Mouse Eya Genes Are Expressed During Limb Tendon Development and Encode a Transcriptional Activation Function

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 11974–11979, October 1997 Developmental Biology

Mouse Eya genes are expressed during limb tendon development and encode a transcriptional activation function (Eyes absent͞Splotch͞tendon patterning͞transcription factor)

PIN-XIAN XU*†,JANE CHENG*†‡,JONATHAN A. EPSTEIN*§, AND RICHARD L. MAAS*†¶

*Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, and †Howard Hughes Medical Institute, Boston, MA 02115

Communicated by Philip Leder, Harvard Medical School, Boston, MA, August 22, 1997 (received for review May 15, 1997)

ABSTRACT Vertebrate limb tendons are derived from The growth and patterning of the tendons have been connective cells of the lateral plate mesoderm. Some of the suggested to be independent of skeletal elements (9). For developmental steps leading to the formation of vertebrate example, although removal of the apical ectodermal ridge limb tendons have been previously identified; however, the (AER) produced truncated digits missing one or two phalan- molecular mechanisms responsible for tendinous patterning ges, the extensor and flexor tendons of the missing phalanges and maintenance during embryogenesis are largely un- extended distally, joining each other over the regions that known. The eyes absent (eya) gene of Drosophila encodes a corresponded to the normal digits (9). Previous studies have novel nuclear protein of unknown molecular function. Here also suggested that the pattern of the musculature appears to we show that Eya1 and Eya2, two mouse homologues of be controlled by the connective tissue (10). For example, when Drosophila eya, are expressed initially during limb develop- pieces of unsegmented thoracic mesoderm are transplanted in ment in connective tissue precursor cells. Later in limb place of branchial somites, the transplanted mesoderm can development, Eya1 and Eya2 expression is associated with cell form an appropriate branchial musculature (11), suggesting condensations that form different sets of limb tendons. Eya1 that the connective tissue in some way governs both tissue and expression is largely restricted to flexor tendons, while Eya2 cellular patterning. These facts point to the existence of precise is expressed in the extensor tendons and ligaments of the embryonic mechanisms controlling the patterning and main- phalangeal elements of the limb. These data suggest that Eya tenance of the limb long tendons. genes participate in the patterning of the distal tendons of the Hox genes are differentially expressed in mesenchymal cells limb. To investigate the molecular functions of the Eya gene that give rise to limb skeletal structures, and probably also to products, we have analyzed whether the highly divergent PST connective tissue (12). However, although they are important (proline-serine-threonine)-rich N-terminal regions of Eya1–3 for specification of skeletal elements (12, 13), Hox genes have function as transactivation domains. Our results demonstrate not been reported to be expressed during limb tendon forma- that Eya gene products can act as transcriptional activators, tion. Recently, two members of a highly divergent family of and they support a role for this molecular function in con- homeobox genes, Six1 and Six2 (homologues of the sine oculis gene of Drosophila), have been shown to be expressed in nective tissue patterning. different sets of developing tendons (14), raising the prospect that patterning of connective tissue may be controlled by A major question in development is how precursor cells of specific transcription factors. Several other molecules have various tissue types become organized and arranged appro- also been found to be expressed in developing limb tendons, priately. Limb development has been extensively studied as a such as the Eph-related receptor tyrosine kinase gene Cek-8 model for analyzing mechanisms involved in patterning and and bone morphogenic protein-7 (Bmp-7) (15, 16). However, morphogenesis. Most studies have focused on the early stages their roles in tendon morphogenesis remain unclear. of limb morphogenesis, such as the formation of limb skeletal Three mouse Eya genes, homologues of the eyes absent (eya) elements and, more recently, formation of muscle (1–3). gene of Drosophila, have been recently been isolated, and their Mechanisms responsible for the formation of the limb tendons expression during embryogenesis has been studied (17). Eya1 have received less attention. and Eya2 are widely expressed in cranial sensory placodes and The morphological events of tendon formation in the avian at the sites of inductive tissue interactions during organogen- limb have been described previously (4, 5). Limb tendons are esis, often in complementary or overlapping patterns (17). derived from connective cells of the lateral plate mesoderm. These features suggest major roles for Eya genes in the The distal tendons of the foot initially consist of single dorsal development of vertebrate organs and sensory systems. Fur- and ventral blastemas. As development proceeds, the dorsal thermore, it has been shown that the expression of Eya1 and blastema differentiates into the extensor tendons, which insert Eya2 in prospective lens and nasal ectoderm is undetectable in into the base of each phalange, while the ventral blastema Pax6-deficient Small eye (Sey) mutant embryos, indicating that undergoes cleavage into the distinct flexor tendons of the Eya1 and Eya2 require Pax6 function for their expression in the digits. As with other components of the limb, both growth and lens and nasal ectoderm (17). Besides the cranial placodes and differentiation of the tendons progress in a proximodistal developing eye, the Eya genes are widely coexpressed with Pax sequence. Previous studies have shown that the distal tendons and Six genes in many tissues during organogenesis, suggesting of the limb can be formed in the absence of muscles (2, 6, 7); possible interactions between their gene products and the however, in the chick, muscles are required for tendon sur- existence of a conserved Pax–Six–Eya regulatory hierarchy. vival—otherwise they undergo degeneration (6, 8). Abbreviations: En, embryonic day n; CAT, chloramphenicol acetyl- The publication costs of this article were defrayed in part by page charge transferase. ‡Present address: Biotransplant, Inc., Charlestown, MA 02129. payment. This article must therefore be hereby marked ‘‘advertisement’’ in §Present address: Department of Medicine, University of Pennsylva- accordance with 18 U.S.C. §1734 solely to indicate this fact. nia, School of Medicine, Philadelphia, PA 19104. © 1997 by The National Academy of Sciences 0027-8424͞97͞9411974-6$2.00͞0 ¶To whom reprint requests should be addressed. e-mail: maas@ PNAS is available online at http:͞͞www.pnas.org. rascal.med.harvard.edu.

