En1 and Wnt7a Interact with Dkk1 During Limb Development in the Mouse

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Developmental Biology 272 (2004) 134–144 www.elsevier.com/locate/ydbio

En1 and Wnt7a interact with Dkk1 during limb development in the mouse

Maja Adamska, Bryan T. MacDonald, Zubair H. Sarmast, Edward R. Oliver, and Miriam H. Meisler*

Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109-0618, USA Received for publication 21 January 2004, revised 12 April 2004, accepted 13 April 2004 Available online 25 May 2004

Abstract

Wnt signaling plays an essential role in induction and development of the limb. Missing digits are one consequence of the reduced Wnt signaling in Wnt7a null mice, while extra digits result from excess Wnt signaling in mice null for the Wnt antagonist Dkk1. The extra digits and expanded apical ectodermal ridge (AER) of Dkk1-deficient mice closely resemble En1 null mice. To evaluate the in vivo interaction between En1 and the canonical Wnt signaling pathway, we generated double and triple mutants combining the hypomorphic doubleridge allele of Dkk1 with null alleles of En1 and Wnt7a. Reducing Dkk1 expression in Dkk1d/+Wnt7aÀ/À double mutants prevented digit loss, indicating that Wnt7a acts through the canonical pathway during limb development. Reducing Dkk1 levels in Dkk1d/dEn1À/À double mutants resulted in severe phenotypes not seen in either single mutant, including fused bones in the autopod, extensive defects of the zeugopod, and loss of the ischial bone. The subsequent elimination of Wnt7a in Dkk1d/dEn1À/ÀWnt7aÀ/À triple mutants resulted in correction of most, but not all, of these defects. The failure of Wnt7a inactivation to completely correct the limb defects of Dkk1d/dEn1À/À double mutants indicates that Wnt7a is not the only gene regulated by En1 during development of the mouse limb. D 2004 Elsevier Inc. All rights reserved.

Keywords: Limb development; Wnt signaling; AER; Dkk1; En1; Wnt7a

Introduction null mice, footpads develop on the dorsal as well as ventral surface, forming ‘double-ventral’ paws. Posterior Wnt signaling is one of the key pathways in vertebrate digits are often missing, indicating that Wnt7a is required limb development (Church and Francis-West, 2002; Martin, for maintenance of posterior limb tissue (Parr and McMa- 2001). Early in limb development, canonical Wnt signaling hon, 1995). The loss of posterior limb tissue is preceded through beta-catenin governs formation of the apical ecto- by reduced expression of Shh in the zone of polarizing dermal ridge (AER), the signaling center that regulates limb activity (ZPA), which directs anterior–posterior specifica- outgrowth. Absence of Wnt3 or beta-catenin in limb ecto- tion during limb development. derm results in defective establishment of the AER (Barrow Overactivation of Wnt signaling by expression of a gain- et al., 2003; Soshnikova et al., 2003). In mice deficient for of-function mutation of beta-catenin results in dramatic both Lef1 and Tcf1, transcription factors mediating canonical expansion of the AER (Soshnikova et al., 2003). A null Wnt signaling, limb development is arrested at the early allele of the Wnt antagonist Dkk1 also leads to expansion of limb bud stage (Galceran et al., 1999). the AER (Mukhopadhyay et al., 2001). doubleridge is a Wnt7a is expressed in dorsal limb ectoderm and hypomorphic allele of Dkk1, designated Dkk1d, with ex- governs formation of dorsal limb structures. In Wnt7a pression levels reduced to 10% of normal in developing limbs (MacDonald et al., 2004). Dkk1d/d mice develop a broad AER and also display postaxial polysyndactyly in the forelimbs, as seen in the Dkk1 null mice (Adamska et al., * Corresponding author. Department of Human Genetics, University of Michigan, 4808 Medical Science II, 1241 East Catherine Street, Ann Arbor, 2003, Mukhopadhyay et al., 2001). MI 48109-0618. Fax: +1-734-763-9691. The En1 homeobox transcription factor is expressed E-mail address: [email protected] (M.H. Meisler). in the ventral limb ectoderm, where it represses expres-

