Biological Institute, Graduate School of Life Science, Tohoku University, Aobayama Aoba-ku, Sendai 980-8578, Japan
*Author for correspondence (e-mail: tam{at}biology.tohoku.ac.jp)
Accepted 3 October 2001
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SUMMARY |
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Key words: Tbx5, Tbx4, Limb bud, Specification, Determination, Chick
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INTRODUCTION |
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Limbs of a tetrapod emerge from four presumptive limb regions as four limb buds (a set of two forelimb and hindlimb buds). The forelimb and hindlimb buds arise at particular levels of the lateral region along the primary body axis at almost the same time. At the beginning of limb development, structural differences in forelimb and hindlimb buds are not obvious. In chick embryos, the shapes and sizes of forelimb and hindlimb buds appear identical for about 16 hours after the onset of budding. Each limb bud has common regions necessary for limb morphogenesis, such as the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER) (Saunders and Gasseling, 1968; Saunders, 1948; Saunders et al., 1976). Moreover, many genes involved in limb pattern formation are expressed at similar timings and regions in forelimb and hindlimb buds, suggesting that they share common mechanisms and signaling pathways through key molecules in order to construct the limb structure. Nevertheless, structures of the forelimb and hindlimb in most tetrapods become completely different with the progress of development. In the chick, forelimb buds finally grow into wings with feathers, and hindlimb buds develop into legs with scales and claws as epithelial structures. The wings and legs also have distinct patterns of skeletal elements, muscles and tendon attachments as mesenchymal features. Many intriguing issues regarding limb-type identification during development remain unresolved: e.g., when the specification and determination of limb identity occurs, whether tissue interactions, inductions or inhibitions from other tissue(s) are involved in the establishment of limb-type identity, and, if so, which tissue(s) and what molecular mechanism are involved in it. Even the results of the following recent interesting studies on Tbx genes showing the molecular nature of limb identity have not resolved these issues.
Limb-type-specific expression of Tbx5/Tbx4 has been postulated to establish the forelimb/hindlimb identity. In normal chick development, Tbx5 and Tbx4 are exclusively expressed in the presumptive forelimb region (and later in the forelimb bud) and the presumptive hindlimb region (and in the hindlimb bud), respectively (Gibson-Brown et al., 1998; Isaac et al., 1998; Logan et al., 1998; Ohuchi et al., 1998). Early expression of Tbx5 or Tbx4 in an ectopic limb bud induced by exogenous FGF corresponds with the later wing or leg phenotype of the additional limb. These limb-type-specific expressions of Tbx5/Tbx4 are well conserved in other tetrapod embryos, including human (Li et al., 1997), mouse (Chapman et al., 1996; Gibson-Brown et al., 1996), Xenopus (Takabatake et al., 2000) and newt embryos (Simon et al., 1997). Paired fin buds in teleost fish have the same character of Tbx gene expression (Ruvinsky et al., 2000; Tamura et al., 1999). Furthermore, with chick manipulation systems, it has been demonstrated that wings with leg-like characters and legs with wing-like characters were observed when Tbx4 and Tbx5 were introduced into the presumptive hindlimb and forelimb regions, respectively (Rodriguez-Esteban et al., 1999; Takeuchi et al., 1999). These studies strongly suggest that Tbx5 and Tbx4 in the presumptive limb regions and limb buds are responsible for the distinct identities of limbs. However, the upstream mechanism restricting the Tbx5/Tbx4 expression in each limb bud is not known.
We have examined conditions for the determination of limb identity by using transplantation methods and tissue culture systems with chick embryos. Interestingly, the medial tissues at the anterior level inhibit Tbx4 expression in the forelimb region as well as activate Tbx5 expression, and in the meantime, the posterior medial tissues are likely to have repressive action toward Tbx5 expression in the hindlimb region. Our data presented here enabled us to predict the timing and conditions needed to specify, determine and exhibit each limb identity and the role of medial tissues for determination of the identity.
