Doubleridge, a mouse mutant with defective compaction of the apical ectodermal ridge and normal dorsal–ventral patterning of the limb

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doubleridge, a mouse mutant with defective compaction of the apical ectodermal ridge and normal dorsal–ventral patterning of the limb Maja Adamska,1 Bryan T. MacDonald,1 and Miriam H. Meisler* Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109-0618, USA Received for publication 20 September 2002, revised 26 November 2002, accepted 6 December 2002

Abstract doubleridge is a transgene-induced mutation characterized by polydactyly and syndactyly of the forelimbs. The transgene insertion maps to the proximal region of chromosome 19. During embryonic development of the mutant forelimb, delayed elevation and compaction of the apical ectodermal ridge (AER) produces a ridge that is abnormally broad and flat. Fgf8 expression persists in the ventral forelimb ectoderm of the mutant until E10.5. Strong expression of Fgf8 and other markers at the borders of the AER at E11.5 gives the appearance of a double ridge. At E11.5, apoptotic cells are distributed across the broadened ridge, but at E13.5, there is reduced apoptosis in the interdigital regions. The Shh expression domain is widely spaced at the posterior margin of the AER. The doubleridge AER is morphologically similar to that of En1 null mice, but the expression of En1 and Wnt7a is properly restricted in doubleridge, and the dorsal and ventral structures are correctly determined. doubleridge thus exhibits an unusual limb phenotype combining abnormal compaction of the AER with normal dorsal/ventral patterning. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Limb development; AER; Dorsal–ventral patterning; Fgf8; En1; Polydactyly; Syndactyly; Mouse

Introduction Development of the vertebrate limb depends on establishment of three axes: proximal– distal (shoulder to fingertips), anterior–posterior (thumb to small finger), and dorsal– ventral (knuckle to palm). Patterning of these axes is one of the best understood developmental systems (Capdevilla and Izpisu´a Belmonte, 2001), and many of the required genes have been identified. We describe here a new mouse mutant that will facilitate better understanding of the interactions between the components of this pathway. The apical ectodermal ridge (AER) is a distinct stripe of stratified squamous epithelium on the distal margin of the limb bud that controls proximal– distal growth of the limb. AER activity is mediated by expression of FGFs, and FGFsoaked beads can substitute for the removed AER (Niswan-

* Corresponding author. Fax: ⫹1-734-763-9691. E-mail address: [email protected] (M.H. Meisler). 1 These authors made equal contributions and should be considered co-first authors.

der et al., 1993). Fgf8 expression is induced by Fgf10 from the lateral plate mesoderm, and mice lacking Fgf10 are born without limbs (Min et al., 1998; Sekine et al., 1999). Elimination of Fgf8 expression in the AER results in limbs that are reduced in size and missing skeletal elements (Lewandoski et al., 2000; Moon and Capecchi, 2000). Limbs deficient in both Fgf4 and Fgf8 do not develop at all (Sun et al., 2002). FGFs from the AER induce proliferation and maintain the undifferentiated state of the underlying mesenchyme (reviewed by Martin, 1998). Recent evidence indicates that proximal– distal patterning is prespecified early in the developing limb bud (Dudley et al., 2002; Sun et al., 2002; see also Chiang et al., 2001). The zone of polarizing activity (ZPA), a cluster of mesenchymal cells expressing Shh that is located directly below the posterior margin of the AER, is a source of posteriorizing signals. Expression of Shh in the ZPA depends on FGFs from the AER (Sun et al., 2002). Extra digits can be induced by grafting the ZPA or SHH-soaked beads into the limb bud (reviewed by Sanz-Ezquerro and Tickle, 2001). Several mutants with preaxial polydactyly display an ec-

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M. Adamska et al. / Developmental Biology 255 (2003) 350 –362