0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.04.026 M. Adamska et al. / Developmental Biology 272 (2004) 134–144 135 sion of Wnt7a. En1 null limbs display an expanded 323-bp fragment from the null allele; primers Wnt7aF AER, polydactyly, and syndactyly (Loomis et al., 1996; and Wnt7aR1 amplify a 175-bp fragment from the wild- Wurst et al., 1994).InEn1À/ÀWnt7aÀ/À double mutants, type allele. Both fragments are obtained from heterozy- there is no expansion of the AER or polysyndactyly, gous DNA. demonstrating that the limb phenotype of En1 null mice is mediated by ectopic expression of Wnt7a in the Other methods ventral limb ectoderm (Cygan et al., 1997; Loomis et al., 1998). Embryos were genotyped by PCR of genomic DNA The limb abnormalities of En1 and Dkk1 mutant mice from embryonic membranes. E0.5 was considered to be are strikingly similar. The similarity could result from noon of the day when the vaginal plug was found. direct regulation of one gene by the other or from similar Whole mount in situ hybridization was carried out with effects of both on a common downstream pathway. To a digoxygenin-labeled probe (Bober et al., 1994) using examine the in vivo interaction of Dkk1 and En1,we BM Purple (Roche) as the substrate for alkaline phos- combined the En1 null genotype with Dkk1 alleles phatase. Antisense mRNA probes for Dkk1 (Glinka et producing a range of Dkk1 expression. We also generated al., 1998), En1 (Wurst et al., 1994), Fgf8 (Crossley and triple mutant Dkk1d/dEn1À/ÀWnt7aÀ/À mice to determine Martin, 1995), Wnt7a (Parr and McMahon, 1995), Shh whether elimination of Wnt7a expression would compen- (Echelard et al., 1993), and Wnt5a (Yamaguchi et al., sate for the synergistic effects in the Dkk1d/dEn1À/À 1999) were prepared as described. Cartilage and bone double mutants. were stained with Alcian blue and Alizarin red (Kimmel and Trammell, 1981). Images of cleared skeletons and whole embryos were captured with a DEI 750 Optronics Methods digital camera and processed using Adobe Photoshop.

Mice Results The doubleridge mutant arose in this laboratory and was previously described (Adamska et al., 2003; Mac- Expression of Dkk1 and En1 in wild-type and single mutant Donald et al., 2004). En1 null mice (Hanks et al., 1995) mice were obtained from Dr. A. Joyner. The Wnt7aUM null allele arose at the University of Michigan on an ICR Expression of Dkk1 and En1 in developing limb of background. The triple mutant mice were generated by wild-type mice has been previously described (Loomis intercrossing Dkk1d/dEn1+/ÀWnt7a+/À mice. All experi- et al., 1998; Monaghan et al., 1999; Mukhopadhyay et ments were performed on a mixed genetic background. al., 2001). At E9.5, En1 and Dkk1 are coexpressed in Animals of the same genotype displayed similar pheno- the ventral ectoderm, and Dkk1 is also expressed in types independent of the specific cross in which they the ventral mesenchyme. At E10.5, En1 continues to were generated. All experiments were carried out with the be expressed in the ventral ectoderm and is also approval of the University of Michigan animal use strongly expressed in the ventral portion of the AER. committee and in accordance with NIH requirements. At E10.5, Dkk1 transcripts are present throughout the AER and in the posterior and anterior mesenchyme of Genotyping the limb bud. To determine whether the expression of En1 is The En1 null allele was detected by amplification of regulated by Dkk1, we analyzed mice homozygous for a 200-bp band with primers specific for the LacZ the hypomorphic allele. The expression of En1 was not sequence: LaczF, GCG ATG TCG GTT TCC GCG altered in Dkk1d/d limbs at E9.5 (Adamska et al., 2003) AG, and LaczR, GTA CCA CAG CGG ATG GTT or at E10.5, when En1 transcripts are present in the CGG (A. Joyner, personal communication). The wild- AER and the ventral ectoderm (Fig. 1A). Thus, no effect type En1 allele was detected by amplifying a 125-bp of Dkk1 deficiency on En1 expression was detected. fragment with the primers En1F, AGC CGG AGC CTA To determine whether En1 influences expression of AAA GTC AG, and En1R, CAC GCT GTC TCC ATC Dkk1, we examined En1 null embryos. Expression of GCT. Dkk1 was genotyped as described by MacDonald Dkk1 did not differ between mutant and wild-type limbs et al. (2004). The Wnt7aUM null allele was genotyped in at E9.5, when there is no morphological difference a three primer assay containing the forward primer between wild-type and En1 null limbs (Fig. 1B).AtE Wnt7aF, GAG CAT CTG CCA TTA GCA AG, and 10.5, Dkk1 was expressed in the expanded AER of En1 the reverse primers Wnt7aR1, GCA CAG CCA TCT null limbs at a level comparable to wild type (Fig. 1C). CAT TAG CT, and Wnt7aR2, TGT GCA CTC AAG Dkk1 was also present at comparable levels in anterior GCT CTT GA. Primers Wnt7aF and Wnt7aR2 amplify a and posterior mesenchyme of mutant and wild-type limb 136 M. Adamska et al. / Developmental Biology 272 (2004) 134–144

