytoplasmic C-terminal domain of the receptor (C. Tan, M.A. Deardorff, J.-P. Saint-Jeannet and P.S. Klein, submitted). Kermit is an intracellular PDZ-domain protein that is expressed in the 7 nervous system and developing eye tissue in a spatial and temporal pattern very similar to that of Xfz3, and appears to be required to transduce signaling through Xfz3. Here we implicate frizzled signaling in the regulation of eye formation. We demonstrate that overexpression ofXfz3 is sufficient to promote complete ectopic eye development and to promote ectopic expression of the eye regulatory genes Xpax6, Xrx and Xotx2. In addition, overexpression of a soluble inhibitory form ofXfz3 blocks endogenous eye development and blocks endogenous expression of the same eye regulatory genes. Finally, inhibition of signaling through Xfz3 by overexpression of Kermit, a protein that binds to the C-terminal intracellular region ofXfz3, also blocks endogenous eye development. In summary, these findings demonstrate a requirement for wnt/frizzled signaling in regulating vertebrate eye development. Materials and Methods Constructs and microinjection of RNA Xfz3 (Shi et aI., 1998) was independently isolated in a screen to identify Xenopus frizzled homologs (Deardorff and Klein, 1999). The open reading frame was subc10ned into pCS2+ adding EcoRI and XhoI sites by PCR. To generate Nxfz3, the region encoding amino acids 1-202 was subc10ned into pCS2+MT using BamHI and ClaI sites added by PCR (M.A.D. and P. K., manuscript submitted). Capped RNA was synthesized in vitro by SP6 transcription from pCS2+-J;fo3, pCS2MT -Nxft3, pCS2+Kermit or pCS2+-nuclear /3-galactosidase (n-.Ral) template DNA using a Message Machine kit from Ambion. For eight-cell stage injections, RNA was injected in a volume of 1 nl into one of the dorsal animal blastomeres in the following amounts (unless otherwise indicated): Xft3 8 (2 ng), Nxfz3 (2 ng), and n.$al (0.1 ng). For the rescue experiments 1ng ofXfz3 RNA was coinjected with 2 ng ofNxfz3 RNA. Kermit RNA was injected at the four-cell stage into the animal pole region of a dorsal blastomere (0.25-2 ng). The embryos were staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1994) and fixed in MEMFA (Harland, 1991) at the stages indicated in the text. X-gal staining was performed on embryos injected with ~-galactosidase RNA as previously described (Turner and Weintraub, 1994). Sectioninll and immunohistochemistry For immunohistochemistry, embryos were fixed at stage 42-45, sectioned on a cryostat at a thickness of 14_ m.· Immunohistochemistry was performed using a mouse anti-rhodopsin antibody (Adamus et aI., 1991) (1: 100), and rabbit anti-Pax6 antibody (Mastick et aI., 1997) (1 :250) using methods described previously (Turner and Weintraub, 1994). The primary antibodies were detected with an Alexa fluor 488 goat anti-mouse IgG antibody or an Alexa Fluor 568 goat anti-rabbit IgG antibody (Molecular Probes; 1 :300). The sections were stained with Hoechst dye (30_ M in PBS) to visualize the nuclei, and coverslippe,d using Fluoromount-G mounting media (Southern Biotechnology Associates). Fluorescent images were acquired on a Nikon E800 microscope using a Xillix PMI camera and Openlab software. For sections that were not immunostained, embryos were fixed at stage 42-45, embedded in paraffin, sectioned at a thickness of 14_ m, then dewaxed and coverslipped using Pro-Texx mounting medium (Baxter Diagnostics, Inc.). 9 In situ hybridization Digoxigenin (DIG)-labeled antisense RNA probes were generated for XPax6 (Hirsch and Harris, 1997), Xrx (Mathers et al., 1997) and Xotx2(Pannese et aI., 1995) using a Maxiscript RNA synthesis kit (Ambion) and digoxigenin-labeled UTP (Boehringer Mannheim). Whole-mount in situ hybridization was performed on albino Xenopus embryos.as previously described (Harland, 1991), except that either BM purple (Boehringer Mannheim) or magenta-phos (Biosynth International) were used as the substrate for the alkaline phosphatase reaction. Results Overexpression of Xfz3 promotes ectopic eye formation and perturbs endogenous eye development In the developing Xenopus nervous system Xfz3 is expressed in the anterior neural plate overlapping the region fated to give rise to eyes (Shi et al., 1998). To determine whether Xfz3 can influence eye development, we injected RNA encoding Xfz3 along with RNA for the lineage tracer ~-galactosidase into a dorsal animal (D!) blastomere of an eight-cell stage embryo, which makes a major contribution to head ectodermal and mesodermal structures (Moody and Kline, 1990). When embryos were scored at stage 41-45 in multiple independent experiments we observed several eye-related phenotypes. In a representative experiment 11 % (9/79) of the embryos had two endogenous eyes along with a third ectopic eye that was roughly spherical, surrounded with dense pigment and associated with lens-like tissue (Figure 1a,b). The ectopic eyes' appeared on the dorsal aspect of the embryo at or just lateral to the