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MATERIALS AND METHODS |
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As graft tissues for transplantations, we used the lateral plate mesoderm of the presumptive limb region and the overlying ectoderm taken from stage 9-14 embryos. Methods of transplantation and relative positions of the host and the graft are shown in Fig. 3A. First, host dorsal medial ectoderm at the level of somite 15 (boundary between the neck and the forelimb) or somite 26 (boundary between the flank and the hindlimb) was cut and partially peeled toward the level of somite 19/20 or 30/31 so as to be separated from underlying tissues. The donor graft was inserted in the cavity of the host and placed between the dorsal medial ectoderm and the underlying neural tube and somites at the level of the forelimb or hindlimb region. After manipulations, the eggs were resealed and allowed to develop in an incubator at 38°C for subsequent analyses by in situ hybridization and skeletal pattern observation. For skeletal pattern observation, embryos were incubated for 7 days after the operation and then fixed in 10% formalin, stained with 0.1% Alcian Blue in 70% acid alcohol, dehydrated in ethanol, and cleared in methyl salicylate.
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Relative quantitative RT-PCR
RNA was isolated from embryos and explants using RNeasy total RNA isolation kit (Qiagen) and analyzed by RT-PCR essentially as described previously (Kimura and Ide, 1998). Tbx5- and Tbx4-specific primers yielding product sizes as indicated are: Tbx5 (369 bp) [forward primer, 5'-AACCCCTACCC GATCTCCCAGG-3' (22 mer); reverse primer, 5'-GTGAAGTGGGCAGAGAAATG-3' (20 mer)], Tbx4 (306 bp) [forward primer, 5'-GCTTCACTTATATGG-TACTCAG-3' (22 mer); reverse primer, 5'-CACGGTCAA-TGGGGGAAGAAGG-3' (22 mer)], and ß-actin (167 bp) [forward primer, 5'-TCTGACTGACCGCGTTACTC-3' (20 mer); reverse primer, 5'-CCAT CACACCCTGATGTCTG-3' (20 mer)]. These primer sets were based on the chick Tbx5 mRNA sequence (GenBank No. AF069396), Tbx4 mRNA sequence (GenBank No. AF069395) and ß-actin mRNA sequence (GenBank No. L08165), respectively. Southern blotting and detection were performed with respective DIG-labeled probes according to the procedure described in the manufacturers instructions, DIG System Users Guide for Filter Hybridization (Roche). Signal detection was performed with anti-digoxigenin-AP Fab fragments and its substrate, CDP-star (Roche). For quantitative assay, chemiluminescence intensity of reacting CDP-star in each PCR product was estimated as photostimulated luminescence (PSL) using Molecular Imager System (GS525, BioRad).
To determine the appropriate cycle number for amplification of each gene in the RT-PCR assay, a series of PCRs with several cycles (15-36) was done with cDNA derived from the stage 13 presumptive wing region for Tbx5 and ß-actin and the stage 13 presumptive leg region for Tbx4 (Fig. 1A). For quantitative comparison of these gene expressions, the proportions of the intensity of the band for Tbx5 and Tbx4 to that for ß-actin were calculated from respective PSL values. On standard samples of each figure, the values of the ratio were fixed as 1. Gene expressions in samples were compared using Students matched-pair t-test.
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RESULTS |
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Expression of Tbx5/Tbx4 in cultured explants of the presumptive limb region
The results in Fig.1 demonstrate that Tbx5/Tbx4 begins to be expressed at stage 13 in the presumptive forelimb/hindlimb region. Before limb identity is exhibited as expression of Tbx5/Tbx4, specification and subsequent determination of limb identity should occur. In order to examine when the specification of limb identity occurs, we next observed Tbx5/Tbx4 expression in isolated and cultured limb-region explants by RT-PCR (Fig. 2A). Presumptive limb regions at stages 9-14 were excised to isolate them from surrounding tissues and cultured for 24 hours. The relative amounts of Tbx5/Tbx4 in each sample were calculated and plotted in Fig. 2B and 2C. The amount of Tbx5/Tbx4 expression in non-cultured presumptive wing/leg region at stage 9 was defined as the standard. In explants derived from stage 9-12 presumptive wing regions, in which normal Tbx5 expression had not yet been up-regulated (see Fig. 1D), Tbx5 expression become abundant after they were cultured for 24 hours, increasing to a level similar to that in non-cultured stage 14 wing explants (Fig. 2B). A high level of Tbx5 expression in stage 13-14 presumptive wing regions was maintained for 24 hours in the culture system. In the same way, in explants Tbx4 expression (cultured for 24 hours) in the presumptive leg region at all stages analyzed was much more abundant than that in the non-cultured stage 9 presumptive leg region (Fig. 2C), and each relative amount was comparable to that in non-cultured stage 14 leg explants. These results clearly show that the presumptive wing/leg regions after stage 9 have the inherent potential to express Tbx5/Tbx4, suggesting that each limb-type identity has already been specified by stage 9.