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In this report, we show that Eya1 and Eya2 transcripts are initially found in connective tissue precursor cells in the limb buds at embryonic day 10.5 (E10.5). Later, Eya1 and Eya2 expression patterns become differentially localized to specific subsets of limb tendons, with Eya1 largely restricted to the flexor tendons (similar to Six2 expression) and Eya2 largely restricted to the extensor tendons (similar to Six1 expression). Our results suggest that the Eya genes function along with the Six genes to establish dorsal͞ventral patterning in limb tendon morphogenesis. In addition, we provide insight into the previously unknown molecular function of the Eya gene products by showing that their N-terminal PST domains can activate Eya1 transcription. We propose that the Eya gene products function as transcription factors involved in specifying connective tissue identity.

MATERIALS AND METHODS In Situ Hybridization. Whole-mount and tissue section in situ hybridizations, washing, and RNase treatment were performed as described (17). Genotypes. Genotype analysis of Splotch (Sp) embryos at E11.5 was performed as described previously (18). GAL4͞Eya Expression Plasmid. The GAL4͞Eya1–3 expression plasmids were constructed in pBXG1 (19), which ex- Eya2 presses the DNA-binding domain of GAL4 (amino acids 1–147) under the control of simian virus 40 enhancer͞ promoter (ori). Fragments encoding the N-terminal regions of Eya1–3 (amino acids 2–320 for Eya1, amino acids 27–261 for Eya2, and amino acids 2–145 for Eya3) were amplified from mouse Eya1–3 cDNA clones (17). These were cloned as EcoRI fragments into a polylinker immediately downstream of and in frame with the GAL4 DNA-binding domain. All constructs FIG.1. Eya1 and Eya2 are expressed in myoblast and connective were confirmed by DNA sequencing. tissue precursors. (A–F) Transverse sections of 35S-labeled in situ Chloramphenicol Acetyltransferase (CAT) Assay. P19 cells hybridization in wild-type and Sp͞Sp mutant limbs. (A–D) The dorsal were plated in 60-mm Petri dishes and transfected with plasmid (d) and ventral (v) Eya1- and Eya2-expressing cell populations. At DNA coated with Transfectam (Promega) according to the E11.5, Eya1 expression levels in the ventral domain are higher than manufacturer’s instructions. The pG5ECAT reporter plasmid that of Eya2 and the expression extends more distally (C). In contrast, contains five GAL4 binding sites inserted upstream of the Eya2 expression in the distal region is broader than that of Eya2 at adenovirus E1b minimal promoter driving the CAT gene (20). E11.5 (D). (E and F) Eya1 and Eya2 expression in the limbs of Sp͞Sp mutant embryos. The dorsal and ventral expression domains are lost Each dish was transfected with 2 ␮gofpG5ECAT DNA and ␮ in the mutant limbs. The residual expression of Eya1 and Eya2 (as 0.01–1 g of a GAL4 expression plasmid. The plasmid ex- indicated by arrows) corresponds to connective tissue precursors. dm, pressing the GAL4 DNA-binding