Fig. 1. En1 and Dkk1 expression in wild-type and mutant mice. (A) Normal expression of En1 in ventral limb ectoderm of Dkk1d/d limb bud. (B) Normal expression of Dkk1 in E9.5 En1 null limb bud. (C) Expression of Dkk1 in the expanded AER of En1 null limb bud at E10.5. buds (Fig. 1C). Thus, there is no apparent effect of En1 Early development of limb abnormalities in Dkk1d/dEn1À/À deficiency on Dkk1 expression. double mutants

Novel limb phenotype due to combined deficiency of Dkk1 To study the molecular basis for the limb defects in and En1 double mutant mice, we examined the expression of markers for the three limb signaling centers: Fgf8 (AER), Shh (ZPA), In Dkk1d/d and En1À/À single homozygotes, limb and Wnt7a (dorsal limb ectoderm). In Dkk1d/d and En1À/À abnormalities are restricted to the autopod (Figs. 2A– single mutants, delayed compaction of the AER is C). Postaxial polysyndactyly is more severe in Dkk1d/d evidenced by expansion of Fgf8 expression in the ventral mice than in En1À/À. To investigate genetic interaction limb ectoderm. In doubleridge limbs, the two Shh domains between these genes, we studied double mutant mice are separated by the expanded AER; while in En1 null obtained from several crosses between Dkk1d/d and limbs, the single Shh domain extends below the AER. En1+/À mice. All classes of progeny were recovered in Wnt7a is restricted to dorsal limb ectoderm in wild-type the expected Mendelian frequencies. The double hetero- and Dkk1d/d limbs, but in En1À/À mice it is ectopically zygotes did not exhibit any abnormalities. expressed in the ventral limb ectoderm below the AER Double homozygotes displayed extensive and severe (Adamska et al., 2003; Cygan et al., 1997; Loomis et al., abnormalities of forelimbs and hindlimbs (Figs. 2E and 1998). F). In most cases, digits were not externally visible. The In En1À/À and Dkk1d/d single mutant embryos at E10.5, autopod and zeugopod of Dkk1d/dEn1À/À limbs were the Fgf8-positive AER encompasses half of the ventral severely malformed. The ulna, radius, fibula, and tibia forelimb ectoderm (Fig. 3A).InDkk1d/dEn1À/À double were thickened and bent, and ossification centers were homozygotes, Fgf8 expression extends through the entire much smaller than in littermate and age-matched controls ventral limb ectoderm from E10 through E11.5 (Figs. 3A (Figs. 2E and F). The position of the deltoid tuberosity and 6C). Shh expression in double mutant limbs is similar to was shifted to the proximal end of the humerus (Fig. 2E), that in Dkk1d/d at E10.5 (Fig. 3B), but at E11.5 Shh is and in 8/10 Dkk1d/dEn1À/À mice the ischial bone of the limited to two weak expression domains in the posterior and pelvic girdle was missing (Fig. 2F). The hindlimb stylo- ventral mesenchyme (Fig. 3C). Wnt7a is correctly excluded pod and pectoral girdle were normal, and the only other from the AER of double homozygotes but is abnormally skeletal defect in the double mutants was the sternal expressed in the proximal region of the ventral limb bud defect previously observed in En1À/À single mutants (Fig. 3D). (Wurst et al., 1994). The forelimbs of double homozygous embryos are En1À/ÀDkk1d/+animals displayed a phenotype interme- visibly smaller than littermates at E11.5 (Fig. 3E). diate between En1À/À null mice and double homozygotes. Apoptotic cells, detected by TUNEL staining, were Severe bone syndactyly was often present in the forelimb. limited to the AER, as in wild-type limbs. No increase The ulna and radius were thick and bent, and the location in the intensity of TUNEL staining was observed, of the deltoid tuberosity was shifted (Fig. 2D). Except for indicating that increased cell death is not responsible occasional ventral digits, the hindlimbs were normal (not for the reduced size (Fig. 3F). The relative size of the shown). Dkk1d/dEn1+/À mice were identical to Dkk1d/d, mutant limb bud decreases as development progresses. with postaxial polysyndactyly of forelimbs (Adamska et By E12.5, there is a striking difference in shape and size al., 2003, and Fig. 2B). Syndactyly was sometimes limited of the limb in double homozygotes and their littermates to soft tissue, but bone was affected in other individuals. (Fig. 3G). M. Adamska et al. / Developmental Biology 272 (2004) 134–144 137