topic Shh expression domain in the anterior limb bud (Chan et al., 1995; Sharpe et al., 1998). Dorsal–ventral polarity is determined by the nonridge limb bud ectoderm. Expression of En1 in the ventral ectoderm represses Wnt7a, which is restricted to the dorsal ectoderm. Wnt7a induces Lmx1b, a transcription factor promoting dorsal fate, in the dorsal mesenchyme. In En1 null mice, the ectopic expression of Wnt7a in the ventral ectoderm and Lmx1b in the ventral mesenchyme results in limbs with double-dorsal characteristics (Cygan et al., 1997; Loomis et al., 1996, 1998). The limbs of Wnt7a null mice lack distal expression of Lmx1b and are double ventral (Parr and McMahon, 1995; Parr et al., 1998). The AER forms on the border between ventral cells expressing En1 and dorsal cells expressing Wnt7a. AER markers are initially expressed in a broad ventral region of the mouse limb bud, and it has been suggested that the AER forms by compaction of this region (Loomis et al., 1998). In En1 null mice, the ventral ectoderm fails to compact, leading to expanded ventral expression of AER markers, such as Fgf8 and Dlx2. The AER is normal in Wnt7a null mice and also in Wnt7a, En1 double mutants (Cygan et al., 1997; Loomis et al., 1998). The doubleridge mutant has early defects in formation of the AER. We have characterized the expression of early markers in the limb buds of the mutant mice, and have mapped the gene responsible for the developmental abnormality.

Materials and methods Generation of transgenic mice As part of a study of epilepsy, a transgene containing the neuron-specific enolase promoter (Kearney et al., 2001) was modified by replacement of the sodium channel cDNA with a calcium channel ␤4 subunit cDNA containing wildtype or mutant sequence (Escayg et al., 2000). A 6.5-kb NotI fragment containing the transgene was gel purified and microinjected into fertilized (C57BL/6 ⫻ SJL/J)F2 eggs in the University of Michigan Animal Model Core (www.med. umich.edu/tamc/). Founders were identified by PCR amplification of a 290-bp transgene fragment from genomic DNA using the transgene-specific primers F2 (5⬘-TCC TGA AGC ACA GGT TTG ATG GGA G) and R6 (5⬘-AAC CTT TGG GGA CGA GAC CTT CAC G) under the following PCR conditions: incubation at 94°C for 2 min followed by 33 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 1 min, followed by a final extension of 7 min at 72°C. Founders were bred to strain C57BL/6J, and transgenic lines were maintained by backcrossing to C57BL/6J. Twenty-nine independent transgenic lines were generated. One of 10 lines carrying the R482X mutation (Escayg et al., 2000) was designated T3 and exhibited a mutant limb phenotype. Mice

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with the mutant phenotype were intercrossed to generate a recombinant inbred line whose genome is 75% C57BL/6J and 25% SJL/J. Seventeen of the 29 transgenic lines expressed the transgene cDNA in brain. T3 is a nonexpressing line. Fluorescence in situ hybridization (FISH) Spleen lymphocytes from a homozygous affected male were cultured and analyzed at SeeDNA (Windsor, Ontario) as previously described (Heng and Tsui, 1994). A 6.5-kb transgene probe was biotinylated by using a BioNick labeling kit, hybridized to metaphase chromosomes, and visualized by using avidin–fluorescein isothiocyanate (FITC). Gbanded chromosomes were identified after counterstaining with 4⬘,6-diamidine-2-phenylindol dihydrochloride (DAPI). Genetic mapping and complementation analysis Genotypes for the microsatellite markers D19Mit16, D19Mit86, D19Mit88, and D19Mit119 (Dietrich et al., 1994) were determined by agarose gel electrophoresis and ethidium bromide staining. The Krd deletion was genotyped by PCR as previously described (Ji et al., 1999). Southern blot Southern blotting was performed as previously described (Kearney et al., 2001). In order to estimate transgene copy number, genomic DNA was digested with EcoRI or DrdI and probed with the 290-bp transgene fragment described above. The probe was gel purified and labeled with the Megaprime DNA Labeling System (Amersham). Embryos Noon of the day when the vaginal plug was found was considered to be E0.5. For embryos younger than E10.5, somites were counted to obtain precise staging (Kaufman, 1993). doubleridge homozygous embryos were obtained by mating two affected parents. Control embryos were obtained from C57BL/6J or (SJL/J ⫻ C57BL/6J) F1 mice. At E11.5, when homozygotes could be recognized morphologically, and dblr/dblr ⫻ dblr/⫹ crosses were also used. The inferred genotypes of embryos from these crosses were dblr/dblr for affected embryos and dblr/⫹ for unaffected embryos. These two classes were obtained in equal numbers. The unaffected heterozygotes served as age-matched controls. Probes for in situ hybridization Antisense mRNA probes were transcribed as previously described for Fgf8 (Crossley and Martin, 1995), En1 (Wurst

