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First published online November 11, 2004
doi: 10.1242/10.1242/dev.01520

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Zebrafish Dapper1 and Dapper2 play distinct roles in Wnt-mediated developmental processes

Joshua S. Waxman^1,2,*, Anne M. Hocking^2,, Cristi L. Stoick^2,3 and Randall T. Moon^2,

¹ Molecular and Cellular Biology Program, University of Washington School of Medicine, Seattle, WA 98195, USA
² Howard Hughes Medical Institute, Department of Pharmacology and Center for Developmental Biology, Box 357750, University of Washington School of Medicine, Seattle, WA 98195, USA
³ Neurobiology and Behavior Graduate Program, University of Washington School of Medicine, Seattle, WA 98195, USA

View larger version (11K): [in a new window]   Fig. 1. Relationship of Dpr family members. Four Dpr sequences had been published previously, while five others were determined from a combination of the GenBank sequence databases. (A) Neighbor joining phylogenetic analysis of 11 Dpr members reveal two monophyletic groups. Each vertebrate except Xenopus has two paralogous Dpr genes. XDpr1 (XDpr1a) and FRODO (XDpr1b) were found to be a Xenopus specific duplication. Zebrafish Dvl2 was defined as the outgroup. Species are: Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Xl, Xenopus laevis; Fr, Fugu rubripes; Dr, Danio rerio. (B) The conserved domains of Dpr family members. The number above the conserved domains is the percent identity and overall similarity (brackets-taking into account conservative changes) from comparison of zebrafish Dpr1 and Dpr2. CD, conserved domain; L2, leucine zipper; P, PDZ binding domain.

View larger version (53K): [in a new window]   Fig. 2. RT-PCR and in situ hybridization analysis of zebrafish dpr1 and dpr2. (A,B) RT-PCR analysis (25 embryos per stage) of dpr1 (A) and dpr2 (B). Zebrafish max is used as a loading/positive control. –RT is a control for genomic DNA contamination. (C,D) In situ hybridization for expression of dpr1 (C, parts a-f) and dpr2 (D, parts a-f), with embryos shown that are representative of at least 25 analyzed per stage. Stages: (a) sphere stage; (b) shield stage; (c) 80% epiboly; (d) tailbud; (e) five somites (in C), three somites (in D); (f) 12 somites. Orientation: (a,b) animal views, dorsal is rightwards; (c,d) dorsal views, anterior is upwards; (e,f) anterior is leftwards. (C, part b) Arrow indicates margin and arrowhead indicates shield. (C, part d) dpr1 (blue), pax2.1 (red, midbrain/hindbrain boundary) and krox20 (red, –r3 and 5). Black arrow indicates dpr1 expression in brain. Black arrowhead indicates dpr1 expression in anterior spinal cord. Red arrow indicates pax2.1. Red asterisk indicates krox20. (C, part e) Black arrow is posterior limit of dpr1(blue) expression. Red arrow is posterior limit of pax6.1(red) expression. Arrowheads indicate r3 and r5. (C, part f) Arrow indicates anterior older somite. Arrowhead indicates staining of posterior, younger somite. Asterisk indicates presomitic mesoderm. Yellow arrow indicates tailbud. (D, part b) Arrow indicates margin and arrowhead indicates shield. (D, part d) Brackets indicate mediolateral banding pattern of staining in lateral mesoderm. (D, part e) Arrow indicates boundary of krox20 (red) and dpr2 (blue) staining. (D, part f) Arrow indicates staining in the anterior of the older somites. Arrowhead indicates presomitic mesoderm.