Fig. 2. Combined loss of En1 and deficiency for Dkk1 results in a novel, severe limb phenotype. Top: right forelimb, dorsal view. Bottom: limbs were stained for cartilage (blue) and bone (red). Note the increased severity with decrease of Dkk1 expression. (B and C) Only autopod elements are affected in Dkk1 and En1 single mutants. (D) Dkk1d/+En1À/À forelimbs display defects in the ulna (u) and radius (r) and proximal shifting of the deltoid tuberosity (dt). (E) In double homozygotes, the tibia (t) and fibula (f) are thick, bent, and poorly ossified. (F) Arrow indicates missing ischial bone in Dkk1d/dEn1À/À pelvic girdle.

Since Wn5a null mice display shortening of limb 3I). The relatively normal appearance of the initial mesen- bones similar to that in Dkk1d/dEn1À/À double homozy- chyme condensation of the zeugopod in Dkk1d/dEn1À/À gotes (Topol et al., 2003; Yamaguchi et al., 1999),we embryos at E13.5, compared with E16.5, indicates that examined expression of Wnt5a at E11.5 (Fig. 3E). Dkk1 and En1 are required for chondrogenesis during the Although the shape of the limbs was altered in the intervening 3 days. double mutants, the level of Wnt5a expression appeared normal. Identification of a spontaneous null allele of Wnt7a To determine whether the zeugopod malformations in neonatal limbs reflect earlier defects in condensation of the Mice with phenotypes strikingly similar to Wnt7a null mesenchyme, E13.5 and E16.5 limbs were stained with mice were identified in an ICR line maintained by Alcian blue and Alizarin red. At E13.5, the zeugopod brother–sister mating. Phenotypes included dorsal foot- elements were close to normal, while autopods already pads, posterior digit loss, and infertility of both males and displayed severe deformations (Fig. 3H). At E16.5, dramat- females. Amplification of genomic DNA from affected ic deformations of the zeugopod were clearly visible (Fig. individuals, using primers flanking the four exons of 138 M. Adamska et al. / Developmental Biology 272 (2004) 134–144