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Fig. 1. Polydactyly and syndactyly in dblr/dblr forelimbs. Skeletal preparations from neonates at P1 (A, B) and an adult at 6 months (C). Cartilage is stained with alcian blue, and bone is stained with alizarin red. (D–F) External limb morphology. Dorsal (hair) and ventral (footpad) structures are correctly developed in the mutant limbs. Right limbs are consistently more severely affected. The most posterior digit is numbered 5 or 6. L, left; R, right. Fig. 2. Hindlimb postaxial polydactyl. The forelimb is affected in 100% of doubleridge mutants, while the hindlimb is affected in only 13%. (A) Skeletal preparation of hindlimbs from a P1 homozygote. (B) Right hindlimb of an adult homozygote.

et al., 1994), Wnt7a (Parr and McMahon, 1995), Shh (Echelard et al., 1993), Lmx1b (Chen et al., 1998), Bmp4, and Msx2 (Ahn et al., 2001). The Jagged2 (Serrate2) IMAGE clone 3598850 was linearized with EcoRI and transcribed with T7 polymerase.

digoxigenin-labeled Shh or Wnt7a probes. The digoxigeninlabeled probe was first stained with BM Purple substrate. After removal of the antibody (Hauptmann and Gerster, 1994), embryos were incubated with anti-fluorescein AP conjugate (Roche). Color was developed with Fast Red substrate (Roche).

Whole-mount in situ hybridization Detection of apoptosis Whole-mount in situ hybridization with a single digoxygenin-labeled probe was performed as described (Bober et al., 1994) by using BM Purple (Roche) as the substrate for alkaline phosphatase. In double-labeling experiments, the fluorescein-labeled Fgf8 probe was cohybridized with

TUNEL staining was performed with ApopTag Red Kit reagents (Intergene). The embryos were fixed, treated with proteinase K, and postfixed by the protocol used for wholemount in situ hybridization. Limbs were incubated with

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Fig. 3. Chromosomal location of the transgene insert. (A) Hybridization of metaphase chromosomes with a transgene probe. (B) Chromosomes were banded by counterstaining with DAPI. (C) Genetic map of the doubleridge phenotype. The observed haplotypes are indicated. Solid symbols, C57BL/6J allele; open symbols, SJL/J allele. For the transgene locus, the open symbol indicates presence of the transgene, and the solid symbol indicates absence. Transgenepositive animals were identified by PCR and classified as tg/⫹ or tg/tg on the basis of their limb phenotype. (D) Resulting genetic map of markers on proximal chromosome 19. Tg, transgene.

equilibration buffer for 10 min at room temperature, followed by overnight treatment with working dilution of TdT enzyme at 37°C, then washed two times with Stop Solution for 15 min, and three times with TBST for 5 min. Preblocking, antibody incubation, post-antibody washes, and color detection were performed as for whole-mount in situ hybridization.

described (Kimmel and Trammell, 1981). Images of cleared skeletons, whole embryos, and tissue sections were captured with a DEI 750 Optronics digital camera and processed by using Adobe Photoshop.

Other methods

Abnormal limb phenotype in homozygous transgenic mice

Cryosections (50 ␮m) were produced as described by Andre´ et al. (1998). Cartilage and bone were stained as

The doubleridge (dblr) mouse mutant was identified in a screen for recessive insertional mutants. Homozygous tg/tg

Results

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Table 1 Visible phenotypes of doubleridge mutant mice Phenotype Postaxial polysyndactyly of: both forelimbs right hindlimb Inner digit syndactyly of: both forelimbs right forelimb only left forelimb only neither forelimb Kinked tail Microphthalmia or anophthalmia one or both eyes

Number of animals

Percent

40 5

100 13

11 23 1 5 12

28 58 3 12 30

14

35

mice were generated by crossing tg/⫹ heterozygotes from the 29 independent transgenic lines described in Materials and methods. In a single line, designated T3, abnormal forelimbs were observed in 25% of the offspring of heterozygous parents, suggesting the presence of an autosomal recessive mutation in this line. None of the other 28 lines exhibited visible abnormalities. All affected animals from line T3 have a sixth digit in the postaxial region of both forelimbs, branching from and fused with the fifth digit (Fig. 1A–F). In addition to the extra digit, most homozygotes exhibit soft tissue fusion of inner digits (Fig. 1 and Table 1). Skeletal preparations revealed that only the soft tissues are fused in most animals (Fig. 1B). An example of skeletal fusion of inner digits is shown in Fig. 1C. Kinked tail and eye defects were observed in one-third of affected progeny. No additional abnormalities were seen in whole-skeleton preparations. Affected animals were fully viable and fertile. The forelimb is abnormal in all affected individuals, but abnormalities of the hindlimb are rare. Examples of postaxial polydactyly of the hindlimb are shown in Fig. 2. The incidence of morphological defects is summarized in Table 1. Recessive inheritance of the limb abnormality To determine the mode of inheritance of the disorder, three genetic crosses were analyzed (Table 2). In crosses between two affected parents, all of the offspring were