midline, and were 10 always positioned rostral to the spinal cord and caudal to the endogenous eyes. Twenty­five percent (20/79) of the embryos showed defects in the endogenous eye on the injected side of the embryo. These defects included eyes in which the retinal pigment epithelium streamed towards the midline (Figure 1 c) and eyes that were placed more medially (Figure 1d,e). In 15% (12/79) of the errlbryos we observed isolated dense patches of pigment that resembled ectopic retinal pigment epithelium. These patches of ectopic pigment were fairly unrestricted in their location on the embryo, and the positions in which they appeared depended upon the blastomere injected. Twenty-four percent (19/79) of the embryos had no eye phenotypes, but showed a shortened anterior-posterior axis and were often also bent dorsally similar to what has been reported for injection of Xfz3 RNA at the two-cell stage (Shi et aI., 1998), and 24% (19/79) of the embryos appeared morphologically normal. We observed none of the above eye phenotypes when Xfz3 RNA was injected at the two-cell stage. In addition, no ectopic eyes or defects in the endogenous eyes were observed when Xfz3 RNA was injected into ventral animal blastomeres (0/84 embryos), which make a much smaller contribution to anterior neural structures (Moody and Kline, 1990). However, 6% (5/84) of these embryos showed patches of ectopic pigment. Finally, no effects were observed when equivalent levels of RNA encoding ~-galactosidase or green fluorescent protein (OFP) were injected (data not shown). Notably, the eye phenotypes obtained with Xfz3 overexpression were remarkably similar in nature and frequency to those observed with Pax6 overexpression in Xenopus, including the formation of complete ectopic eyes and perturbation of endogenous eye formation (Chow et aI., 1999). Overall, these findings suggest that Xfz3 is activating a specific signaling pathway that is sufficient to promote eye development. 11 Ectopic eyes induced by Xfz3 overexpression were frequently located in the roof of the fourth ventricle, with photoreceptors invariably located in the outermost layer and oriented towards the lumen of the ventricle (Figure If). The photoreceptors were always organized into a defined layer, had morphologically distinct outer segments (Figure 1 f, see inset) and were labeled with anti-rhodopsin antibodies (data not shown; 5/5 embryos). Inclusion of RNA encoding the lineage tracer ~-galactosidase in the injections showed that the ectopic eyes were derived exclusively from tissue expressing Xfz3, demonstrating that Xfz3 acted cell autonomously to promote eye formation (Figure If). The laminar organization of the ectopic eyes was similar to endogenous eyes (compare Figures Ig & Ih). The retina was surrounded by retinal pigment epithelium and adjacent to this were rhodopsin-positive photoreceptors with outer segments. Pax6-positive cells located more internally indicated the presence of retinal ganglion cells and amacrine cells (Figure 1h). In addition, morphologically distinct lens tissue was often associated with the ectopic eye (Figure 1h). These findings demonstrate that Xfz3 can function cell autonomously to promote the formation of fully differentiated ectopic eyes. Overexpression of Xfz3 activates ectopic expression of Xpax6, Xrx and Xotx2 The ability of Xfz3 to promote eye development suggested that it could be acting upon the network of transcription factors that regulates the earliest stages of eye development. We therefore examined the expression of the homeodomain transcription factors Xpax6 and Xrx, which are expressed in the early eye field in vertebrates and are required for normal eye development (Hill et aI., 1991; Mathers et aI., 1997). We also examined the expression of Xotx2, which begins expression earlier and is critical for 12 Figure 1: Overexpression of Xfz3 promotes ectopic eye formation and causes proximal eye defects. a) Stage 42 embryo showing an ectopic eye in the dorsal head region (white arrow). Inset shows a higher magnification view of the ectopic eye. b) Stage 45 embryo with an ectopic eye in the dorsal midline of the head. c) Stage 42 embryo showing streaming of the retinal pigment epithelium from the endogenous eye towards the midline. d) Stage 45 embryo with abnormally positioned eye on the injected side (black arrow), marked with p-galactosidase (blue). e) Section through an embryo similar to the one shown in d showing the eye adjacent to the neural tube on the injected side (left). f) Section through a stage 45 embryo showing an ectopic eye in the roof of the fourth ventricle that is fully labeled with the tracer p-galactosidase (blue). Inset shows a high magnification view from an adjacent section showing a row of morphologically distinct photoreceptor outer segments in the ectopic eye (arrowheads). g,h) Immunolabeling of a section through a normal eye (g) and ectopic eye (h) with anti-Pax6 antibodies (red) and anti-rhodopsin antibodies (green). Nuclei are