domain only was used as a Dermomyotome; so, somites. negative control. A plasmid expressing the GAL4 DNA- binding domain and activation region II (amino acids 768–881) appears to correlate with the two major ventral and dorsal (21) and a human GAL4-PAX6 PST expression plasmid (19) collections of myoblast cells (Fig. 1 A and B), and it progresses were used as positive controls. The amount of plasmid trans- distally by E11.5 (Fig. 1 C and D). The ventral expression fected into each dish was equalized by the addition of domain of Eya1 is broader than that of Eya2 at E10.5, and its pGEM3Z DNA. Cell extracts were prepared after 48 h and expression level is higher than that of Eya2 at E11.5 (Fig. 1 A assayed for CAT activity as described (22). CAT assays were and C). To determine whether the Eya1 and Eya2 limb performed a minimum of three times, and extracts were expression specifically resided in myoblast precursor cells, diluted as necessary to measure activity in the linear range. Eya1 and Eya2 expression was analyzed in Pax3-deficient The percent acetylation was quantified directly from thin-layer Splotch mutant embryos (26). In Sp͞Sp mutant embryos, chromatography plates by using a PhosphorImager (Molecular myoblast precursor cells do not migrate into the limb and Dynamics) and multiplied by a dilution factor to give relative myogenic genes are not expressed there (27, 28). The dorsal transcriptional activity. and ventral expression domains of Eya1 and Eya2 are not observed in the limbs of Sp͞Sp mutant embryos (Fig. 1 E and RESULTS F). However, in Sp͞Sp mutant embryos, some expression of Eya1 and Eya2 Are Initially Expressed in Myoblast and Eya1 is detected in both proximal and distal regions (Fig. 1E, Connective Tissue Precursors in the Limb Buds. Both Eya1 indicated by arrows). In addition, Eya2 expression was ob- and Eya2 are expressed in somites at stage E9.5 (17), and their served in discontinuous patches in the limbs of Sp͞Sp mutant expression was detected in the dermomyotome at stages embryos (Fig. 1F, indicated by arrows). Although our data do E10.5–11.5 (Fig. 1 and data not shown). Myoblast precursor not prove that Eya1 and Eya2 are expressed in myogenic cells for the limb musculature are derived from the lateral precusors, the findings that Eya1 and Eya2 are expressed in portion of the dermomyotome adjacent to the limb bud. These somites, lateral dermomyotome, and dorsal and ventral limb cells migrate into the limb-forming region of the embryo mesenchyme and that the latter expression domains are lost in (23–25). Expression of Eya1 in the developing limb was first Sp͞Sp mutant embryos all support this interpretation. In observed at E10.0 (data not shown), several hours earlier than addition, Eya1 and Eya2 are coexpressed with myogenin later Eya2 expression. At E10.5, the expression of Eya1 and Eya2 in limb muscle development (data not shown). We thus Downloaded by guest on September 24, 2021 11976 Developmental Biology: Xu et al. Proc. Natl. Acad. Sci. USA 94 (1997)