affected. In crosses between affected animals from line T3 and nontransgenic animals from strain C57BL/6J or SJL/J, none of the offspring was affected. In crosses between affected individuals and unaffected tg/⫹ individuals, half of the offspring were affected. The data are consistent with autosomal recessive inheritance of the limb abnormality. All offspring of affected mice were positive for the transgene, indicating that the affected parents were homozygous for the transgene (tg/tg). Cytogenetic mapping of the transgene insertion site The chromosomal location of the transgene insert was determined by fluorescence in situ hybridization (FISH). Metaphase chromosomes from cultured cells isolated from an affected animal were hybridized with a biotinylated transgene probe and visualized with avidin–FITC. Fluorescent signals were detected in both copies of chromosome 19, indicating that the transgene is located on chromosome 19 and that the affected animal was homozygous tg/tg (Fig. 3A and B). G-banded patterns obtained by counterstaining chromosomes with DAPI demonstrated that the transgene insertion site is located on chromosome band 19C (Fig. 3B). Each insertion site contains between six and eight copies of the 6.5-kb transgene, as estimated by Southern blotting of genomic DNA with the 290 bp transgene probe, and comparison with copy standards (data not shown). Genetic mapping of the limb phenotype to chromosome 19 The locus responsible for the limb phenotype was genetically mapped to an interval of chromosome 19 by crossing the (C57BL/6J ⫻ SJL/J-tg/⫹) F2 transgenic founder of line T3 to C57BL/6J nontransgenic mice. Transgenic offspring were identified by PCR and genotyped for the chromosome 19 markers D19Mit16, D19Mit86, D19Mit88, and D19Mit119. All of the tg/⫹ offspring were heterozygous for C57BL/6J and SJL/J alleles of these markers. The observed transmission of the transgene together with chromosome 19 alleles from SJL/J indicates that the transgene inserted into an SJL/J-derived chromosome in the founder. To determine whether the limb phenotype was linked to the chromosome 19 insertion site, tg/⫹ offspring were intercrossed and the progeny were genotyped for chromosome 19 markers and

Table 2 Recessive inheritance of limb abnormalities in line T3 Genetic cross

1. Affected ⫻ affected 2. Affected ⫻ nontransgenic 3. Affected ⫻ unaffected tg/⫹

Affected offspring

Unaffected offspring

% Affected Observed

Predicted

92 0 14

0 91 15

100 0 50

100 0 50

Note. The incidence of postaxial polysyndactyly in offspring from three crosses is indicated. The nontransgenic mice were from strain C57BL/6J or SJL/J. All offspring of affected mice in cross 2 carried the transgene, indicating that affected animals were homozygous. The data are in agreement with the prediction for inheritance of an autosomal recessive mutation that cosegregates with the transgene.

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Fig. 4. Time course of Fgf8 expression in normal and dblr/dblr limb buds. ⫹/⫹ controls were obtained from matings of nontransgenic mice. dblr/⫹ controls are littermates of affected embryos from (tg/tg ⫻ tg/⫹) matings. Genotypes of the normal controls are indicated in each panel. Developmental stage is indicated at the left. The two panels at the right are at higher magnification.

examined for limb morphology. The limb phenotype and the transgene cosegregated in these animals (Fig. 3C). The locus responsible for the limb phenotype was designated

doubleridge (gene symbol, dblr). Analysis of recombinants localized the transgene insertion site and the dblr locus to the 8 cM interval between D19Mit86 and D19Mit88.

