Induction of Additional Limb at the Dorsal–Ventral Boundary of a Chick Embryo

DEVELOPMENTAL BIOLOGY 182, 191 –203 (1997) ARTICLE NO. DB968476

Induction of Additional Limb at the Dorsal– Ventral Boundary of a Chick Embryo Mikiko Tanaka,* Koji Tamura,* Sumihare Noji,† Tsutomu Nohno,‡ and Hiroyuki Ide* *Biological Institute, Tohoku University, Aoba, Sendai 980-77, Japan; †Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, 2-1 Minami-Josanjima, Tokushima 770, Japan; and ‡Department of Pharmacology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-01, Japan

In the early chick embryo, an apical ectodermal ridge (AER) is formed from the overlying ectoderm of the presumptive limb bud region at the dorsal –ventral (DV) boundary. We report here that the ectopic DV boundary formed in the presumptive wing, flank, and leg fields induces an ectopic AER structure. Dorsal tissue (ectoderm and mesoderm) from the presumptive wing field of stage 10 to 17 embryos was inserted into a slit in the somatopleure of the future ventral side of host embryos. The same method was used to implant ventral tissue into the future dorsal side of host embryos. After the implantation, ectopic AER was induced and an additional limb or limb-like structure developed. In related experiments, ectoderm-free presumptive wing tissue was implanted, which resulted in a considerably decreased frequency of ectopic AER formation. Further analysis of chick and quail chimeras suggests that the ectopic AER was formed from the ectodermal cells overlying the boundary of host and graft mesodermal cells. These results indicate that the DV boundary organizes the AER structure in the limb bud field of early-stage chick embryos and that the ectoderm of the grafted tissues plays an important role in this process. q 1997 Academic Press

INTRODUCTION Limb bud formation depends on signals from the anterior– posterior (AP), proximal –distal (PD), and dorsal– ventral (DV) axes. AP identity is regulated by signals from the zone of polarizing activity (ZPA), a specialized region at the posterior margin of the limb bud (Saunders and Gasseling, 1968). PD outgrowth is controlled by the apical ectodermal ridge (AER) at the distal tip, which acts on progress zone cells. DV identity is regulated by ectodermally derived signals. Rotation of the limb bud ectoderm along the DV axis induces a corresponding inversion in the pattern of mesodermally derived structures (MacCabe et al., 1974). Less is known about the molecular mechanisms of DV axis specification. However, Wnt-7a, which is expressed in the dorsal ectoderm of the limb bud in mouse (Gavin et al., 1991; Parr et al., 1993; Parr and McMahon, 1995) and chick (Dealy et al., 1993), has recently been identified as an ectodermally derived signal participating in the regulation of DV limb patterning. Further, genes which exhibit DV restriction in the limb mesoderm were found. Chick Lmx-1, a LIM homeodomain transcription factor, is expressed in

the dorsal mesoderm of the developing limb bud and its expression is regulated by the overlying ectoderm where Wnt-7a mRNA is localized (Riddle et al., 1995. Vogel et al., 1995). Molecules that are expressed in the ventral ectoderm are also known. Transcripts of En-1 (Davis et al., 1991; Wrurst et al., 1994) and Wnt-5a (Parr et al., 1993) are detected in the ventral ectoderm of the mouse limb bud. Recently, it was reported that loss of En-1 function in mice results in the dorsal transformation of ventral structures and also in altered expression of dorsal- and AER- specific markers (Loomis et al., 1996). In Drosophila, formation of wing imaginal discs also needs signals of three axes. AP polarity depends on the function of hedgehog (hh) (Basler and Struhl, 1994). In the case of DV polarity, dorsal cells which express fringe (fng) and Serrate (Ser) controlled by the apterous (ap) gene and ventral cells interact to induce the expression of vestigial (vg) and wingless (wg), wing-patterning genes along the DV boundary (Blair et al., 1994; Cohen et al., 1992; Dias-Benjumea and Cohen, 1993; Williams et al., 1994; Couso et al., 1994). The PD outgrowth is induced by the interaction of signaling molecules which control AP and DV polarity respectively (Basler and Struhl, 1994; Dias-Benjumea et al., 1994). These

0012-1606/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

AID DB 8476

/

6x19$$$$21

191

01-16-97 01:35:42

dba

192

Tanaka et al.

studies give powerful evidence that the boundary model postulated by Meinhardt (1983) is correct in the case of insects. His model includes the hypothesis that molecules that control patterning are produced by cooperative interactions between cells in differently determined territories. As to vertebrate limbs, he thought the AER is produced along the DV boundary by the interaction between dorsal and ventral cells. In the present study, we show the induction of ectopic AER and additional limb cartilage by the formation of new DV boundaries in the presumptive limb and flank fields of stage 10/11 to 16/17 chick embryos.

MATERIALS AND METHODS Operations Chick and quail embryos were staged according to Hamburger and Hamilton (1951). The experiments were performed on stage 9– 18 embryos. The ectoderm and mesoderm representing the presumptive wing and leg region (Chaube, 1959) was excised and transferred to Tyrode’s solution. The tissues were further subdivided to isolate the dorsal and ventral region. (Fig. 1). The dorsal was the region adjacent to the somites, while the ventral was the region adjacent to the extraembryonic region (Rosenquist, 1971). The intermediate region was discarded. For the grafting of ectoderm-free tissues, the ectoderm was mechanically removed after trypsinization. The AP direction of implanted tissue was not changed.

Alcian Blue Staining Embryos were incubated until Day 9 and then fixed overnight in 10% Formalin in Tyrode’s solution, stained with 0.1% Alcian blue in 70% ethanol with 0.1 N HCl at 377C for 6 hr, dehydrated, and cleared in methyl salicylate.

FIG. 1. Schematic diagram of implantation experiment. Excised presumptive limb tissues from donor embryos were divided into the future dorsal side and ventral side and the intermediate region was discarded. The future dorsal or ventral tissue was inserted into a longitudinal slit in the site of the future dorsal or ventral side. Future ventral (dorsal) region was inserted into a slit in the future dorsal (ventral) site.