Fig. 3. Development of limb defects in Dkk1d/dEn1À/À embryos. (A) No compaction of the AER in forelimb buds of double homozygous at E10.5. (B and C) Shh expression level is normal at E10.5 but dramatically reduced at E11.5. (D) Wnt7a expression is correctly excluded from the expanded AER of E11.5 hindlimb bud. (E) Normal expression of Wnt5a. (F) TUNEL staining reveals no increase in apoptosis at E10.5. (G) The autopod of Dkk1d/dEn1À/À limb is dramatically smaller than in the littermate controls at E12.5. The AER is marked by Fgf8 expression. (H) Alcian blue staining of cartilage in double mutants at E13.5 shows severe defects of mesenchyme condensation in the autopod and nearly normal zeugopod bones. (I) Alcian blue (cartilage) and Alizarin red (bone) staining of E16.5 forelimbs reveal dramatically misshapen zeugopod elements in the double mutant.

Wnt7a (Parr et al., 1998), demonstrated that exons 3 and 4 Wnt7a and Dkk1 cooperate in establishing digit number were deleted. A 54-kb deletion that includes exons 3 and 4 of Wnt7a, but does not disrupt adjacent genes, was Dkk1d/d mice were crossed to Wnt7a+/À mice and prog- detected by PCR analysis of 150 kb of genomic DNA eny were intercrossed or backcrossed to generate single and from the region. The precise position of the deletion was double mutants. Most of the Wnt7aÀ/À single mutant confirmed by amplification of a 2-kb fragment from offspring, 30/38, lacked posterior digits in one or both genomic DNA of homozygous affected mice using a forelimbs (Fig. 4B), consistent with previous reports (Parr forward primer terminating at 91,798,090 bp and a reverse and McMahon, 1995). In the Dkk1d/+Wnt7aÀ/À offspring, primer terminating at 91,854,167 bp on mouse chromo- the partial reduction of Dkk1 was sufficient to restore some 6 (Ensembl v. 16.30.1 6 May 2003—Mouse Genome normal digit number in all of the animals obtained (37/37) Assembly NCBI 30, , http://www.ensembl.org/Mus_mus- (Fig. 4D). culus). The new allele, designated Wnt7aUM, was used to In single mutant Wnt7aÀ/À limbs, decreased expression investigate the genetic interaction of Dkk1 and Wnt7a. of Shh is evident at E10.5, and there is reduction of tissue in M. Adamska et al. / Developmental Biology 272 (2004) 134–144 139

Fig. 4. Dkk1 and Wnt7a interact to establish digit number. (A–D) Reduction of Dkk1 expression prevents reduction of Shh expression, abnormal shape of limb bud, and digit loss in Wnt7a nulls. (E and F) Loss of Wnt7a does not prevent the expansion of AER and syndactyly in Dkk1d/d limbs but inhibits formation of extra digit. Left: dorsal view of adult forelimbs; middle: distal and ventral views of E11.5 forelimbs hybridized with Fgf8 probe; right: posterior view of E10.5 forelimbs hybridized with Shh probe. the posterior part of the limb bud (Fig. 4B).InDkk1d/+ with an occasional small postaxial skeletal element (Figs. 4F Wnt7aÀ/À double mutants, the expression of Shh and the and 5C, arrow). shape of the limb bud were corrected (Fig. 4D). Two phenotypes were not altered in double mutants. In Single mutant Dkk1d/d mice have six digits in their Dkk1d/dWnt7aÀ/À embryos, there was no correction of the forelimbs (Adamska et al., 2003).InDkk1d/dWnt7aÀ/À ventrally expanded AER seen in Dkk1d/d mice (Figs. 4E double homozygotes, digit number was restored to five, and F). In addition, the ectopic dorsal footpads character- 140 M. Adamska et al. / Developmental Biology 272 (2004) 134–144