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Complementation of doubleridge and Krd The Krd mutation on mouse chromosome 19 is a 5 cM deletion that includes two genes involved in limb development, Fgf8 and Fbxw4 (Ji et al., 1999; Keller et al., 1994; Sidow et al., 1999). To test these genes as candidates for the doubleridge mutation, we crossed an affected tg/tg individual with a heterozygous Krd/⫹ animal. Among the 11 offspring obtained, 5 were double heterozygotes, consistent with the Mendelian prediction of 50%. All of the Krd/⫹, tg/⫹ double heterozygotes exhibited normal limb morphology, demonstrating complementation of the two mutations. These results exclude Fgf8 and Fbxw4 as candidate genes for doubleridge. Abnormal Fgf8 expression in AER of affected embryos To investigate the developmental basis for polydactyly and syndactyly in mutant mice, we examined gene expression during limb development between E9.0 and E11.5 by whole-mount in situ hybridization. Expression of Fgf8 in doubleridge embryos did not differ from wild type at E9.0 to E9.5 (17–23 somites), when the Fgf8 expression domain encompassed the entire ventral forelimb ectoderm (Fig. 4A–D). However, at E10 (26 –33 somites), when Fgf8 expression was restricted to less than half of the proximal forelimb ectoderm in wild type embryos, it encompasses all of the ventral ectoderm in doubleridge embryos (Fig. 4E– H). By E10.5, Fgf8 is restricted to the AER in wildtype embryos (Fig. 4I and K), but in mutant embryos, the expression domain is expanded and the distal portion of the mutant limb bud is morphologically broad and flat (Fig. 4J and L). After E10.5, Fgf8 expression in mutant embryos is strongest at the borders of the broad AER, giving the impression of a double ridge (Fig. 4N and P). Transgene heterozygotes (tg/⫹) exhibited normal morphology and gene expression throughout this time period (e.g., Fig. 4M and O), as expected from the recessive inheritance of the mutation. The developmental abnormalities in hindlimbs of affected embryos were more subtle. The overall shape of the limb bud and the AER was normal. A bifurcation or broadening of the posterior portion of the Fgf8 domain was visible in some affected embryos and was always more severe in the right hindlimb (Fig. 4Q–T).

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and dorsal markers Wnt7a and Lmx1b. Expression of these markers is correctly restricted in the E9.5 limb bud (Fig. 5). This is consistent with the normal appearance of dorsal (hair) and ventral (footpad) structures in adult limbs (Fig. 1). Abnormal expression of additional markers in the AER To ascertain whether the broad Fgf8 expression described above represents an expansion of the AER, we examined the expression of additional AER markers. At E11.5, the expression domains of Bmp4, Msx2, and Jagged2 (Serrate2) was expanded similarly to Fgf8 (Fig. 6). The AER domain of En1 was also wider at this stage. Some of the markers appeared to stain more strongly at the borders of the AER. The distribution of apoptotic cells in the AER, detected by TUNEL staining, was also expanded (Fig. 6K and L). Comparison of left and right forelimb (Figs. 4P and 6) demonstrated that the broadening of the AER extended along the entire distal margin of the right limb buds (R), while it was restricted to the posterior portion in the left limb bud (L). Abnormal morphology of the AER Longitudinal sections of forelimb buds from E11.5 were examined after whole-mount hybridization of Fgf8, En1, and Wnt7a. The shape of the mutant limb buds was thicker than normal, and the expansion of the AER was evident (Fig. 7). In the doubleridge forelimb, there was strong expression of En1 in the broad AER. The Wnt7a domain was correctly restricted to the dorsal ectoderm. Fgf8 expression appeared to be concentrated at the borders of the AER in the mutant. Shh expression in mutant limbs During normal development, Shh is expressed in two areas below the posterior end of the AER (Fig. 8A and B). In mutant forelimbs, these two Shh domains are more widely separated due to the increased width of the AER (Fig. 8C and D). The anterior–posterior axis of the mutant limb appears to be correctly patterned, since Shh domains are present only in the posterior margin (Fig. 8D).

Dorsal–ventral patterning is not affected by the doubleridge mutation

Reduced apoptosis in the interdigital regions of doubleridge forelimbs

To assess the patterning of the ventral and dorsal limb ectoderm, we studied expression of the ventral marker En1

To determine whether syndactyly in doubleridge mice is a consequence of reduced apoptosis, we stained E13.5 fore-

Fig. 5. Normal expression of dorsal and ventral markers of limb development at E9.75. Dorsal markers, Wnt7a and Lmx1b; ventral marker, Enl. v, ventral; d, dorsal. Fig. 6. Expression of AER markers in forelimbs of E11.5 embryos. All markers exhibit an abnormally broad expression domain in the mutant embryos. The broadened expression domains in the right mutant limbs (R) extend further in the anterior direction than the left limbs (L). Apototic cells detected by TUNEL staining are also broadly dispersed in the mutant limb. (A–J) Posterior view. (K, L), Distal view.