In Situ Hybridization To obtain a DNA fragment of L-CAM, PCR was carried out in a similar manner to that described previously (Suzuki et al., 1991; Sano et al., 1993). The reaction products (Ç450 bp) were separated by agarose gel, extracted, and subcloned into the cloning site of pCRII by TA-cloning system (Invitrogen). Sequencing of several clones was carried out according to the dideoxynucleotide chain termination method, using a sequencing kit from Amersham and a DNA autosequencer (Hitachi, SQ5500). After the DNA sequence analysis, XhoI-linearized plasmid of L-CAM was transcribed with SP6 RNA polymerase for making an antisense probe. cDNA fragments of Fgf-8, Shh, and Lmx-1 were cloned in the same manner as L-CAM, respectively. The Fgf-8 cDNA was subcloned into pBluescript (SK0 ). Shh and Lmx-1 cDNA was subcloned into pBluescript (SK/ ). They were linearized with appropriate restriction enzymes to prepare antisense probes. Whole-mount in situ hybridization of L-CAM in the chick embryo was performed after Wilkinson (1992) with slight modifications (Yonei et al., 1995). To examine the expression patterns of the Fgf-8, Shh, and Lmx1, ectopic limb buds, which had been formed by the implantation of the presumptive wing ventral tissue into the presumptive dorsal

field at stage 15, were fixed with 4% paraformaldehyde at 36 hr after implantation. The 4% paraformaldehyde was replaced by 10% sucrose– PBS and embedded in OCT compound (Miles) and frozen in liquid nitrogen. Frozen sections at 10 mm in thickness were spread on glass slides. The expression of the Fgf-8, Shh, and Lmx1 was probed in alternate sections by means of non-RI in situ hybridization according to the methods of Yokouchi et al. (1991a). Three to six embryos were used for each hybridization experiment.

Chimera Analysis Dorsal or ventral tissues at the 15 –20 somite level representing the presumptive wing region were excised from stage 12 quail embryos and grafted into slits made in the presumptive wing ventral or dorsal limb region of chick hosts. Two days after the operation, the embryos were fixed in 4% paraformaldehyde in PBS, embedded in OCT compound (Miles) and serially sectioned at 10 mm. The distribution of host chick cells was observed using A223 antibody. A223 antibody stains only chick cells (Yokouchi et al., 1991b; Ide et al., 1994).

Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.

AID DB 8476

/

6x19$$$$22

01-16-97 01:35:42

dba

193

AER Induction at the DV Boundary

FIG. 2. Induction of AER with L-CAM expression from new DV boundary of wing bud. (a) Dorsal view of the normal wing bud. (b) Dorsal view of the wing bud 36 hr after the implantation of presumptive dorsal wing tissue to the dorsal region of presumptive wing bud of stage 12/13 chick embryo. The appearance is normal. (c) Dorsal view of the wing bud 36 hr after implantation of presumptive dorsal wing tissue to the ventral region of presumptive wing bud of stage 12/13 chick embryo. Ectopic AER was indicated by arrowheads.

RESULTS Induction of Ectopic Limbs in the Presumptive Wing Field As a marker of ectopic limb formation, we first examined the expression of L-CAM mRNA in the limb bud with a whole-mount in situ hybridization. L-CAM mRNA expression occurred only in AER at stages 17 to 30, but no expression was observed in the other ectodermal tissues of the limb bud. Figure 2a shows L-CAM mRNA expression in the limb bud. A detailed expression pattern of the L-CAM mRNA will be reported elsewhere (Tamura et al., submitted). We examined L-CAM mRNA expression in the limb buds 36 hr after the implantation. In control embryos which received presumptive dorsal wing tissue in the same presumptive dorsal field at wing bud level (D r D), almost all limb buds (17/18) did not form ectopic AER with L-CAM mRNA expression and the morphology was normal (Fig. 2b; Table 1). However, the presumptive wing ventral field that received presumptive dorsal wing tissue (D r V) formed an ectopic AER at the DV boundary with strong expression of L-CAM (Fig. 2c). The ability to induce ectopic AER changed depending on the stages of the grafts and the hosts (Table 1). At stages 10/11 through 16/17, ectopic L-CAM expression occurred at high frequencies (more than 50%). It is worth noting that the ectopic AER was induced after stage 10/11, when the polarizing activity could be first measured along the flank region at 12 to 22 somite level including the wing field (Hornbruch and Wolpert, 1991). After stage 17, when the wing bud starts to elongate, no ectopic expression of LCAM was observed.

When the presumptive ventral wing tissue was implanted into the presumptive wing dorsal field (V r D), additional AER with ectopic expression of L-CAM was also observed in stage 10/11 to 16/17 embryos at high frequencies (more than 40%; Table 1). Ectopic expression of L-CAM was observed at the highest frequency in stages 12/13 and 14/15 presumptive wing buds (67 and 63%, respectively). When presumptive wing ventral tissue was implanted into the wing ventral field (V r V), no ectopic AER with L-CAM mRNA expression was formed at any stage (Table 1). Next we examined ectopic cartilage formation by heterotopic transplantation. In the case of stage 10 to 16/17 embryos, which retained the ability to induce AER, ectopic cartilage pattern was induced after the heterotopic transplantation (D r V and V r D). Figure 3a shows a cartilage pattern obtained after D r V grafting at stage 12/13. An ectopic, although incomplete, wing with one humerus, one ulna, and two digits (digit pattern; 34) was formed. Several types of ectopic cartilage patterns were observed at stylopod to autopod level. However, no ectopic wings with a complete pattern were observed. Sometimes unidentified wing cartilage elements were formed. Moreover, in some cases, not only the ectopic, but also host wing pattern was often incomplete (Table 2). It is surprising that no additional wing cartilage elements were formed after the V r D operation in stage 14/15 and 16/17 embryos despite the high frequency of ectopic AER induction at these stages.

Expression of Fgf-8, Shh, and Lmx1 in the Ectopic Wing Bud Fgfs are known to be expressed characteristically in the AER (Niswander and Martin, 1992; Savage et al., 1993; Hei-

Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.

AID DB 8476

/

6x19$$$$22

01-16-97 01:35:42

dba

194

Tanaka et al.