labeled with Hoechst (blue). The ectopic eye in h shows a similar arrangement of retinal layers as the normal eye in g. 13 normal development of structures derived from the anterior neural plate (Acampora et aI., 1995; Matsuo et al., 1995). Embryos were injected at the 8-cell stage with a mixture of RNA for Xfz3 and ~-galactosidase, which was used to mark the region of the embryo derived from the injected blastomere. The embryos were collected for whole-mount in situ hybridization analysis at stage 14, to determine whether Xfz3 influences the expression of these genes in the early eye field, and at stage 28 to determine whether the later pattern of expression of these genes was affected. At stage 14, overexpression of Xfz3 caused no change in Xpax6 expression (17117 embryos; Figure 2b); however, we observed weakly expanded or ectopic Xrx expression in 25% of embryos (4116; Figure 2d) and expanded Xotx2 expression in 13% of embryos (3/23; Figure 2f). At stage 28, 11 % of embryos (3/28) showed ectopic Xpax6 expression (Figure 2h), 21 % of embryos (6/29) showed ectopic Xrx expression (Figure 2j) andl2% of embryos (2/17) showed ectopic Xotx2 expression (Figure 21), The frequency of ectopic gene activation roughly paralleled the frequency of ectopic eye formation observed with Xfz3 overexpression. In addition, the ectopic gene expression at stage 28 was often located in the dorsal part of the embryo in the general location where ectopic eyes frequently formed. At stage 28, when ~-galactosidase expression directly overlay the endogenous eye, we observed some reduction of Xrx expression (7/29 embryos) and Xotx2 expression (5/17 embryos) on the injected side (data not shown) consistent with Xfz3 overexpression causing proximal eye defects (e.g., misplaced eyes). In summary, these findings demonstrate that overexpression of Xfz3 is sufficient to cause ectopic expression of eye regulatory genes. This was most apparent at later stages of development, consistent with either later Figure 2: Overexpression of Xfz3 promotes ectopic expression of genes regulating eye development. Whole-mount in situ hybridization analysis of stage 14 embryos (a-f) that , are either uninjected (a,c,e) or Xfz3 injected (b,d,f) then labeled for Xpax6 (a,b), Xrx (c,d) or Xotx2 expression (e,f). Arrows indicate modest expansion of Xrx or Xotx2 expression (d,f). Stage 28 embryos (g-l) are either uninjected (g,i,k) or Xfz3 injected (h,j,l) then labeled for Xpax6 (g,h), Xrx (i,j) or Xotx2 expression (k,l). Arrows indicate ectopic expression in injected embryos (h,j,l). B-galactosidase (sky blue) marks the regions of the embryo derived from the injected blastomere. 14 15 availability of wnt ligand to activate the frizzled receptor or a delay in the competence of the responding tissue. Overexpression of an inhibitory form of Xfz3 blocks endogenous expression of Xpax6. Xrx and Xotx2 and blocks normal eye development The ability of Xfz3 overexpression to induce ectopic expression of eye regulatory genes suggested that Xfz3 could playa role in regulating their expression during normal development. To determine whether Xfz3 function was required for the expression of Xpax6, Xrx and Xotx2 we overexpressed an inhibitory form of Xfz3 (Nxfz3) by RNA injection into a dorsal blastomere of 8-cell stage embryos. Nxfz3 consists of the extracellular cysteine-rich domain (CRD) of the Xfz3 protein, a region in frizzled receptors that has been implicated in the binding of wnt ligand (Bhanot et aI., 1996). Nxfz3 can block interaction of wnt ligands with the endogenous Xfz3 receptor and has been shown to interfere with signaling through Xfz3 (M.A. Deardorff, 1.-P. Saint-leannet and P.S. Klein, submitted). Following injection of RNA for Nxfz3 along with RNA encoding p-galactosidase embryos were grown until stage 14 (open neural plate stage) or stage 18-20 (late neurula) and analyzed by whole-mount in situ hybridization for expression ofXpax6, Xrx or Xotx2. At stages 18-20, in embryos where p-galactosidase signal was localized in the region of the developing eye field (anterior neural tube) there was reduced or absent Xpax6 expression on the injected side in 38% (9/24) of the embryos (Figure 3b). Similarly, Xrx and Xotx2 expression was reduced or absent in 48% (15/31) and 33% (7121) of the embryos respectively (Figure 3d, f). In embryos where p-galactosidase expression was not localized to the developing eye field, Xpax6, Xrx and 16 a c e Figure 3: Overexpression of Nxfz3 suppresses expression of genes regulating eye development. Whole-mount in situ hybridization analysis of stage 18-20 embryos that are either uninjected (a,c,e) or Nxfz3 injected (b,d,f) then labeled for Xpax6 (a,b), Xrx (c,d) or Xotx2 e"xpression (e,f). ~-galactosidase (sky blue) marks the regions of the embryo derived from the injected blastomere. 