conclude that a component of Eya1 and Eya2 expression, likely latest stage examined (Fig. 1H). Besides the developing ten- corresponding to myogenic precursor cells, is absent from the dons, Eya1 expression is also observed in the zones of joint limbs of Sp͞Sp mutant embryos and that the residual expres- formation (Fig. 2I), and in the interdigital regions at E13.5 as sion of Eya1 and Eya2 corresponds to connective tissue well as in the perichondrium at E15.5 (data not shown). precursors. This view implies that, in the absence of myogenic Eya2 expression is strongly detected in the condensing precursors, connective tissue precursors exhibit anomalous mesenchyme flanking the cartilage of individual digits at patterns within limb mesenchyme, thus accounting for the E12.0–13.5 (Fig. 3 A, C, and F), and in regions flanking the dispersed patches of Eya expression. phalangeal components of the digits at E14.5 (Fig. 3 B, D, E, Eya1 and Eya2 Are Expressed in Distinct Sets of Tendons G, and H). (As in Fig. 2 C–E, the ectoderm staining shown in During Limb Development. To study the role of Eya genes in Fig. 3 C–E is artifactual.) At E14.5, Eya2 expression is also connective tissue patterning during limb development, we observed dorsally in the condensing mesenchymal cells that further analyzed their expression during tendon morphogen- will form extensor tendons (Fig. 3 E, arrows, and H). Although, esis. At E12.5, high levels of Eya1 expression are observed the condensed dorsal extensor blastemas are smaller than ventrally in the foot plate (Fig. 2A). At E13.5, individual digits ventral flexor blastemas, Eya2 expression remains in these are visible and cartilage begins to condense. Eya1 expression structures at E17.5, the latest stage examined (data not shown). is found in the developing tendons (Fig. 2F). By E14.5, Eya2 transcripts were also found in the perichondrium at different digits are well formed and separated. Eya1 expression E14.5–15.5, but not in the interdigital regions (data not remains strong in the developing tendons (Fig. 2 B and G). shown). Our data show that the Eya2-expressing cells in the Transverse sections reveal that the expression of Eya1 in limbs correspond to tendons and probably also to ligaments of condensing mesenchymal cells is associated with the develop- the phalangeal elements of the developing digits. Interestingly, ment of flexor tendons (Fig. 2 C–E). (In Fig. 2 C–E, the Eya2 is expressed in the extensor tendons, whereas Eya1 is ectoderm staining is artifactual and arises because the whole expressed in the flexors. mount was intentionally overdeveloped to show mesenchymal Transcriptional Activation Function of the Eya N-Terminal Eya1 expression.) Eya1 expression is initially observed in a PST Domain. The molecular functions of the Eya proteins are broad domain (Fig. 2C). As cartilage condensation proceeds, currently unknown. The highly divergent Eya N termini consist the broad domain of Eya1 expression becomes concentrated of 35–40% proline, serine, and threonine residues and resem- and predominantly mesenchymal (Fig. 2 D and E and data not ble the proline–serine–threonine (PST) transactivation do- shown). Eventually, Eya1-expressing cells are seen below the mains found in other transcription factors (29, 30). To deter- cartilage elements, and these tendon-like structures become mine whether the PST domains of Eya1–3 can function as localized toward the center of the limb (Fig. 2G). Eya1 transactivation domains, we constructed a series of expression expression remains strong in the flexor tendons at E17.5, the plasmids in which the N-terminal region of Eya1, -2, or -3 was

FIG.2. Eya1 is expressed in tendons of the limb. Whole mount (A–E) and radioactive (F–I) in situ hybridization in the developing limb. (A and B) Ventral views of whole-mount limbs, showing the expression in the foot plates at E12.0–14.0. (C–E) Transverse sections of whole-mount limbs show ventral (v) expression initially in a broad domain and in condensing mesenchyme flanking the cartilage condensations, as indicated by arrows. Arrows in D point to two domains of expression. The ectoderm staining is artifactual and results because the whole-mount in situ hybridization was intentionally overdeveloped to show the mesenchymal Eya1 expression. (F–H) Longitudinal sections show the expression in the developing tendons (t) at E13.5–17.5. (I) Longitudinal section shows the expression of Eya1 in the zone of joint formation (arrows) at E14.5. (F–I) Anterior is to the left and distal is up. Other abbreviations: a, anterior; p, posterior; d, dorsal. Downloaded by guest on September 24, 2021 Developmental Biology: Xu et al. Proc. Natl. Acad. Sci. USA 94 (1997) 11977

FIG.3. Eya2 is expressed in tendons and ligaments of the limb. Whole-mount (A–E) and radioactive (F–H) in situ hybridization in the developing limb. (A and B) Dorsal views of whole-mount limbs, showing the expression anteroposteriorly in the mesenchymal cells flanking the cartilage (A, arrows) at E12.0, and flanking the phalangeal components of the digits (B, the regions indicated by the arrow) at E14.0 in the foot plates. (C–E) Transverse sections of whole-mount limbs show the expression of Eya2 anteroposteriorly in the condensing mesenchyme flanking the cartilage condensations at E13.0 (C, arrow), flanking the phalangeal components of the digits at E14.0 (D, arrow, and E) and in the dorsal condensed mesenchyme that will form extensor tendons (E, arrows). The ectoderm staining is artifactual and results because the whole-mount in situ hybridization was intentionally overdeveloped to show the mesenchymal Eya2 expression. (F–H) Longitudinal sections show the expression anteroposteriorly in the condensing mesenchyme flanking the cartilage at E13.5 (F), in the mesenchymal cells flanking the phalangeal components of the digits, and in the developing tendons (t) at E14.5 (G and H). In F–H anterior is to the left and distal is up. Other abbreviations: a, anterior; d, dorsal; p, posterior; v, ventral.