Fig. 5. Loss of Wnt7a results in partial rescue of the Dkk1d/dEn1À/À phenotype. (A) Dkk1d/dEn1À/À mice display severe fusions of autopod elements, thickening and poor ossification of the ulna (u), radius (r), fibula (f), and tibia (t), proximal shifting of the deltoid tuberosity (dt), and loss of the ischial bone (i). (B) In triple Dkk1d/dEn1À/ÀWnt7aÀ/À mutants, zeugopod elements of the hindlimb are normal, the ischial bone is present, and fusions of autopod elements are less severe. The ulna and radius are partially corrected, forelimb digits are severely fused, and the first digit of the hindlimbs is missing. The olecranon process (o) is not fully attached to the ulna. (C) Dkk1d/dWnt7aÀ/À limbs are characterized by a normal zeugopod and normal digit number in hindlimbs and forelimbs. A small postaxial element is sometimes present in the forelimb (arrow). istic of Wnt7aÀ/À mice were not affected by Dkk1 genotype 1998). To determine whether inactivation of Wnt7a could (Fig. 4). correct the abnormalities of Dkk1d/dEn1À/À limbs, we generated triple homozygous mice. All genotypes were recov- Partial correction of Dkk1, En1 phenotypes by removal of ered at the predicted frequencies (Table 1).Thesevere Wnt7a abnormalities were largely corrected in the triple mutants, including restored size of the limb, shape of the zeugopod The expanded AER, syndactyly, and polydactyly ob- elements, morphology of the digits, and presence of the served in En1 null limbs can be completely prevented by ischial bone (Fig. 5B). Other abnormalities were not com- inactivation of Wnt7a (Cygan et al., 1997; Loomis et al., pletely corrected (Fig. 5). Forelimbs of the triple mutants M. Adamska et al. / Developmental Biology 272 (2004) 134–144 141

Table 1 Genotypes of offspring from crosses generating double and triple mutants Cross Offspring En1+/+ En1+/À En1À/À (n) Wnt7a+/+ Wnt7a+/À Wnt7aÀ/À Wnt7a+/+ Wnt7a+/À Wnt7aÀ/À Wnt7a+/+ Wnt7a+/À Wnt7aÀ/À Dkk1d/dEn1+/ÀWnt7a+/À 120 9 10 6 19 38 15 5 11 7 intercross Dkk1d/dEn1+/ÀWnt7a+/À Â 34 1 7 1 5 10 4 1 4 1 Dkk1d/+En1+/ÀWnt7a+/À Dkk1d/dEn1+/ÀWnt7a+/À Â 67 3 10 4 10 10 5 8 10 7 En1+/ÀWnt7a+/À En1+/ÀWnt7a+/À intercross 129 6 16 4 20 34 15 7 16 11 Expected ratio 1 2 1 2 4 2 1 2 1 Genotype frequencies did not differ from the Mendelian predictions indicated in the bottom row. displayed severe bone and soft tissue fusions similar to these than the Dkk1d/dEn1À/À double mutant but are more severe in Dkk1d/dEn1À/À limbs, and nails were missing in some than the Dkk1d/dWnt7aÀ/À double mutant (Fig. 6D). The individuals. The bones of the zeugopod were often thick and forelimb AER occupies approximately half of the ventral misshapen like those of Dkk1d/+En1À/À forelimbs (Fig. 5B). ectoderm, with two well-defined borders, while the expan- The olecranon process of the ulna was partly detached and sion of the hindlimb AER is restricted to the most proximal sometimes free-floating (Fig. 5B). All of the triple homo- limb region (Fig. 6D). zygotes displayed loss or reduction of the most anterior digit of the hindlimb, a phenotype not observed in double mutations (Fig. 5B). In addition, the dorsal footpads char- Discussion acteristic of Wnt7aÀ/À mice were present in all four limbs of the triple mutants. Removal of Wnt7a thus compensated for Dkk1 and En1 act synergistically during mouse limb some, but not all, of the abnormalities of Dkk1d/dEn1À/À development mice. Mice deficient for Dkk1, an inhibitor of the canonical Morphology of the AER in triple mutants Wnt signaling pathway, develop a ventrally expanded AER, syndactyly, and polydactyly (Adamska et al., 2003; Mac- To determine whether the AER is expanded in the triple Donald et al., 2004; Mukhopadhyay et al., 2001). En1 null mutants, we examined Fgf8 expression at E11.5 (Fig. 6). mice also display ventral expansion of the AER, syndactyly, The AER of wild-type limbs is represented by a thin line of and polydactyly, which are combined with dorsal–ventral Fgf8 expression (Fig. 6A).InDkk1d/d single mutants and patterning defects (Cygan et al., 1997; Loomis et al., 1998; Dkk1d/dWnt7aÀ/À double mutants, the forelimb AER is Wurst et al., 1994). To investigate in vivo interactions broader than wild type, with strong Fgf8 expression in between En1 and the canonical Wnt signaling pathway, the well-defined dorsal and ventral borders (Figs. 6B and we examined the phenotypes of double and triple mutant E).InDkk1d/dEn1À/À limbs, the greatly expanded AER mice, as summarized in Table 2. occupies entire ventral portion of the limb and lacks a Combining the En1 null allele with reduced expression visible ventral border, with Fgf8 expression gradually of Dkk1 in Dkk1d/dEn1À/À mice increased the severity of decreasing in the proximal ectoderm (Fig. 6C). The AER autopod defects and produced novel abnormalities in other abnormalities in the triple mutant are clearly less extensive elements of the limb. The domain of Fgf8 expressing cells