TABLE 1 Ectopic AER Formation by Implantation of Presumptive Wing Tissue Host stage

Wing dorsal r wing ventrala Ectopic AER Total number of cases Percentage of ectopic AER Wing dorsal r wing dorsalb Ectopic AER Total number of cases Percentage of ectopic AER Wing ventral r wing dorsalc Ectopic AER Total number of cases Percentage of ectopic AER Wing ventral r wing ventrald Ectopic AER Total number of cases Percentage of ectopic AER

9

10/11

12/13

14/15

16/17

18

0 5 0

3 6 50

6 12 50

7 8 88

3 6 50

0 5 0

0 3 0

0 5 0

1 6 17

0 4 0

4 9 44

6 9 67

5 8 63

2 5 40

0 5 0

0 5 0

0 6 0

0 4 0

0 7 0

0 7 0

a The frequency of ectopic AER formation after the implantation of presumptive wing dorsal tissue in the wing ventral region of 9 to 18 chick embryos. b The frequency of ectopic AER formation after the implantation of presumptive wing dorsal tissue in the wing dorsal region of 10/11 to 16/17 chick embryos. c The frequency of ectopic AER formation after the implantation of presumptive wing ventral tissue in the wing dorsal region of 9 to 18 chick embryos. d The frequency of ectopic AER formation after the implantation of presumptive wing ventral tissue in the wing ventral region of 10/11 to 16/17 chick embryos.

kinheimo et al., 1994; Ohuchi et al., 1994; Crossley and Martin, 1995; Mahmood et al., 1995) and presumably required for normal limb development. Especially, Fgf-8 is expressed throughout the entire AER. Fgf-8 mRNA is readily detectable in the ectoderm at stage 16, in a patchy, longitudinal stripe within the prospective limb territory. By stage 17, the domain of Fgf-8 expression has extended to cover most of the prospective limb territories. At stage 18, Fgf-8 RNA is detected along the entire AER (Fig. 4a). Thus, we confirmed the induction of AER by examining Fgf-8 expression in the ectopic limb bud. When the presumptive wing ventral tissue was implanted to the presumptive wing dorsal field at stage 15 and fixed after 36 hr, Fgf-8 signal is observed in the entire AER of ectopic limb bud (Fig. 4b). Since the induction of AER was detected with both L-CAM and Fgf-8, we used L-CAM as AER marker in the following experiments. As to the AP and DV polarity of the ectopic wing bud, the expressions of Shh and Lmx1 in the ectopic limb buds were examined 36 hr after the grafting of presumptive ventral tissue into the dorsal field at stage 15. Shh is known as a marker of ZPA, posterior margin of limb buds (Riddle et al., 1993; Fig. 4c), and Lmx1 is a homeobox gene (German et al., 1992) expressed in the dorsal mesenchymal cells of limb buds (Fig. 4e) under the influence of the dorsal ectoderm (Riddle et al., 1995). As shown in Fig. 4d, Shh was

/

6x19$$$$23

01-16-97 01:35:42

stage stage stage

expressed in the posterior mesenchyme of the ectopic limb bud, indicating essentially no changes in AP polarity in the ectopic limb bud. Lmx1 RNA was detected in the dorsal half of ectopic limb bud (Fig. 4f) or in the ventral half of it.

Induction of Ectopic Limbs in the Presumptive Leg Field Heterotopic transplantation at the presumptive leg region also induced ectopic limb formation. The results are summarized in Table 3. With the implantation of presumptive dorsal leg tissue into the presumptive ventral leg field, ectopic AER was induced in stage 12/13 embryos (2/7), and the frequency of ectopic AER formation decreased rapidly after stage 16/17 (4/7 at stage 16/17 and 1/6 at stage 18). Figure 3c shows a cartilage pattern obtained after the implantation of presumptive dorsal leg tissue into the ventral side of presumptive leg field. Very few cases of ectopic LCAM expression were seen after the implantation of presumptive dorsal leg tissue into the presumptive dorsal leg field (Fig. 3d and Table 3). When presumptive ventral leg tissue was implanted into the presumptive leg dorsal field (V r D) at stage 10/11, 4 of 6 embryos formed ectopic AER. This high frequency of ectopic AER formation continued up to stage 16/17. In the control experiments, the implantation of presumptive ventral leg tissue into the presumptive

Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.

AID DB 8476

stage

dba

195

AER Induction at the DV Boundary

FIG. 3. Dorsal view of the ectopic wing and leg cartilage patterns produced by the implantation of presumptive dorsal limb tissue to the presumptive limb bud region at stage 12/13. (a) Implantation of presumptive dorsal wing tissue into the presumptive ventral wing region. Additional limb was formed in the ventral (lower) side. Digit pattern of the additional limb 3 4; one ulna in zeugopod. (b) Implantation of presumptive dorsal wing tissue to the dorsal region of presumptive wing bud; normal pattern. (c) Implantation of presumptive dorsal leg tissue to the ventral side of presumptive leg field. Additional limb was formed in the ventral (upper) side, which contained proximal elements (femur and tibia). (d) Implantation of presumptive dorsal leg tissue to the dorsal region of the leg; normal pattern. R, radius; U, ulna; Fe, femur; Fi, fibula; T, tibia.

ventral leg field did not induce ectopic AER formation with L-CAM expression at any stages (Table 3).

Implantation of Ectoderm-Free Presumptive Dorsal (or Ventral) Wing Tissue into the Presumptive Ventral (or Dorsal) Wing Field As shown in Table 4, the frequency of ectopic AER formation after the implantation of ectoderm-free wing tissue in stage 12/13 and 14/15 embryos is considerably low. No AER was induced after the implantation of the ectoderm-free (i.e., mesoderm only) presumptive dorsal wing tissue into the presumptive ventral wing field. When presumptive ventral wing mesoderm was implanted into the dorsal wing field, only 2 of 13 embryos formed an ectopic AER in the dorsal side of the host wing bud. Furthermore, ectopic AER did not form after the implantation of ectoderm alone.

Induction of Additional AER in the Presumptive Flank Field Implantation of presumptive dorsal (ventral) wing tissue into the flank ventral (dorsal) field led to the development of additional limbs (Figs. 5a– 5c). The grafting was carried out using stage 12/13 embryos and L-CAM expression in the ectopic AER and limb cartilage pattern was observed (Table 5). After the implantation of presumptive dorsal wing tissue into the flank ventral field, 4 of 8 embryos showed ectopic L-CAM expression and 9 of 13 embryos had additional limb cartilage pattern (Table 5). In 2 of 9 cases, a duplicated leg with additional digits was observed. No additional limbs were observed after the implantation of presumptive dorsal wing tissue into the dorsal flank region (0/5). When presumptive ventral wing tissue was implanted into the flank dorsal field, 2 of 7 embryos showed ectopic expression of L-CAM (Fig. 5b) and 4 of 7 embryos formed

Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.