fused in frame to the 147-amino acid DNA-binding domain of than that of Eya2 (Fig. 1). In the developing tendons, Eya1 is yeast GAL4 (Fig. 4A) and cotransfected them into P19 em- expressed ventrally in mesenchymal cells (the flexor blaste- bryonic carcinoma cells with a reporter plasmid containing the mas) flanking the cartilage condensations (Fig. 2). In contrast, CAT gene (see Materials and Methods). The GAL4-Eya1 and the cells expressing Eya2 are found anteroposteriorly in the -Eya2 fusion proteins strongly stimulated CAT expression over regions flanking the cartilage condensations, and later on in a wide range of plasmid concentrations (Fig. 4B), at a level the regions flanking the phalangeal components of the digits approximately one-fifth that of the GAL4(II) positive control and in the developing extensor tendons (Fig. 3). The cells and similar to the level of the GAL4-PAX6 PST positive expressing Eya2 probably also correspond to the ligaments of control (Fig. 4C). In contrast, the Eya3 PST domain expression the phalangeal elements of the developing digits. The Eya1 and construct stimulated CAT expression relatively weakly, with Eya2 expression patterns in the dorsal and ventral limb, and 8-fold less activity compared with Eya1 and Eya2 (Fig. 4 B and their proximodistal extension, coincide with tendon blastema C). Similar data were obtained from three independent ex- formation (9). The distal limb tendons initially consist of a periments. We conclude that the Eya proteins are transcrip- single dorsal and ventral blastema, which upon further subdi- tional activators and their activation function resides within the vision will give rise to the different tendons that insert in the N-terminal PST domains. digit phalanges. As with other limb components, the growth and differentiation of tendons occurs in a proximodistal DISCUSSION direction. The expression of Eya1 specifically in the flexor blastemas and Eya2 in the extensor blastemas indicates that Eya Genes and Connective Tissue Patterning. In Splotch these genes may pattern the flexors and extensors of the mutant embryos, the Eya1 and Eya2 expression patterns in the phalangeal elements. In addition, Eya1 and Eya2 may specify limb buds appear to include both myoblast precursors and cell type precursors involved in connective tissue development. mesenchymal cells. Eya1 expression in the developing limb was Interestingly, haploinsufficiency for human EYA1 has recently detected several hours earlier than Eya2 expression (data not been found responsible for the branchio-oto-renal (BOR) shown). At E10.5–11.5, both genes are expressed in the dorsal syndrome (31), a dysmorphosis not known to involve connec- and ventral limb, where the expression level of Eya1 is higher tive tissue. However, this result does not discount a role for Downloaded by guest on September 24, 2021 11978 Developmental Biology: Xu et al. Proc. Natl. Acad. Sci. USA 94 (1997)