Fig. 6. Intermediate expansion of the AER in triple mutant Dkk1d/dEn1À/ÀWnt7aÀ/À mice. E11.5 limbs were hybridized with an Fgf8 cDNA probe. The ventral view of the right limb bud is shown. 142 M. Adamska et al. / Developmental Biology 272 (2004) 134–144

Table 2 Phenotypes of single, double, and triple mutants Genotype Ischial bone Zeugopod abnormalities Digit number missing Dkk1 En1 Wnt7a Forelimb Hindlimb Forelimb Hindlimb Single mutants d/d +/+ +/+ – – – 6 5 +/+ À/À +/+ – – – 5+ 5 +/+ +/+ À/À –––45 Double mutants +/+ À/ÀÀ/À –––45 d/d +/+ À/À –––5+5 d/d À/À +/+ + + + fused fused Triple mutant d/d À/ÀÀ/À – + – fused 4 More than eight animals of each genotype were examined. The indicated phenotypes were present in z75% of individuals. was expanded to include most of the ventral ectoderm of To evaluate genetically the interaction of Wnt7a with the limb. The skeletal elements of the autopod were Dkk1 during limb development, we analyzed mice that were severely fused, resulting in apparent digit loss. The bones null for Wnt7aÀ/À and also had reduced expression of Dkk1. of the zeugopod were dramatically misshapen, thickened, The limb abnormalities in Wnt7a null mice include loss of and poorly ossified. Since there was no delay in the initial posterior tissue in the limb bud, missing posterior digits, and mesenchymal condensation of zeugopod elements, it ventralization of the limbs with formation of footpads on appears that the effects of combined En1 and Dkk1 dorsal and ventral surfaces (Chen and Johnson, 2002; Parr deficiency occurred later during the outgrowth of the and McMahon, 1995; Parr et al., 1998). Although the zeugopod bones. During this period, Dkk1 is known to expression domains of Wnt7a and Dkk1 do not overlap, be expressed in cells lining the perichordium and in cells their expression is closely juxtaposed in the AER. Digit loss involved in mineralization of bone (Monaghan et al., was rescued by the modest reduction of Dkk1 in Dkk1d/+ 1999). However, En1 transcripts have not been detected Wnt7aÀ/À limbs. It has been proposed that Wnt7a maintains in developing bones (Davis and Joyner, 1988, and un- posterior limb tissue by up-regulation of Shh, which in turn published observations). The zeugopod phenotype may promotes expression of Fgfs in the AER (Niswander et al., therefore reflect changes in gene expression early in 1994; Parr and McMahon, 1995). Dkk1d/+limbs do not development that only become evident later as the long display up-regulation of Shh expression, but the reduction bones grow. of Shh expression seen in Wnt7a null limbs was prevented Dkk1d/+mice have normal AER and develop normal in Dkk1d/+Wnt7aÀ/À mice. The effects of Dkk1 on Shh limbs, but Dkk1d/+En1À/À double mutants exhibit zeugopod expression and digit loss in the Wnt7a double mutants abnormalities not present in the En1À/À single mutant. provide evidence that derepression of the canonical Wnt Exacerbation of the En1À/À phenotype in this case is clearly pathway can compensate for Wnt7a deficiency during not due to superimposition of two mutant phenotypes but development of the mouse limb. This is consistent with reflects underlying interactions between En1 and the canon- the model that Wnt7a acts