17 Xotx2 expression was normal. Inhibition of gene expression was apparent from the onset of eye field formation, since at stage 14 expression ofXpax6 (9/18 embryos), Xrx (10/25 embryos) and Xotx2 (6/23 embryos) was suppressed on the injected side (data not shown). These findings demonstrate that Nxfz3 is able to interfere with the expression of key regulatory genes controlling eye deve:lopment and suggest that normal frizzled signaling is required for early expression of these genes. Since loss of Pax6, Rx or Otx2 expression results in disrupted eye development in mammals (Hill et aI., 1991; Acampora et aI., 1995; Matsuo et aI., 1995; Mathers et ai., 1997), we examined whether overexpression of Nxfz3 in Xenopus embryos would interfere with normal eye development. 630/0 (62/98) of the embryos analyzed at stage 45 that were injected with Nxfz3 RNA at the 8-cell stage showed reduced or malformed eyes (Figure 4b), and 7% (7/98) were missing eyes entirely (Figure 4c) with only residual disorganized pigment in their place (Figure 4d). There were no general effects on anterior development since the olfactory pits and cement gland on the injected side were intact (data not shown). In addition, the neural tube in Nxfz3 injected embryos looked morphologically normal (Figure 4d). To demonstrate that the effect of Nxfz3 on' endogenous eye development was due to interference with Xfz3 function, we rescued the effects of Nxfz3 by co-expressing full-length Xfz3 and observed that 88% (43/49) of the embryos were normal, with only 12% (6/49 embryos) showing reduced or missing eyes. No effects on eye development were observed when the tracer ~-ga1actosidase was not localized to the head region. These findings demonstrate that normal frizzled signaling, potentially through Xfz3, is required for endogenous eye development. Overexpression of Kermit. an Xfz3-interacting protein, inhibits endo~enous eye development To further test whether signaling through Xfz3 is required for endogenous eye development we have assessed whether Kermit, a component of the signaling pathway 18 immediately downstream of Xfz3, can modulate this process. Overexpression of Kermit inhibits signaling through Xfz3, likely by swamping out interactions between the receptor and downstream signaling components. Kermit was overexpressed by injecting RNA into the animal pole region of a dorsal blastomere of four-cell stage embryos along with RNA encoding ~-galactosidase. The majority of embryos analyzed at stage 40-45 that were injected with Kermit RNA at the eight-cell stage showed reduced eyes (58%, 193/331 embryos; Figure 4f), and some were missing eyes entirely (7%, 24/331 embryos), whereas only 1/67 uninjected embryos had a reduced eye. These findings provide independent evidence that perturbing wntlfz signaling can disrupt normal eye development. Discussion We show that wntlfz signaling can regulate normal eye development and can also initiate ectopic eye fonnation. Overexpression of Xfz3 is sufficient to initiate expression of eye specific genes and promote the formation of differentiated retinal tissue. We also provide evidence that frizzled signaling is required for endogenous eye development. Overexpression of a soluble inhibitory form of Xfz3 blocks endogenous eye development and blocks endogenous expression of Xpax6, Xrx and Xotx2. In addition, inhibition of signaling through Xfz3 by overexpression of Kermit, a protein that binds specifically to 19 Figure 4: Overexpression of Nxfz3 or Kermit inhibits endogenous eye formation. a) Uninjected embryo at stage 41. b,c) Nxfz3-injected embryos at stage 41 showing a reduced (b) or absent eye (c) on the injected side (white arrow). d) Section through an embryo with a missing eye, showing the loss of normal eye structure on the injected side with only residual disorganized pigment remaining (black arrow). The tissue derived from the injected blastomere is marked with ~-galactosidase (sky blue). e) Uninjected embryo at stage 43. f) Kermit-injected embryo at stage 43 showing a reduced eye on the injected side (arrow). 20 the C-terminal intracellular region of Xfz3, also blocks endogenous eye development. Our data provide the first evidence of an extracellular signaling pathway that regulates normal and ectopic eye formation. Our data suggest that Xfz3 is the frizzled receptor involved in eye formation during normal development. Xfz3 is expressed in the anterior neural plate in the region of the early eye field in concert with other early eye markers (Shi et aI., 1998). Other members of the frizzled family such as Xenopus frizzled 2 (Xfz2) and zebrafish frizzled 9 (Zfz9) can also promote ectopic eye formation at similar frequencies to Xfz3 (J.T.R., M.S.R. and M.L.V., unpublished, University of Utah, Dept. Neurobiology and Anatomy). However, Fz9 is not expressed in the developing eye in any species so far examined (T. Van Raay, J.T.R., M.S.R. and M.L.V., unpublished, University of Utah,Dept. Neurobiology and Anatomy), making it an unlikely candidate, and Xfz2 is expressed in the eye at later developmental stages after eye formation has been initiated. The ability of these receptors to promote ectopic eye formation