View larger version (55K): [in a new window]   Fig. 3. Zebrafish dpr1 and dpr2 morphant phenotypes. (A) RT-PCR analysis of efficacy of dpr1 MO. dpr1 splice blocking MO induces the predicted shift to an 1700 band, indicating it abrogates proper splicing of the transcript. Spliced band is at 500 bp. U, uninjected; I, injected. (B) 5'-Dpr2-luciferase construct is blocked in an in vitro transcription/translation reaction. ß-Galactosidase was used as an internal control. (C) dpr1 morphants (b, injected with 12 ng MO) are discernibly smaller but have no major change of cell fates compared with control (a). dpr2 morphants (c, injected with 8 ng MO) have convergent extension defects, which are more apparent in whole embryos (D, parts c,d). However, the notochord is noticeably wider – compare distances between the small posterior arrowheads in c with a and b. dpr1+dpr2 morphants (d) do not have novel phenotypes, indicating they are not redundant. White arrow indicates anterior limit of head. Black arrows indicate distance between opl and en2. Small arrowhead indicates somite. Posterior arrowheads indicate width of notochord. Embryos were flatmounted, with anterior towards the left. (D) Whole-mount in situ hybridizations of dpr2 morphants indicate they are shorter (compare arrowheads in a and c) and the somites and notochord are wider (compare myod staining in b and d), but major specification events are not affected. For C and D, the experiments were repeated four times with comparable results, see text for penetrance of phenotypes. In situ probes used from anterior to posterior were: cathepsinL (catL-hatching gland), opl (telencephalon), en2 (midbrain/hindbrain boundary), krox20 (rhombomeres 3 and 5) and myod (adaxial cells and somites).

View larger version (55K): [in a new window]   Fig. 4. Dpr1 is an enhancer of Wnt/ß-catenin signaling required for ventral and posterior cell fates. (A) Representative embryos for scoring of ventroposteriorization at 24 hours. 0, wild type; 1, slight enlargement of the telencephalon (arrow); 2, strong anteriorization phenotype, enlargement of the head and reduction of the tail; and 3, very strong anteriorization phenotype, super enlargement of the telencephalon (arrowhead) and major loss of trunk and tail. Graph shows percentage of embryos with the respective phenotypes (numbers in brackets indicate the number of embryos used). (B) dpr1, dpr2 and wnt8MO2 morphant phenotypes at the one- to two-somite stage. In situ markers used were opl (telencephalon), pax2.1 (midbrain/hindbrain boundary) and tbx6 (ventrolateral mesoderm). Arrowheads indicate distance between anterior limit of opl and posterior limit of pax2.1. Asterisks indicate anterior limit of tbx6. (a,c,e,g,i,k,m,o) Dorsal is towards the right. (b,d,f,h,j,l,n,p) Dorsal is out of the plane of the page. In all figures, anterior is upwards. Suboptimal dose of wnt8 MO2 is 0.45 ng each MO (0.9 ng total). dpr1 MO dose is 12 ng. dpr2 MO dose is 8 ng. The experiments were repeated five times with comparable results; see text for penetrance of phenotypes.

View larger version (64K): [in a new window]   Fig. 5. dpr2 morphants phenocopy strabismus/trilobite (stbm) morphants, and functionally interact with stbm and wnt11/silberblick. (A) dpr2 morphants (middle-injected with 7.5 ng dpr2 MO) phenocopy stbm morphants (right-injected with 3 ng). Arrow indicates flattened, non-chevron-shaped somite. Arrowhead indicates narrowed eyes. (B) Scoring of embryos that were co-injected with suboptimal doses of stbm and dpr2 MOs. 0, wild type; 1, moderate CE defects; 2, severe CE defects. (C) Percentage of embryos injected with MOs exhibiting phenotype. (D) Scoring of cyclopia index of embryos co-injected with dpr2 MO and wnt11 MO (a-f). dpr2 MO (normal dose, g,h). 0, wild type; 1, narrowing of the eyes; 2, fusion of the lens. (E) Percentage of embryos injected displaying respective phenotypes. Suboptimal (hypomorphic) dose of dpr2 MO is 3 ng, stbm MO is 0.4 ng and wnt11 MO is 1.5 ng. All injections were balanced with control MO. The numbers in brackets indicate the numbers of embryos used.