through the canonical pathway to ical Wnt signaling pathway. regulate development of the posterior digits. The defective dorsal or ventral polarity in Wnt7a null Wnt7a maintains posterior limb tissue through the limbs was not corrected in the Dkk1/Wnt7a double mutant, canonical Wnt pathway consistent with the lack of Dkk1 expression in the dorsal limb. The present experiments thus do not address the The phenotype of Dkk1d/dEn1À/À double homozygotes mechanism of Wnt7a action on Lmx1b in the dorsal limb. may reflect synergistic hyperactivation of Wnt signaling due to ectopic expression of Wnt7a combined with reduced Restriction of Wnt7a expression is not the only function of expression of the antagonist Dkk1. It is not established En1 during limb development whether Wnt7a acts through the canonical Wnt pathway during development of the limb. In the chicken limb, over- Wnt7a is misexpressed in the ventral limb ectoderm in expression of Wnt7a did not affect formation of the AER, the absence of En1, leading to expansion of the AER. In although treatment with Wnt3a did produce expansion of the En1À/À Wnt7aÀ/À mice, the lack of misexpressed Wnt7a AER (Kengaku et al., 1998). In the mouse, the loss of Lrp6 prevents the expansion of the AER seen in En1 null mice generates multiple defects that combine the effects of single (Cygan et al., 1997; Loomis et al., 1998). This observation mutations in Wnt1, Wnt3a, and Wnt7a (Pinson et al., 2000), has been interpreted as indicating that the sole function of suggesting that Wnt7a is acting through the canonical En1 during embryonic limb development is repression of pathway. Direct biochemical interaction of Wnt7a with Wnt7a expression in the ventral limb ectoderm. If that Lrp6 and Dkk1 was recently demonstrated in rat PC12 cells were the case, then the limbs of triple mutant Dkk1d/d (Caricasole et al., 2003). En1À/ÀWnt7aÀ/À mice would be undistinguishable from M. Adamska et al. / Developmental Biology 272 (2004) 134–144 143 the Dkk1d/dWnt7aÀ/À double mutants. In fact, removal of Church, V.L., Francis-West, P., 2002. Wnt signalling during limb develop- Wnt7a expression resulted in significant but not complete ment. Int. J. Dev. Biol. 46, 927–936. d/d À/À Crossley, P.H., Martin, G.R., 1995. The mouse Fgf8 gene encodes a family rescue of the complex Dkk1 En1 phenotype (Table 2). of polypeptides and is expressed in regions that direct outgrowth and The defects of the hindlimb zeugopod and ischial bone were patterning in the developing embryo. Development 121, 439–451. corrected, but the anterior digit was missing or severely Cygan, J.A., Johnson, R.L., McMahon, A.P., 1997. Novel regulatory inter- reduced in the triple mutant. The forelimbs of the triple actions revealed by studies of murine limb pattern in Wnt-7a and En-1 mutant were similar to Dkk1d/+En1À/À mice, with severe mutants. Development 124, 5021–5032. Davis, C.A., Joyner, A.L., 1988. Expression patterns of the homeo box- fusions of autopod elements, and thickened ulna and radius. containing genes En-1 and En-2 and the proto-oncogene int-1 diverge d/d À/À Since some of the Dkk1 En1 defects were not mediated during mouse development. 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