is likely due to overlap between frizzled receptors with respect to ligand binding specificity and the activation of downstream ,; signaling pathways. Overexpression of Xfz3 promoted the formation of complete ectopic eyes. To date, only a few proteins, all homeobox-containing transcription factors, have been shown to be sufficient to induce ectopic eye development in vertebrate embryos. In Xenopus and medaka, these include Xpax6~ Six3/Xsix3 and the related factor Xsix6 (Xoptx2) (Chow et aI., 1999; LoosH et aI., 1999; Bernier et al., 2000). Our data suggest that the pathv/ay used for ectopic eye formation following Xfz3 overexpression is similar to that used during endogenous eye development. The frequency of complete ectopic eye 21 formation (11 %) is similar to that obtained with overexpression of Xpax6 (Chow et aI., 1999). In addition the formation of ectopic pigment and effects on endogenous eye development were also observed with Xpax6 overexpression, arguing that Xfz3 acts in a common pathway with this gene to regulate eye development. In support of this, overexpression of Xfz3 was sufficient to promote ectopic expression of Xpax6, Xrx and Xotx2. This ectopic expression occurred in regions of the embryo where the ectopic eyes formed, arguing that expression of these genes preceded and contributed to ectopic eye formation. In addition, disruption of signaling through Xfz3 using Nxfz3 blocked endogenous expression of Xpax6,Xrx and Xotx2 in the anterior neural plate and blocked endogenous eye development. These data suggest that Xfz3 functions to control the expression of these eye regulatory genes. Our findings provide the first clue in any species as to what is potentially required upstream of these transcription factors to initiate eye development. Additional experiments will determine whether the regulation by Xfz3 is direct or acting in parallel overlapping pathways. The ectopic eyes were comprised of cells that were overexpressing Xfz3, suggesting that it is a cell autonomous effect. Of interest was our observation that the ectopic eyes induced by Xfz3 overexpression appeared at or near the midline in proximity to the midbrainlhindbrain junction and often in the roof of the fourth ventricle. It is possible that there is a local source of wnts available to activate the Xfz3 receptor when overexpressed. For example, Xwnt-l and Xwnt-3A are highly expressed in the midbrainlhindbrain region of the developing Xenopus nervous system (W olda and et aI., 1993), and Xwnt-l0 is expressed in the dorsal hindbrain (Wolda and Moon, 1992). Alternatively, there may be requisite cooperating factors that are spatially restricted. For 22 example, in some cases wnts can synergize with members of the fibroblast growth factor family to regulate patterning events (McGrew et aI., 1997). Finally, there may be a restricted subset of tissues competent to form eye tissue. Overexpression of Six3 or Six6 results in transformation of midbrain and rostral hindbrain tissue into retina, and it was suggested that this region of the nervous system is uniquely competent to form retinal tissue, perhaps due to endogenous expression of Xotx2 (Loosli et aI., 1999; Bernier et aI., 2000). Additional experiments will be necessary to distinguish between these possibilities. The observation that Xfz3 can initiate ectopic eye formation identifies wnt signaling as the first identified extracellular signaling pathway that regulates eye formation. Several wnts, including Xwnt-l, Xwnt-3A and Xwnt-8 are expressed in the anterior neural plate in a region that overlaps the eye fields (Christian et aI., 1991; Wolda and et aI., 1993). In addition, Xwnt-1 is much more potent than any other wnt ligand in synergizing with Xfz3 to promote both axis duplication and neural crest induction (M.A. Deardorff, J.-P. Saint-Jeannet and P.S. Klein, submitted), However, given the relative promiscuity of binding and the number of wnts present in the developing nervous system it remains to be determined which wnt regulates eye development in vivo. Frizzled activation can lead to signaling either through a canonical pathway involving ~-catenin, or noncanonical pathways that regulate planar cell polarity in Drosophila and possibly vertebrates (Vinson et aI., 1989; Mlodzik, 1999; Tada and Smith, 2000), as well as calcium mobilization and protein kinase C activation in Xenopus and zebrafish (Kiihl et aI., 2000). The signaling pathway utilized by Xfz3 to promote eye development has not yet been defined, although limited evidence points to the 23 noncanonical planar cell polarity (PCP) pathway. Overexpression of Xfz3 alone (unlike Xfz8) does not lead to axis duplication, a phenotype linked to activation of the canonical signaling pathway, although coexpression of Xfz3 with X wnt 1 can promote efficient axis duplication (M.A. Deardorff, J.