AID DB 8476

/

6x19$$$$23

01-16-97 01:35:42

dba

196

Tanaka et al.

TABLE 2 Effects of Heterotopic Grafting of Chick Presumptive Wing Regions on the Skeletal Pattern Host stage

Dorsal r ventrala Total number of cases Ectopic wing (Percentage) Sup Stylopodium Zeugopodium Autopodium Unidentified

11

12/13

14/15

16/17

10 4 40

8 4 50

11 8 73

7 4 57

4 1 25

Miss

S

1

1 2

1 2 1

Ventral r dorsalb Total number of cases Ectopic wing (Percentage)

10

8 3 38 Sup

Stylopodium Zeugopodium Autopodium Unidentified

M

2

S

M

5 5 7 1

1 3 4

8 2 25 Miss

S

M

S

1

3 4 3

2

M

S

10 0 0 M

S

M

1 1

2 2 2

12 7 58

1 1

2 2

S

7 0 0 M

S

M

1 3 3

Note. Abbreviation for categories under wing cartilages: Sup and S, number of wings with cartilage element duplication; Miss and M, number of wings with cartilage element deletion. a Ectopic cartilage formation after the implantation of presumptive wing dorsal tissue in the wing ventral region of stage 10 to 16/17 chick embryos. b Ectopic cartilage formation after the implantation of presumptive wing ventral tissue in the wing dorsal region of stage 10 to 16/17 chick embryos.

additional limb cartilage pattern (Fig. 5c). In Fig. 5c, the additional wing formed a symmetrically duplicated digit pattern 43234. When the presumptive wing ventral tissue was implanted into the flank ventral region, no additional limbs were formed (0/5). To know the AP and DV polarity of the ectopic limb formed in the flank, the expression of Fgf-8, Shh, and Lmx1 was examined 36 hr after the grafting of presumptive wing ventral tissue into the flank dorsal field at stage 12. Fgf-8 signal was observed in the AER of all the ectopic limb buds formed in the flank. Shh RNA was detected both in the posterior and anterior mesenchyme of some ectopic limb buds. These results show close agreement with the additional wing cartilage pattern, which formed a duplicated digit pattern. Lmx1 was expressed in the dorsal or ventral half of the ectopic limb buds. We also cross-transplanted left and right flank tissue including the DV boundary, remaining the AP polarity unchanged, at stage 12/13. We did not observe any addi-

tional limb formation in these embryos, although a new DV boundary was formed, suggesting that the limb bud formation may require not only a DV boundary but also some molecules which exist in the presumptive limb field or tissue.

Chimera Analysis Chimera analysis was performed to examine the distribution of graft cells in the host flank (Fig. 6). Stage 12 quail presumptive wing dorsal and ventral tissue was implanted into stage 12 chick flank ventral and dorsal fields, respectively. Thirty-six hours after implantation, induced ectopic limb buds were sectioned and stained with A223. In both cases, when the sections through the middle of the additional limb bud were examined, the ectopic AER was formed from the ectodermal cells overlaying the boundary of host and graft mesodermal cells. Quail cells which were of presumptive wing dorsal tissue origin were observed

Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.

AID DB 8476

/

6x19$$$$23

01-16-97 01:35:42

dba

197

AER Induction at the DV Boundary

FIG. 4. Distribution of Fgf-8, Shh, and Lmx1 transcripts in the limb buds. (a, c, e) Transverse section of the normal wing buds. (b, d, f) Transverse section of the ectopic limb buds which was formed by the implantation of presumptive ventral wing tissue to the dorsal side of presumptive wing bud region at stage 15. (a, b) Distribution of Fgf-8 transcripts in the AER (arrowheads). (c, d) Distribution of Shh transcripts in the distal posterior mesenchyme (arrowheads). (e, f) Distribution of Lmx1 transcripts in the dorsal half of the limb bud (arrowheads). D, dorsal; V, ventral; A, anterior; P, posterior regions.

to occupy the ventral half of the limb bud (Fig. 6c) and quail cells which were of presumptive wing ventral origin were observed to occupy the dorsal half of the limb bud (Fig. 6e). In both cases, the transplanted quail ectoderm did not occupy the ectoderm of the ectopic limb bud when measured 36 hr after implantation. The distribution of A223-positive cells and Lmx1-expressing cells in adjacent sections of the ectopic limb bud (Fig. 7) was examined. The ectopic limb bud was fixed 48 hr after the implantation of presumptive wing ventral tissue into the wing dorsal field at stage 15. In this case, graft tissue occupied ventral half and had ventral polarity.

DISCUSSION Juxtaposition of DV Forms an AER In both the presumptive wing bud and leg bud regions, when new DV juxtaposition was made by implanting the dorsal tissue into the ventral field, ectopic AER with strong L-CAM expression was formed in the ventral side of host limb buds. Ectopic AER with L-CAM expression was also induced in the dorsal side by implanting ventral tissue to the dorsal field. Since L-CAM expression occurred in the AER of limb buds of stage 17 to 30 and the induction of

Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.

AID DB 8476

/

6x19$$$$23

01-16-97 01:35:42

dba

198

Tanaka et al.

TABLE 3 Ectopic AER Formation by Implantation of Presumptive Leg Tissue Host stage

Leg dorsal r leg ventrala Ectopic AER Total number of cases Percentage of ectopic AER

9

10/11

12/13

14/15

16/17

18

0 5 0

0 4 0

2 7 29

3 9 33

4 7 57

1 6 17

0 4 0

0 5 0

1 6 17

0 3 0

4 6 67

6 9 67

4 9 44

2 4 50

0 3 0

0 5 0

0 5 0

0 4 0

Leg dorsal r leg dorsalb Ectopic AER Total number of cases Percentage of ectopic AER Leg ventral r leg dorsalc Ectopic AER Total number of cases Percentage of ectopic AER

0 5 0

Leg ventral r leg ventrald Ectopic AER Total number of cases Percentage of ectopic AER

0 5 0

a The frequency of ectopic AER formation after the implantation of presumptive leg dorsal tissue in the leg ventral region of stage 9 to 18 chick embryos. b The frequency of ectopic AER formation after the implantation of presumptive leg dorsal tissue in the leg dorsal region of stage 10/ 11 to 16/17 chick embryos. c The frequency of ectopic AER formation after the implantation of presumptive leg ventral tissue in the leg dorsal region of stage 9 to 18 chick embryos. d The frequency of ectopic AER formation after the implantation of presumptive leg ventral tissue in the leg ventral region of stage 10/ 11 to 16/17 chick embryos.