View larger version (46K): [in a new window]   Fig. 6. Dpr immunoprecipitation, localization and activation of Wnt/ß-catenin target genes. (A) Dpr1-myc and (B) Dpr2-myc immunoprecipitate with Dvl2-HA from Xenopus lysates. (C) Dpr1-myc and (D) Dpr2-myc localization in Xenopus animal caps. Small arrow in D indicates nuclear staining. Large arrow indicates membrane staining. Scale bar: 20 µm in C,D. (E) Injection of 4 ng dpr1 RNA induces ß-catenin target genes, but 4 ng dpr2 RNA does not. (F) Dpr1 and Dpr2 synergize with suboptimal dose of zebrafish Dvl2. dpr1 and dpr2 low dose is 0.25 ng, medium dose is 0.5 ng and high dose is 1.0 ng. ß-galactosidase RNA dose is 1.0 ng. (G) Dpr1 and Dpr2 with internal control ß-gal in TnT reaction (upper). Dpr1-myc and Dpr2-myc western from Xenopus lysates with internal control GFP (lower). (H) Dpr1-Dvl2 synergy is inhibited by Axin. (I) Dpr1 and Dpr2 do not inhibit Wnt8 activation of ß-catenin target genes. (J) Dpr1 and Dpr2 synergize with Dvl2 in activating a ß-catenin-dependent luciferase reporter in HEK 293T cells. The experiment is representative of three independent transfections, with each data point being conducted in triplicate. Unless noted otherwise, 1 ng of dpr1-myc, dpr2-myc and ß-galactosidase RNA, and 0.4 ng dvl2 RNA were used. For I, 1 pg of wnt8 RNA, and 4 ng of dpr1 and dpr2 RNA were used. For J, 400 ng of Dpr1, Dpr2 or ß-galactosidase, and 20 ng of zebrafish Dvl2 were transfected. AU, arbitrary light units.

View larger version (39K): [in a new window]   Fig. 7. Dpr synergizes with Dvl-interacting kinases in Xenopus animal caps. For all injections, unless noted, 1 ng of dpr1, 0.1 ng of ck1, 0.1 ng of ck1-DN, 1 ng of par1, 2 ng of par1-KN, 0.4 ng of dvl2, 1 ng of ck2, 1 ng of ck2ß, 1 ng of gbp, 1 ng of ß-galactosidase, 0.5 ng of dvlpgb RNA were used. Low dose of dpr1 RNA is 0.5 ng for B and E. (A) Dvl2 synergizes with Dpr1, CK1, CK2, GBP and Par1. (B) Zebrafish Dpr1 synergizes with CK1, but not CK1-DN. (C) CK2 can synergize with Dpr1 (Dvl2 is shown as a positive control). (D) Dpr1 synergizes with Par1, but not Par1-KN. Low dose of dpr1 RNA is 0.5 ng. (E) Dpr1 does not synergize well with GBP. (F) Dpr1 does not synergize with the Dvl2PGB.

View larger version (31K): [in a new window]   Fig. 8. Dpr synergy requires CK1 or CK2. (A) Dvl2 or ß-catenin activation of canonical target genes does not require CK1, but Dvl2-CK2 synergy does require CK1. Lanes 1 and 2, 0.4 ng of dvl2, 1 ng of ck2 and 1 ng of ck2ß RNA; lanes 3 and 4, 0.8 ng dvl2 RNA; lanes 5 and 6, 0.1 ng stabilized ß-catenin RNA. (B) Dpr1-Dvl2 and Dpr1-Par1 synergy requires CK1, but Dpr1-CK2 synergy does not. The amounts of RNA injected were 1 ng of dpr1, 1 ng of par1, 0.4 ng of dvl2, 1 ng of ck2 and1 ng of ck2ß. CKI-7 (5 nl of 2.5 mM) was co-injected in experiments in A and B. (C) Table of Dpr1 synergistic interactions and requirement on CK1. Asterisk indicates higher doses of Dvl2 RNA that could activate alone did not require CK1, but Dvl2-CK2 synergy did require CK1.

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