-P. Saint-Jeannet and P.S. Klein, submitted). Expression of the closely related homolog Mfz3 in Xenopus enlbryos results in protein kinase C activation but not expression of siamois and Xnr3, which are downstream effectors in the canonical pathway (Sheldahl et aI., 1999). In addition, activation of the canonical wnt signaling pathway represses anterior neural development, arguing against this pathway mediating the regulation of eye development, although localized activation of the canonical pathway at later stages of development has not been examined. We have used 8-cell RNA injection to overexpress a truncated form of Dsh (Dsh­LlN), which preferentially activates the noncanonical PCP pathway (Tada and Smith, 2000). We found that 28% of the embryos (311110) had dense ectopic pigment at or near the midline in the region of the hindbrain, reminiscent of the phenotype observed with Xfz3 overexpression. Conversely, injection of RNA encoding a truncated form of Dsh (Dsh-DEP+), which preferentially inhibits the noncanonical PCP pathway (Tada and Smith, 2000), resulted in reduced or missing eyes in 51 % of injected embryos (52/101), similar to what was observed with Nxfz3 overexpression. These findings implicate the noncanonical PCP signaling pathway in regulating eye development, although further experiments will be necessary to confirm this. In summary, our findings suggest a hitherto unsuspected role for wnt/frizzled signaling in regulating eye development and provide the first example of a transmembrane receptor that is capable of promoting ectopic eye formation. Wnt-fz 24 signaling may thus represent a critical link between extracellular patterning events and transcriptional regulation of early eye formation. CHAPTER 3 CONCLUSIONS Previous data shows that Frizzled 3 is expressed at the appropriate time and place to be involved in eye formation (Shi et aI., 1998). The Frizzled 3 overexpression and dominant negative interference experiments described here demonstrate that WntlFrizzled signaling is both necessary and sufficient for eye development. These experiments also show that the Frizzled receptor functions in a cell autonomous manner to produce ectopic eyes that contain fully differentiated cell types arranged in characteristic layers. These data are the first evidence to implicate a cell-signaling pathway in vertebrate eye development and describe an unsuspected function of the Frizzled receptors in retinogenesis. Cell signaling is important for patterning and development of many different tissues and organs within an embryo. The pivotal finding of a cell-signaling pathway in retinogenesis opens up a new avenue of thought about eye development and how cell-cell communication might be tied to other developmental processes within the errlbryo. Recent work has implicated Wnt signaling in development of the ear. Fibroblast Growth Factor (Fgf) signals induce mesoderm which in turn, activates Wnt signaling to direct terminal differentiation of cells within the inner ear (Ladher et aI., 2000). These results suggest that multiple signaling pathways may be functioning in eye development as well. Signaling proteins such as Epidermal Growth Factor (EGF) like molecules and Fgfs are 26 expressed in the presumptive eye field and could participate with WntlFrizzled signaling in eye specification and differentiation (Kuriyama et aI., 2000) (Golub et aI., 2000). Cell signaling cascades, such as the Egfs, Fgfs and WntlFrizzled pathways, can link eye development to development of the rest of the embryo. Although the experiments described here define the involvement of Frizzled signaling in retinogenesis, several outstanding issues need to be resolved. One interesting question involves which pathway Frizzled 3 uses to regulate development of the endogenous and ectopic eyes. As mentioned previously, Frizzled receptors initiate their downstream effects by activating the intermediary protein Dishevelled that in turn activates either the ~-catenin or the PCP pathway. It is equally possible for either pathway to be used by Frizzled 3 during eye formation. In a previous experiment to elucidate Frizzled signaling pathways during Xenopus gastrulation, several modified forms of Dishevelled were tested and found to be capable of selectively activating either the PCP or the ~-catenin pathway (Wallingford et aI., 2000). To resolve which downstream pathway is involved in eye formation, these mutant Dishevelled constructs could be injected into the developing eye region of Xenopus embryos. Production of an ectopic eye upon activation of one pathway or the other by the modified Dishevelled constructs would indicate the functional pathway used for development of the endogenous eyes. It is also possible that a combination of both pathways is used to produce a fully formed eye. For example, the ~-catenin pathway could be used by Frizzled 3 to initially specify eye tissue while later on, the planar polarity pathway could be used to direct differentiation and laminar organization, or vice versa. Recent evidence has shown that the same Frizzled receptor can activate both pathways at different times 27 during development; therefore