AER was detected with both L-CAM and Fgf-8, we used LCAM as an AER marker in the present experiments. However, whether L-CAM mRNA exists in small amounts in non-AER ectoderm remains uncertain, since the L-CAM protein, which is recognized by L-CAM antibody, is expressed not only in the AER but also in the other limb bud

TABLE 4 Ectopic AER Formation by Implantation of Ectoderm-Free Ventral or Dorsal Tissue to the Dorsal or Ventral Region of Presumptive Wing Bud

Ectopic AER Total number of cases Percentage of ectopic AER formation

Dorsal into ventral

Ventral into dorsal

Host stage

Host stage

12/13

14/15

12/13

14/15

0 8

0 6

1 6

1 7

0

0

17

14

ectoderm (Crossin et al., 1985). It is possible that synthesis of both L-CAM protein and mRNA occurs principally in the AER, but because L-CAM protein is more stable than L-CAM mRNA, only L-CAM protein remains in the ectoderm. It may be possible that L-CAM (E-cadherin) supplements the function of N-cadherin in the AER, whose antibody cannot bind to the AER (Hatta et al., 1987). The induction of AER occurred from stage 10/11 to stage 16/17 embryos. It is interesting to note that stage 10/11 corresponds to the stage when polarizing activity can first be detected along the flank lateral field from somite level positions 12 to 22 (Hornbruch and Wolpert, 1991) and stage 16/17 corresponds to the stage when normal limb bud begin to rise. It seems that the stage when both presumptive dorsal and ventral sides begin to develop their identity corresponds roughly to the time when the presumptive wing, flank and leg mesoderm start to exhibit a capacity for limb formation when grafted to the coelom of a host embryo (Dhouailly and Kieny, 1972; Stephens et al., 1989). Despite the ability to induce ectopic AER in stage 10/11 to 16/17 embryos, no additional wing cartilage was observed in stage 14/15 and 16/17 embryos when presumptive ventral wing tissue was implanted into the presumptive dorsal field. In such cases, ectopic limb buds with morphologically

Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.

AID DB 8476

/

6x19$$$$23

01-16-97 01:35:42

dba

199

AER Induction at the DV Boundary

When the presumptive wing ventral tissue was implanted into the presumptive wing dorsal field at stage 15 and examined after 36 hr, the expression of Shh was observed in the posterior mesenchyme of the ectopic limb bud, suggesting that the AP polarity of the additional limb bud was normal. In the present experiment, not only the ectopic limbs but also host limbs were incomplete. This result seems to be caused by the limited number of cells which could participate in the limb bud formation. Fibroblast growth factors (FGFs) and retinoic acid (RA) can also induce additional limb structures. When FGFs (Mima et al., 1995) and RA (Summerbell, 1983; Tickle et al., 1985) form additional limbs, they can extend the limb-forming region. However, in the present experiment, the implantation of presumptive limb bud tissue may not have extended the limb-forming region. Induction of ectopic AER occurred at low frequency after implantation of the dorsal tissue into the dorsal region, although no induction occurred after the implantation of the ventral tissue into the ventral region in the presumptive wing or leg field (Tables 1 and 3). Further, in the case of the implantation of the ectoderm-free grafting, the ectopic AER induction was observed only after the implantation of the ventral tissue into the dorsal region in the presumptive wing field. Thus, it is possible that some indispensable factors for ectopic AER induction in the presumptive dorsal field may exist.

What Is the Role of Ectoderm in the Formation of DV Polarity?

FIG. 5. Additional limb produced by the implantation of presumptive ventral wing tissue into the flank dorsal region of stage 12/13 chick embryo. (a) Ectopic bud (arrows) between wing bud and leg bud. Anterior is up and dorsal is right. (b) Expression of L-CAM (arrowheads) in the AER of additional limb bud. Anterior is up and dorsal is right. (c) Dorsal view of the cartilage pattern of additional wing at 10 days of development. Note duplicated digit pattern, 4 3 2 3 4.

AER-like structure were formed at 36 hr, but they did not develop cartilage structures (data not shown). The expression pattern of Fgf-8 and Shh in such ectopic limb buds seemed to be normal (Fig. 4). The reason for the limb bud degeneration remains unclear, but it is possible that the ectopic AER induced in stage 14/15 and 16/17 wing buds later disappeared because of the deficiency of some AER maintenance factors. Since the limb bud regression occurs at various stages, it is difficult to analyze the degeneration mechanism.

The frequency of the ectopic AER formation decreased considerably when the ectoderm-free presumptive wing tissues were heterotopically implanted. Some signals from the ectoderm have been considered to influence DV patterning (MacCabe et al., 1974; Stark and Searls, 1974), and it was recently demonstrated that Wnt-7a plays an important role in DV patterning because knockout mice of Wnt-7a lacked their dorsal limb structures (Parr and McMahon, 1995). Wnt-7a is expressed in the flank ectoderm of the trunk of the mice at pre-limb bud stages (Riddle et al., 1995), although whether Wnt-7a activity is sufficient to establish dorsal mesodermal fate in the presumptive limb bud field is unclear. Furthermore, it was also reported that En-1 promotes ventral-type skin differentiation and inhibits dorsaltype differentiation in the ventral limb because the mice that have a null mutation in En-1 displayed dorsal transformations of ventral structures (Loomis et al., 1996). Moreover, ectopic AER could not form after the implantation of ectoderm alone (data not shown). These results suggest that the ectoderm of the presumptive limb bud region is important in establishing DV identity, but the interaction with mesodermal cells is also important. Moreover, the following possibilities can be considered. The mesoderm which has its DV identity influenced by the ectodermal signal might maintain its DV identity without continuous ectodermal signal(s), or the mesoderm has an intrinsic DV polar-

Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.

AID DB 8476

/

6x19$$$$23

01-16-97 01:35:42

dba

200

Tanaka et al.