rapidly condenses into a mesenchymal layer. However, one difference is that Eya1 is expressed only ventrally in the condensing mesenchymal cells, whereas Six2 is expressed both dorsally and ventrally in the condensing mesenchymal cells, although stronger ventrally (14). Eya1 is expressed in limb buds one day earlier than Six2 (14). Eya2 shows an expression pattern similar to that of Six1 in the developed phalanges at E14.5 (14). It will be interesting to assess the relation between Eya gene expression and Six gene expression, because the same pair of genes are also widely coexpressed in many other tissues during organogenesis, with Eya1 and Six2 in the craniofacial tissues, nasal placode, gut mesenchyme, nephrogenic cord, genital tubercle, and kidney, Eya2 and Six1 in the craniofacial tissues, nasal placode, branchial arch, tongue, kidney, somite, dorsal root ganglion, and future intercostal muscles. These data suggest the possibility of direct molecular interactions between the Eya and Six gene products during organogenesis. The Eya genes together with Six genes provide a key oppor- tunity to explore the link between patterning mechanisms and tissue formation in developing limbs. Both Eya1 and Eya2 are expressed not only in developing connective tissue but also in somites. Thus, Eya1 and Eya2 may be expressed at the same time both in myoblast precursors migrating from the somite into the limb and in a subpopulation of lateral plate-derived mesenchymal cells that will give rise to the connective tissue. This may ensure that the tendon and muscle precursor cells expressing the same set of genes migrate to appropriate places. Besides in the developing limb, Eya1 and Eya2 are also expressed in other body skeletal muscles and connective tissues (17). In conclusion, we propose that Eya1 and Eya2 are involved in patterning limb connective tissue (tendon and ligaments), and these genes may also subserve this function in other body connective tissues and in skeletal muscles in which they are expressed. The Molecular Functions of Eya Gene Products. On the basis of their amino acid content, the N-terminal regions of Eya1–3 were tested for transcriptional activity. Consistent with the observation that Drosophila Eya is localized in the cell nucleus (32), our data indicate that the N-terminal PST domains of Eya1–3 are transcriptional activators in cultured cells (Fig. 4). Interestingly, the PST domains of Eya1 and Eya2 show strong transcriptional activity, while that of Eya3 is FIG. 4. Transcriptional activation by the N-terminal PST domains comparatively weak (Fig. 4). Eya3 is the most divergent of the of Eya1–3 proteins. (A) Schematic diagram of GAL4 expression three mouse Eya family members, and its expression is differ- constructs. The GAL4 DNA-binding domain (BD; amino acids 1–147) was used as a negative control. The GAL4 activation domain II (AD; ent from that of Eya1 and Eya2 during mouse embryonic amino acids 768–881) and PAX6 PST domains were used as positive development (17). Eya1 and Eya2 are expressed in all cranial controls. N-terminal regions of Eya1 (amino acids 2–320), Eya2 placodes and their derivatives at E9.5, whereas Eya3 is ex- (amino acids 27–261), and Eya3 (amino acids 2–145) are fused to the pressed in the underlying craniofacial and branchial arch GAL4 DNA-binding domain. (B) CAT assay. P19 cells were trans- mesenchyme at the same stage (17). Taken together, these fected with the pG5ECAT reporter and 0.01–1 ␮g of a plasmid data suggest that Eya3 may have distinct developmental roles expressing the GAL4 DNA-binding domain only or with the GAL4 during mouse embryonic development. activation domain, PAX6 PST domain, or the Eya1 PST domain fused to the GAL4 DNA-binding domain. (C) Relative activity of GAL4 The Drosophila Eya N-terminal region has not been tested expression constructs (symbols are defined in A). The PST domain of for transcriptional activation function, but it is also PST-rich 1 Eya1 stimulates transcription approximately ⁄5 as well as the GAL4 and contains a large number of alanine, glycine, and glutamine 3 activation domain and approximately ⁄5 as well as the PAX6 PST residues, suggesting that despite their sequence divergence, the domain. The PST domain of Eya2 stimulates transcription approxi- Drosophila Eya and murine Eya gene products could have a 1 mately ⁄3 as well as the GAL4 activation domain, while Eya3 only conserved molecular function during evolution. Although the weakly stimulates transcription and shows 8-fold less activity compared Eya proteins do not possess a known DNA-binding motif, with either Eya1 or Eya2, although 6-fold higher than the negative analysis of the Eya protein sequences suggests that the highly control (see Inset). conserved Eya domain could interact either with DNA or a DNA-binding protein to activate transcription, since the Eya Eya1 in connective tissue patterning, because such function domain is predicted to be hydrophobic and to contain two might be observed only in the context of complete loss of gene conserved ␣-helical regions. In conclusion, the identification function. of transcriptional activation functions for the Eya proteins Recently, expression of several other genes has been iden- provides insight into the functional role of these gene products tified in the developing long tendons of the autopod (14–16). during mouse embryonic development. The expression of Eya1 in the developing limb tendons shows a pattern of expression similar to that of Six2, with expression This work was supported by National Eye Institute Grant 1RO1 initially confined to a broad sheet under the ectoderm that EY10123 and by the Howard Hughes Medical Institute. Downloaded by guest on September 24, 2021 Developmental Biology: Xu et al. Proc. Natl. Acad. Sci. USA 94 (1997) 11979

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