the pathway activated depends more on which Wnts are expressed than on the specific receptor that is present in a particular location (Medina et aI.,2000). Since multiple Wnts are expressed at the right time and place to be involved in eye development, another important question is which of these ligands signal via the Frizzled 3 receptor (Christian et aI., 1991) (Zakin et aI., 1998). Production of an ectopic eye upon co-injection of a candidate Wnt ligand with low concentrations of the Frizzled 3 receptor would indicate a synergistic effect between the ligand and receptor. This would suggest an in vivo interaction in normal, uninjected embryos. If both downstream pathways are involved, then multiple Wnts might be expected to affect eye development. For example, Wnt 3A is known to activate the ~-catenin pathway while Wnt 5A signals through the planar polarity pathway and both are expressed in the eye field during development (Shimizu et aI., 1997) (Saint-Jeannet et aI., 1997) (Moon et aI., 1993). If both the ~-catenin and the PCP pathways are involved in retinogenesis then overexpression of one Wnt may duplicate the result seen with overexpression of Frizzled 3 while overexpression of the other Wnt may interfere with retinal differentiation and lamination. The experiments presented here are the first to implicate cell signaling in eye development and have opened up a new area of retinogenesis for exploration. The results and implications discussed will likely change the way the field is thought about and studied in the future. Transcription factors, such as those involved in eye development, are ultimately, downstream effectors of signaling cascades initiated by receptors such as Frizzled 3. Deciphering the upstream signaling pathways will allow us to better understand development not only of the eye, but other sensory structures as well. 28 REFERENCES Acampora D, Mazan S, Lallemand Y, Avantaggiato V, Maury M, Simeone A, Brulet P (1995) Forebrain and midbrain regions are deleted in Otx2-/- mutants due to defective anterior neuroectoderm specification during gastrulation. Development 121 :3279-3290. Adamus G, Zam ZS, Arendt A, Palczewski K, McDowell JH, Hargrave PA (1991) Anti­rhodopsin monoclonal antibodies of defined specificity: characterization and application. Vision Res. 131: 17-31. Bernier G, Panitz F, Zhou X, Hollemann T, Gruss P, Pieler T (2000) Expanded retina territory by midbrain transformation upon overexpression of Six6 (Optx2) in Xenopus embryos. Mech. Dev. 93:59-69. Bhanot P, Brink M, Samos CH, Hsieh JC, Wang YS, Macke JP, Andrew D, Nathans J, Nusse R (1996) A new merrlber of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382:225-230. Cadigan KM, Nusse R (1997) Wnt signaling: a common theme in animal development. Genes & Dev. 11:3286-3305. Chow RL, Altmann CR, Lang RA, Hemmati-Brivanlou A (1999) Pax6 induces ectopic eyes in a vertebrate. Development 126:4213-4222. Christian JL, Gavin BJ, McMahon AP, Moon RT (1991) Isolation of cDNAs partially encoding for Xenopus Wnt-llint-1-related proteins and characterization of their transient expression during embryonic development. Dev. BioI. 143:230-234. Deardorff MA, Klein PS (1999) Xenopus frizzled-2 is expressed highly in the developing eye, otic vesicle and somites. Mech. Dev. 87:229-233. Djiane A, Riou J, Umbhauer M, Boucaut J, Shi D (2000) Role of frizzled 7 in the regulation of convergent extension movements during gastrulation in Xenopus laevis. Development 127:3091-3100. Dorsky RI, Moon RT, Raible DW (1998) Control of neural crest cell fate by the Wnt signalling pathway. Nature 396:370-373. Eagleson GW, Harris W A (1990) Mapping of the presumptive brain regions in the neural plate of Xenopus laevis. 1. NeurobioI. 21:427-440. 30 Frisen J, Lendahl U (2001) Dh no, Notch again! Bioessays 23:3-7. Golub R, Adelman Z, Clementi J, Weiss R, Bonasera J, Servetnick M (2000) Evolutionarily conserved and divergent expression of members of the FGF receptor family among vertebrate embryos, as revealed by FGFR expression patterns in Xenopus. Dev Genes EvoI210:345-357. Gradl D, Kuehl M, Wedlich D (1999) Keeping a close eye on Wnt-lIwg signaling in Xenopus. Mechanisms of Development 86:3-15. Halder G, Callaerts P, Gehring WJ (1995) Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267: 1788-1792. Harland RM (1991) In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell BioI. 36:685-695. He X, Saint-Jeannet JP, Wang Y, Nathans J, Dawid I, Varmus H (1997) A member of the Frizzled protein family mediating axis induction by Wnt-5A. Science 275:1652-1654. Heisenberg CP, Tada M, Rauch GJ, Saude L, Concha ML, Geisler R, Stemple DL, Smith JC, Wilson SW (2000) SilberblicklWnt11 mediates convergent extension movements during zebrafish gastrulation. Nature 405:76-81. Hill RE, Favor J, Hogan BL, Ton CC, Saunders GF, Hanson 1M, Prosser J, Jordan T, Hastie ND, van Heyningen V (1991) Mouse small eye results from mutations in a paired­like homeobox-containing gene. Nature 354:522-525. Hirsch N, Harris WA (1997) Xenopus Pax-6 and retinal development. J. NeurobioI. 