TABLE 5 Ectopic AER and Additional Limb Cartilage Elements Formation by Implantation of Presumptive Wing Ventral or Dorsal Tissue to the Flank Dorsal or Ventral Region of Stage 12/13 Chick Empryo

Ectopic AER formation Ectopic AER formation Total number of cases Percentage of ectopic AER Additional limb cartilage elements Additional limb elements formation Total number of cases Percentage of additional limb formation

WD r FV a

WD r FDb

WV r FDc

WV r FVd

4 8 50

0 5 0

2 7 29

0 5 0

9 13 69

4 7 57

a The frequency of ectopic AER formation after the implantation of presumptive wing dorsal tissue in the flank ventral region of 12/13 chick embryos. b The frequency of ectopic AER formation after the implantation of presumptive wing dorsal tissue in the flank dorsal region of 12/13 chick embryos. c The frequency of ectopic AER formation after the implantation of presumptive wing ventral tissue in the flank dorsal region of 12/13 chick embryos. d The frequency of ectopic AER formation after the implantation of presumptive wing ventral tissue in the flank ventral region of 12/13 chick embryos.

ity before the ectodermal signal influences it, as Geduspan and MacCabe reported (1989). Actually, in the present experiment, there were a few cases in which ectopic AER was formed even if the overlying ectoderm was removed from the grafts.

Ectopic DV Boundary in the Flank Region Induces an Ectopic AER Formation In stage 12/13 embryos, after the implantation of the presumptive wing dorsal (ventral) tissue into the presumptive ventral (dorsal) flank field, ectopic AER was induced and additional limbs developed in the non-limb flank region. It seems that flank tissue also has the potential to respond to the implanted tissue. In the chick embryo at stages 11 to 14, presumptive wing, flank, and leg fields exhibit uniform limb-forming ability when grafted to a host coelom (Dhouailly and Kieny, 1972; Stephens et al., 1989). During these stages, the flank tissue might have common DV-specific molecules with the presumptive limb tissue. Often the AP polarity of the additional limb was abnormal (duplicated and reversed). The duplication and reversion of the additional limb might be caused by the higher potential of the polarizing activity in the anterior flank (Hornbruch and Wolpert, 1991). We confirmed that the ectopic AER was formed from the host ectodermal cells overlaying the boundary of host and graft mesodermal cells. No additional limbs were formed after the right– left heterotopic transplantation of flank tissue (ectoderm and mesoderm) including the Wolffian ridge at stage 12/13 with retaining AP polarity (data not shown). This suggests that the formation of limb bud may require not only a DV boundary, but also some molecules which exist in the presumptive limb field.

/

6x19$$$$24

01-16-97 01:35:42

stage stage stage

What Mechanisms Are Operating along the DV Boundaries? We found that the ectopic DV boundary was formed by implantation of quail limb tissue into the presumptive limb field causes ectopic AER formation from stage 10/11 to 16/ 17. The DV boundary which was formed by the implantation of presumptive wing tissue into the flank field also caused ectopic AER formation. As mentioned above, the wing, flank, and leg exhibit a uniform capacity for limb formation at stages 11 – 14. It is possible that DV polarization in the mesoderm is established prior to the expression of such known factors as Wnt-7a and Lmx1. This directly supports the earlier suggestion by Geduspan and MacCabe (1989) that in embryos up to stage 14 the DV polarity of the future limb is determined by the mesoderm rather than the ectoderm. A related study showed that Wnt-7a knock-out mice are ventralized only at the distal part (Parr and McMahon, 1995), which also indicates that a DV pattern exists prior to Wnt-7a expression. We consider that the AER was formed at the DV boundary after the DV polarity of the limb bud was determined through the process described above. This idea was originally postulated in the boundary model by Meinhardt (Meinhardt, 1983; and reviewed by Martin, 1995). The boundary model includes the hypothesis that the molecules which control patterning are produced by interaction between the cells in differently determined territories. He considered that the AER was formed at the border of DV territories. One probable molecule which expresses in the DV boundary is the FGF family. FGF-4 (Niswander et al., 1993) and FGF-2 (Fallon et al., 1994) can substitute for AER. In particular, Fgf-8 expression is observed not only in the AER imme-

Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.

AID DB 8476

stage

dba

201

AER Induction at the DV Boundary

diately after the limb bud is established, but also in the surface ectoderm of the limb field just prior to and during the early stages of limb bud outgrowth in both mouse and chick embryos (Ohuchi et al., 1994; Crossley and Martin, 1995; Mahmood et al., 1995), suggesting that FGF-8 may stimulate initial outgrowth of the limb buds. In the present experiment, Fgf-8 expression was detected in the AER of the ectopic limb bud which formed after the implantation of the presumptive wing ventral tissue into the presumptive wing dorsal field at stage 15, although no expression of Fgf8 was detected in the somatopleure at the presumptive wing bud level. It is worth noting that in many cases only one ectopic AER formed at the DV boundary, even though two AERs should be possible since two DV boundary lines are created by the grafting. In the chimera analysis, only one ectopic AER was formed at the boundary between graft tissue and host tissue, although there were two boundaries (arrows in Fig. 6e). In addition, ectopic and original AERs seem to avoid lying parallel to each other (see Fig. 2c). Thus it is possible that there may be some mechanisms which inhibit further AER formation in the surrounding tissue. It is interesting that ectopic limb buds which were formed after the implantation of the presumptive ventral tissue to the dorsal field exhibited two different DV polarities and the grafted tissues occupied dorsal (Fig. 6e) or ventral half (Fig. 7a) of the ec-

FIG. 7. Serial sections of the ectopic limb bud which was formed by the implantation of presumptive wing ventral tissue into the wing dorsal field at stage 15. (a) Immunohistochemistry with A223. Quail cells (arrows) are not stained with A223. (b) Phase-contrast photograph of a. (c) Distribution of Lmx1 transcripts in the ectopic limb bud (arrows). AER is indicated by arrowheads. FIG. 6. Immunohistochemistry with A223 to show the position of grafted quail cells in the ectopic limb bud. Quail tissue was grafted at stage 12. Three chimeras were analyzed. In all photographs, dorsal is up and ventral is down. Quail cells (arrows) are not stained with antibody A223. (a) Schematic representation of an ectopic limb bud formed between wing bud and leg bud. The line represents the position of sections for b –e. (b, c) Section of the ectopic limb bud which was formed by the implantation of presumptive wing dorsal tissue into the flank ventral region. (c, e) Immunohistochemistry with A223. (b) Phase-contrast photograph of c. (d, e) Section of the ectopic limb bud which was formed by the implantation of presumptive wing ventral tissue into the flank dorsal region. (d) Phase-contrast photograph of e. AER is indicated by arrowheads. Bar in b and d, 200 mm. D, dorsal; V, ventral regions.

topic limb buds. It is conceivable that this difference in DV polarity might depend upon which DV boundary forms the ectopic AER. When the ectopic AER is formed at the dorsal margin of the grafted ventral tissue, the DV axis of the ectopic limb bud will be normal (equal to body DV axis), but when the AER is formed at the ventral margin, the DV axis of the ectopic limb bud will be reversed. Moreover, 48 hr after the implantation, the ectopic AER was not formed on the boundary of host and graft mesodermal cells. Hence, the DV boundary might be essential to form AER in the

Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.