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limbs using the TUNEL assay. Strong domains of apoptosis were visible in normal limbs between the developing digits and in the posterior region (Fig. 9). In the mutant limbs, the level of apoptosis in both regions was clearly reduced. The shape of the fifth digit sometimes appears broader, presaging the development of the 6th ectopic digit (e.g., Fig. 9, left limb). Lack of expression of the transgene in line T3 To determine whether the transgene is ectopically expressed in developing limb buds, whole-mount in situ hybridization was carried out at E9.5 and E10.5 with a Cacnb4 cDNA probe. Both wild type and transgenic embryos were negative even after prolonged staining (data not shown). We conclude that the transgene is not expressed in the developing limb. Therefore, ectopic expression is unlikely to contribute to the limb abnormalities.

Discussion doubleridge is an insertional mutation on chromosome 19 The abnormal limb phenotype described here was identified in one of the 29 independent transgenic lines that were screened for insertional mutants, indicating that the phenotype is related to the specific insertion site in line T3. The insertion site was mapped to chromosome 19C by cytogenetic methods, and the phenotype was mapped to the same chromosome region by genetic linkage analysis. The colocalization of transgene and mutant phenotype, together with the evidence that affected animals are homozygous for the transgene insertion, supports the model that random transgene insertion disrupted a gene required for limb development. Transgene expression was not detected in the developing limb, indicating that ectopic expression of the transgene is not responsible for the developmental abnormalities. The observed mutation rate of 1/29 (3%) in this series of transgenic lines is consistent with other reports. For example, we previously identified 6 visible mutations in a series of 200 transgenic lines (Meisler, 1992). Transgene insertional mutants have been used to isolate genes responsible for a variety of interesting phenotypes (reviewed in Woychik and Alagramam, 1998). Future work on line T3 will be directed toward isolation of the gene responsible for the doubleridge phenotype. The position of the candidate gene Dkk1 in the assembled human genome sequence corresponds to the doubleridge region of mouse chromosome 19. The limb abnormalities in doubleridge are very similar to those in the targeted null mutation of Dkk1 (Mukhopadhyay et al., 2001). Both mutants exhibit syndactyly, polydactyly, and broadening of the domain of Fgf8 expression in the AER. In contrast to doubleridge, the Dkk1 null mutation is lethal as

a result of the complete absence of anterior head structures. We amplified the Dkk1 gene in a single 2-kb fragment from homozygous doubleridge genomic DNA and completely sequenced the exons and introns. No changes were identified when the doubleridge sequence was compared with wildtype cDNA and genomic sequences in GenBank (NM_010051 and NW_00143). In addition, no abnormalities were observed in the lengths of restriction fragments that hybridize with the full-length Dkk1 cDNA probe on Southern blots after digestion of doubleridge genomic DNA with four different enzymes (unpublished observations). The data indicate that the Dkk1 gene was not disrupted by the transgene insertion. Relationship between morphology of the AER and subsequent limb abnormalities doubleridge homozygotes consistently exhibit postaxial polysyndactyly of the forelimb and, at a lower frequency, syndactyly of the inner digits. Since FGF signals from the AER induce proliferation and promote cell survival in the underlying mesenchyme, the expanded domain of Fgf8 expression, which begins at E10.0, may be responsible for the increased thickness of the limb bud at E11.5. The difference in severity of abnormalities between left and right limbs, and between forelimb and hindlimb, is correlated with the extent of alteration of expression domains for Fgf8 and other markers at E11.5 (Figs. 4 and 6). Both syndactyly and polydactyly thus appear to be secondary to the ventral expansion of the hyperplastic AER. The expression of En1 and other AER markers throughout the ridge suggests that it may function as a single AER. The expression of Fgf8, Bmp4, and Jag2 is strongest at the borders of this abnormal AER. The critical role of the width of the AER in limb development is also demonstrated by mice lacking the Notch ligand Jagged2/Serrate2 (Jiang et al., 1998; Sidow et al., 1997). Like doubleridge, these mice exhibit a hyperplastic AER, reduced apoptosis in the interdigital regions, and incomplete separation of the digits (Jiang et al., 1998). Laterality of the doubleridge phenotype Syndactyly of the internal digits of the forelimb, as well as polydactyly of the hindlimb, are more frequent on the right side of doubleridge mice (Table 1). Higher incidence in the right limb was also observed for preaxial polydactyly in Bmp4⫹/⫺ mice (Dunn et al., 1997) and for postaxial polydactyly in Bmp4/glypican3 double heterozygotes (Paine-Saunders et al., 2000). The molecular basis for this preference is not known. doubleridge and En1 En1 null mice and doubleridge mice have a similar delay in compaction of the Fgf8 expression domain and similar