32:45-61. Hsieh JC, Rattner A, Smallwood PM, Nathans J (1999) Biochemical characterization of wnt-frizzled interactions using a soluble, biologically active vertebrate wnt protein [In Process Citation]. Proc Natl Acad Sci USA 96:3546-3551. Kiihl M, Sheldahl LC, Park M, Miller JR, Moon RT (2000) The WntlCa2+ pathway: a new vertebrate Wnt signaling pathway takes shape. TIG 16:279-283. Kuriyama S, Miyatani S, Kinoshita T (2000) Xerl: a novel secretory protein expressed in eye and brain of Xenopus embryo. Mech Dev 93:233-237. Ladher RK, Anakwe KU, Gurney AL, Schoenwolf GC, Francis-West PH (2000) Identification of synergistic signals initiating inner ear development. Science 290: 1965- 1967. LoosH F, Winkler S, Wittbrodt J (1999) Six3 overexpression initiates the formation of ectopic retina. Genes Dev 13:649-654. ---------------- ---~---.---------------- 31 Mastick GS, Davis NM, Andrews GL, Easter SS (1997) Pax-6 functions in boundary formation and axon guidance in the embryonic mouse forebrain. Development 124: 1985- 1997. Mathers PH, Grinberg A, Mahon KA, Jamrich M (1997) The Rx homeobox gene is essential for vertebrate eye development. Nature 387:603-607. Matsuo I, Kuratani S, Kimura C, Takeda N, Aizawa S (1995) Mouse Otx2 functions in the formation and patterning of rostral head. Genes Dev. 9:2646-2658. McGrew LL, Hoppler S, Moon RT (1997) Wnt and FGF pathways cooperatively pattern anteroposterior neural ectoderm in Xenopus. Mech Dev. 69: 105-114. Medina A, Reintsch W, Steinbeisser H (2000) Xenopus frizzled 7 can act in canonical and noncanonical Wnt signaling pathways: implications on early patterning and morphogenesis. Mech Dev 92:227-237. Miller JR, Hocking AM, Brown JD, Moon RT (1999) Mechanism and function of signal transduction by the Wntlbeta-catenin and WntlCa2+ pathways. Oncogene 18:7860-7872. Mlodzik M (1999) Planar polarity in the Drosophila eye: a multifaceted view of signaling specificity and cross-talk. EMBO J. 18:6873-6879. Moody SA, Kline MJ (1990) Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres. Anal. Embryo!. 182:347-362. Moon RT, Campbell RM, Christian JL, McGrew LL, Shih J, Fraser S (1993) Xwnt-5A: a maternal Wnt that affects morphogenetic movements after overexpression in embryos of Xenopus laevis. Development 119:97-111. Niehrs C (1999) Head in the WNT: the molecular nature of Spemann's head organizer. Trends Genet. 15:314-319. Nieuwkoop PD, Faber J (1994) Nonnal table of Xenopus laevis. New York: Garland Publishing, Inc. Oliver G, Mailhos A, Wehr R, Copeland NG, Jenkins NA, Gruss P (1995) Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121:4045-4055. Pannese M, Polo C, Andreazzoli M, Vignali R, Kablar B, Barsacchi G, Boncinelli E (1995) The Xenopus homologue of Otx2 is a maternal homeobox gene that demarcates and specifies anterior body regions. Development 121:707-720. Saint-Jeannet JP, He X, Varmus HE, Dawid IB (1997) Regulation of dorsal fate in the neuraxis by Wnt-1 and Wnt-3a. Proc Nat! Acad Sci USA 94:13713-13718. 32 Sheldahl LC, Park M, Malbon CC, Moon RT (1999) Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner. Curr. BioI. 9:695-698. Shi D-L, Goisset C, Boucaut J-C (1998) Expression of Xfz3, a Xenopus frizzled family member, is restricted to the early nervous system. Mech. Dev. 70:35-47. Shi DL, Boucaut JC (2000) Xenopus frizzled 4 is a maternal mRNA and its zygotic expression is localized to the neuroectoderm and trunk lateral plate mesoderm. Mech Dev 94:243-245. Shimizu H, Julius MA, Giarre M, Zheng Z, Brown AM, Kitajewski J (1997) Transformation by Wnt family proteins correlates with regulation of beta-catenin. Cell Growth Differ 8: 1349-1358. Sumanas S, Strege P, Heasman J, Ekker SC (2000) The putative wnt receptor Xenopus frizzled-7 functions upstream of beta- catenin in vertebrate dorsoventralluesoderm patterning. Development 127: 1981-1990. Tada M, Smith JC (2000) Xwntll is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway. Development 127:2227-2238. Turner DL, Weintraub H (1994) Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev. 8:1434-1447. Vinson CR, Conover S, Adler PN (1989) A Drosophila tissue polarity locus encodes a protein containing seven potential transmembrane domains. Nature 338:263-264. Wallingford JB, Rowning BA, Vogeli KM, Rothbacher U, Fraser SE, Harland RM (2000) Dishevelled controls cell polarity during Xenopus gastrulation. Nature 405:81-85. Wolda SL, et al. (1993) Overlapping expression of Xwnt-3A and Xwnt-1 in neural tissue of Xenopus laevis embryos. Dev BioI. 155:46-57. Wolda SL, Moon RT (1992) Cloning and developmental expression of seven additional members of the Wnt family. Oncogene 7:1941-1947. Yang-Snyder J, et al. (1996) A frizzled homolog functions in a vertebrate Wnt signaling pathway. Curr BioI. 6:1302-1306. Zakin LD, Mazan S, Maury M, Martin N, Guenet JL, Brulet P (1998) Structure and expression ofWnt13, a novel mouse Wnt2 related gene. Mech Dev 73: 107-116.