AID DB 8476

/

6x19$$$$24

01-16-97 01:35:42

dba

202

Tanaka et al.

ectoderm, and after the AER induction, presumptive dorsal cells might proliferate faster than presumptive ventral cells (Geduspan and Solursh, 1992). The results of our experiments show that AER is formed along the DV boundary in the presumptive limb bud and flank region. However, the signaling molecules which participate in this process remain unclear. Further study will be necessary to determine what kind of factors are at work in the process of normal and ectopic limb bud formation.

ACKNOWLEDGMENT This work was supported by research grants from the Ministry of Education, Science and Culture of Japan.

REFERENCES Basler, K., and Struhl, G. (1994). Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368, 208– 214. Blair, S., Brower, D., Thomas, J., and Zavortnk, M. (1994). The role of apterous in the control of dorsoventral compartmentalization and PS integrin gene expression in the developing wing of Drosophila. Development 120, 1805 –1815. Chaube, S. (1959). On axiation and symmetry in the transplanted wing of the chick. J. Exp. Zool. 140, 29 –77. Cohen, B., McGriffin, M. E., Pfeifle, C., Segal, D., and Cohen, S. M. (1992). apterous, a gene required for imaginal disc development in Drosophila encodes a member of the LIM family of developmental regulatory proteins. Genes Dev. 6, 715 –729. Couso, J., Bishop, S., and Martinez-Arias, A. (1994). The wingless signaling pathway and the patterning of the wing margin in Drosophila. Development 120, 621– 636. Crossin, K. L., Chuong, C.-M., and Edelman, G. M. (1985). Expression sequences of cell adhesion molecules. Proc. Natl. Acad. Sci. USA 82, 6942 –6946. Crossley, P. H., and Martin, G. R. (1995). The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121, 439– 451. Davis, C. A., Holmyard, D. P., Millen, K. J., and Joyner, A. L. (1991). Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum. Development 111, 287– 98. Dealy, C. N., Roth, A., Ferrari, D., Brown, A. M. C., and Kosher, R. A. (1993). Wnt-5a and Wnt-7a are expressed in the developing chick limb bud in a mannar suggesting roles in pattern formation along the proximodistal and dorsoventral axes. Mech. Dev. 43, 175–186. Dhoailly, D., and Kieny, M. (1972). The capacity of the flank somatic mesoderm of early bird embryos to participate in limb development. Dev. Biol. 28, 162– 175. Dias-Benjumea, F., and Cohen, S. M. (1993). Interaction between dorsal and ventral cells in the imaginal disc directs wing development in Drosophila. Cell 75, 741– 752. Dias-Benjumea, F., Cohen, B., and Cohen, S. (1994). Cell interaction between compartments establishes the proximal –distal axis of Drosophila legs. Nature 372, 175– 178. Fallon, J. F., Lopez, A., Ros, M. A., Savage, M. P., Olwin, B. B., and

Simandl, B. K. (1994). FGF-2: Apical ectodermal ridge growth signal for chick limb development. Science 264, 104 –106. Gavin, B. J., McMahon, J. A., and McMahon, A. P. (1991). Expression of multiple novel Wnt-1/int-1-related genes during fetal and adult mouse development. Genes Dev. 4, 2319 – 2332. Geduspan, J. S., and MacCabe, J. A. (1989). Transfer of information from mesoderm to ectoderm at the onset of limb development. Anat. Rec. 224, 79 –87. Geduspan, J. S., and Solursh, M. (1992). Cellular contribution of the different regions of the somatopleure to the developing limb. Dev. Dyn. 195, 177–187. German, M. S., Wang, J., Chadwick, R. B., and Rutter, W. J. (1992). Synergistic activation of the insulin gene by a LIM homeodomain protein and a basic helix –loop –helix protein: Building a functional insulin minienhancer complex. Genes Dev. 6, 2165 – 2176. Hamburger, V., and Hamilton, H. (1951). A series of normal stages in the development of chick embryo. J. Morphol. 88, 49–92. Hatta, K., Takagi, S., Fujisawa, H., and Takeichi, M. (1987). Spacial and temporal expression pattern of N-cadherin cell adhesion molecules correlated with morphogenetic processes of chicken embryos. Dev. Biol. 120, 215 –227. Heikinheimo, M., Lawshe, A., Shackleford, G. M., Wilson, D. B., and ManAthur, C. A. (1994). Fgf-8 expression in the post-gastrulation mouse suggests roles in the development of the face, limbs, and central nervous system. Mech. Dev. 48, 129– 138. His, W. (1868). ‘‘Untersuchunger uber die erste Anlage des Wirbelthierleibes.’’ Vogel, Leipzig. Hornbruch, A., and Wolpert, L. (1991). The spatial temporal distribution of polarizing activity in the flank of the pre-limb-bud stages in the chick embryo. Development 111, 725– 731. Ide, H., Wada, N., and Uchiyama, K. (1994). Sorting out of cells from different parts and stages of the chick limb bud. Dev. Biol. 162, 71– 76. Javois, L. C., and Iten, L. E. (1982). Supernumerary limb structures after juxtaposing dorsal and ventral chick wing bud cells. Dev. Biol. 90, 127– 143. Loomis, C. A., Harris, E., Michaud, J., Wurst, W., Hanks, M., and Joyner, A. L. (1996). The mouse Engrailed-1 gene and ventral limb patterning. Nature 382, 360–363. MacCabe, J. A., Errick, J., and Saunders, J. W. (1974). Ectodermal control of the dorso– ventral axis in the leg bud of the chick embryo. Dev. Biol. 39, 69–82. Mahmood, R., Bresnick, J., Hornbruch, A., Mahony, C., Morton, N., Colquhoun, K., Martin, P., Lumsden, A., Dickson, C., and Mason, I. (1995). A role for FGF-8 in the initiation and maintenance of vertebrate limb bud outgrowth. Curr. Biol. 5, 797– 806. Martin, G. R. (1995). Why thumbs are up. Nature 374, 410–411. Meinhardt, H. (1983). A boundary model for pattern formation in vertebrate limbs. J. Embryol. Exp. Morphol. 76, 115–137. Mima, T., Ohuchi, H., Noji, S., and Mikawa, T. (1995). FGF can induce outgrowth of somatic mesoderm both inside and outside of limb-forming regions. Dev. Biol. 167, 617–620. Niswander, L., and Martin, G. R. (1992). Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 114, 755– 768. Niswander, L., Tickle, C., Vogel, A., Booth, J., and Martin, G. R. (1993). FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell 75, 579– 587. Ohuchi, H., Yoshioka, H., Tanaka, A., Kawakami, Y., Nohno, T., and Noji, S. (1994). Involvement of androgen-induced growth factor (FGF-8) gene in mouse embryogenesis and morphogenesis. Biochem. Biophys. Res. Commun. 204, 882– 888.

Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.

AID DB 8476

/

6x19$$$$24

01-16-97 01:35:42

dba

203

AER Induction at the DV Boundary

Parr, B. A., and McMahon, A. P. (1995). Dorsaling signal Wnt-7a required for normal polarity of D –V and A – P axes of mouse limb. Nature 374, 350 –353. Parr, B. A., Shea, M. J., Vassileva, G., and McMahon, A. P. (1993). Mouse Wnt genes exhibit discrete domaines of expression in the early embryonic CNS and limb bud. Development 119, 247 –261. Riddle, R. D., Johnson, R. L., Laufer, E., and Tabin, C. (1993). Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401 –1416. Riddle, R. D., Ensini, M., Nelson, C., Tsuchida, T., Jessell, T. M., and Tabin, C. (1995). Induction of the LIM homeobox gene Lmx1 by WNT7a establishes dorsoventral pattern in the vertebrate limb. Cell 83, 631–640. Rosenquist, G. C. (1971). The origin and movement of the limbbud epithelium and mesenchyme in the chick embryo as determined by radioautographic mapping. J. Embryol. Exp. Morphol. 25, 83– 96. Sano, K., Tanihara, H., Heimark, R. L., Obata, S., Davidson, M., St. John, T., Taketani, S., and Suzuki, S. (1993). Protocadherins: a large family of cadherin-related molecules in central nervous system. EMBO J. 12, 2249 – 2256. Saunders, J. W., Jr., and Gasseling, M. T. (1968). Ectodermal –messenchymal interactions in the origin of limb symmetry. In ‘‘Epithelial–Mesenchymal Interactions’’ (R. Fleishmajer and R. E. Billingham, Eds.), pp. 78 –97. Williams and Wilkins, Baltimore. Savage, M. P., Hart, C. E., Riley, B., Sasse, J., Olwin, B. B., and Fallon, J. R. (1993). Distribution of FGF-2 suggests it has a role in chick limb bud growth. Dev. Dyn. 198, 159– 170. Searls, R. L. (1976). Effect of dorsal and ventral ectoderm on the development of the limb of the embryonic chick. J. Embryol. Exp. Morphol. 35, 369– 381. Stark, R. J., and Searls, R. L. (1974). The establishment of the cartilage pattern in the embryonic chick wing, and evidence for a role of the dorsal and ventral ectoderm in normal wing development. Dev. Biol. 38, 51– 63. Stephens, T. D., Beier, R. L. W., Bringhurst, D. C., Hiatl, S. R.,

Prestridge, M., Pugmire, D. E., and Willis, H. J. (1989). Limbness in the early chick embryo lateral plate. Dev. Biol. 133, 1– 7. Summerbell, D. (1983). The effects of local application of retinoic acid to the anterior margin of the developing chick limb. J. Embryol. Exp. Morphol. 78, 269–289. Suzuki, S., Sano, K., and Tanihara, H. (1991). Diversity of the cadherin family: Evidence for eight new cadherins in nervous tissue. Cell Regul. 2, 261– 270. Tamura, K., Yajima, H., and Ide, H. (Submitted for publication). Tickle, C., Lee, J., and Eichele, G. (1985). A quantitive analysis of the effect of all-trans-retinoic acid on the pattern of chick wing development. Dev. Biol. 109, 82– 95. Vogel, A., Rodriguez, C., Warnken, W., and Izpisua Belmonte, J. C. (1995). Dorsal cell fate specified by chick Lmx1 during vertebrate limb development. Nature 378, 716– 720. Wilkinson, D. G. (1992). Whole-mount in situ hybridization of vertebrate embryos. In ‘‘In Situ Hybridization: A Practical Approach’’ (D. G. Wilkinson, Ed.), pp. 75 –83. Oxford, IRL Press. Williams, J. A., Paddock, S., Vorwerk, L., and Carroll, S. (1994). Organization of wing formation and induction of a wing-patterning gene at a compartment boundary. Nature 368, 298– 305. Wrurst, W., Auerbach, A. B., and Joyner, A. L. (1994). Multiple developmental defects in Engrailed-1 mutant mice: an early midhindbrain deletion and patterning defects in forelimbs and sternum. Development 120, 2065 –2075. Yokouchi, Y., Ohsugi, K., Sasaki, H., and Kuroiwa, A. (1991a). Chicken homeobox genes Msx-1: Structure, expression in limb buds and effect of retinoic acid. Development 113, 431 –444. Yokouchi, Y., Ohsugi, K., Uchiyama, K., and Ide, H. (1991b). Control of the expression of AV-1 protein, a position-specific antigen, by ZPA (zone of polarizing activity) factor in the chick limb bud. Dev. Growth Differ. 32, 173– 180. Yonei, S., Tamura, K., Ohsugi, K., and Ide, H. (1995). MRC-5 cells induce the AER prior to the duplicated pattern formation in chick limb bud. Dev. Biol. 170, 542– 552. Received for publication January 30, 1996 Accepted November 17, 1996

Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.

AID DB 8476

/

6x19$$$$25

01-16-97 01:35:42

dba