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Fig. 7. Correct restriction of dorsal–ventral markers at E11.5. The broadening of the mutant AER is evident in the longitudinal sections of right forelimbs. In the double label experiment, Fgf8 staining is red, and Wnt7a staining is blue. d, dorsal; v, ventral. Fig. 8. Expression of the sonic hedgehog transcript at E11.5. Shh expression is appropriately restricted to the posterior margin of the bud, with increased spacing between the two domains. Purple, Shh; pink, Fgf8. (A, C), Posterior view. (B, D) Transverse sections. a, anterior; p, posterior.

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Fig. 9. Reduced apoptosis in doubleridge forelimbs at E13.5. Regions with apoptotic cells were detected by TUNEL staining. There is diminished interdigital cell death in the mutant. Apoptosis is visible in the posterior region of the wildtype limbs (*), but not the mutant. Dorsal view. Fig. 10. Schematic representation of forelimb patterning. In the wildtype limb, En1 determines ventral identity by repressing Wnt7a. En1 also determines the ventral border of expression of Fgf8 and other AER markers by promoting compaction. FGFs from the AER positively regulate Shh. In the En1 mutant, Wnt7a is expressed in the ventral ectoderm, and formation of the ventral AER border is defective. In the doubleridge limb bud, En1 correctly specifies ventral identity, but fails to correctly establish the ventral AER border. As a result of this failure, the mutant AER is broadened. The three axes are correctly patterned in the doubleridge limbs.

ventral expansion of the AER (Fig. 10; Loomis et al., 1998), but they differ in the dorsal/ventral morphology of the adult limbs. The En1 null limbs are double-dorsal, and the ectopic digits sometimes develop in the ventral portion of the limb. In doubleridge, En1 expression is normal, the dorsal hair and ventral foot pads are correctly positioned, and the ectopic digits are always in the normal plane of the paw (Fig. 1). Since dorsal–ventral patterning and induction of the AER are both likely to be initiated by BMP signaling (Ahn et al. 2001; Pizette et al., 2001), doubleridge appears to act downstream of this effect. The relationships among the expression domains of Fgf8, En1, Wnt7a, and Shh during normal and mutant development are represented in Fig. 10. During normal development, En1 is expressed in the ventral ectoderm, where it represses Wnt7a expression. As a result, Wnt7a is restricted to the dorsal ectoderm, where it induces Lmx1b in the

underlying mesenchyme, leading to development of dorsal structures. In the En1 null mutant, Wnt7a is expressed in the ventral ectoderm, and there is expansion of the AER and increased width of the limb bud. The doubleridge mutant reproduces this expanded AER without affecting expression of En1 or Wnt7a. Thus, doubleridge displays a subset of the abnormalities seen in the En1 null mouse. In both mutants, excess signaling from the AER leads to increased proliferation/survival of the underlying mesenchyme and subsequent syndactyly and polydactyly, that are more severe in forelimb than hindlimb. The similarity of AER abnormalities in the two mutants may be accounted for by a mechanism in which the doubleridge gene acts downstream of En1 in a pathway leading to compaction of the AER. Alternatively, the doubleridge gene product may contribute to restriction of the AER by an independent mechanism. Future isolation of the insertion

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site in the doubleridge mutant will provide molecular access to the affected gene and clarify the mechanism of action on limb development.

Acknowledgments This work was supported by Grant GM24872 from the USPHS and by the March of Dimes (6-FY00-129) for generation of the transgenic lines. B.T.M. acknowledges support from the Michigan Predoctoral Training Program in Genetics (T32 GM07544). We thank Andrew Escayg, Val Drews, and Jennifer Kearney for unpublished information regarding expression of the transgene, and Derek DelMonte for technical assistance. We thank A. Joyner, X. Sun, E.B. Crenshaw, and A. McMahon for providing probes.

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