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Transplantation of the presumptive limb region onto the dorsal midline
The next step after the specification of limb identity should be determination of the identity. After determination it is thought that the tissue identity cannot be altered, and the determined cell will follow its fate even when grafted into another part of the embryo.
We transplanted the presumptive wing or leg bud region onto the dorsal midline in median tissues, and we examined whether the surrounding tissues have the ability to alter Tbx5/Tbx4 expression of the grafted presumptive limb region. The stage 10 presumptive wing/leg region was transplanted onto the dorsal midline at the level of the presumptive leg/wing region (Fig. 3A) and incubated for 48 hours, and Tbx5/Tbx4 expression in the transplanted limb bud growing from the dorsal region were analyzed by whole-mount in situ hybridization. Tbx5/Tbx4 expression in the tissue grafted to the same level (wing region to wing level and leg region to leg level) showed no change (all grafted wing buds having only Tbx5 expression and grafted leg buds having only Tbx4 expression, data not shown). When the stage 10 presumptive wing region was transplanted to the leg level, all limb buds expressed Tbx5 (n=8/8, Fig. 3B) and none expressed Tbx4 (n=7/7, Fig. 3C). However, all grafts of a stage 10 presumptive leg region into the wing level were Tbx4 positive, (n=9/9, Fig. 3F) although the expression was sometimes weak. Surprisingly, most of the leg grafts in the wing level examined with the Tbx5 probe showed significant Tbx5 expression (n=5/8, Fig. 3E), suggesting that the grafted presumptive leg regions were converted to express Tbx5. To confirm that Tbx5 and Tbx4 are expressed in the same leg transplants at the same time, we performed double staining by whole-mount in situ hybridization on operated embryos after the same surgery. When the stage 10 presumptive wing region was transplanted onto the presumptive leg level, all supernumerary limbs expressed Tbx5 (in green, Fig. 3D; n=18/18), but Tbx4 was negative in all specimen (in red, Fig. 3D). However, two types of gene expression pattern were observed in specimens of stage 10 presumptive leg region at the wing level. One was only Tbx4 expression (n=4/16, see Table 1), and the other was Tbx5 expression as well as Tbx4 expression in a supernumerary limb (n=12/16, Fig. 3G) in which Tbx5 and Tbx4 seemed to have a mosaic expression pattern. To further examine the detailed distribution of the Tbx5-expressing region and Tbx4-expressing region in a graft, we prepared serial sections of a supernumerary limb bud derived from the presumptive leg region and stained them with Tbx5 or Tbx4. The region in which Tbx5 expression was induced in the supernumerary limb nearly corresponded to the region in which Tbx4 expression was down-regulated (Fig. 3H,I).
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In summary, we could alter leg-type gene expression and the skeletal phenotype to wing-type until stage 12, but wing-type gene expression and the phenotype were never converted to leg-type throughout the stages we examined. These results suggest that the presumptive leg region is determined as a leg at around stage 12 and that wing determination occurs very early (before stage 9).
Effects of presumptive wing/leg level-median tissues on Tbx5 and Tbx4 expressions in the presumptive leg region
The results of transplantation experiments suggest that the environment of the midline seems to be able to regulate Tbx5/Tbx4 expression in the presumptive limb region. In order to try to determine what controls this, we cultured stage 10 presumptive leg regions combined with tissues form various median levels (Fig. 5A, each level of the median tissue including a neural tube, notochord, somites, intermediate mesoderm, overlying ectoderm, and underlying endoderm) for 48 hours. Tbx5 expression was higher in the combination of the presumptive leg region and presumptive wing level-median tissues (regions 2+5 in Fig. 5C) than in the case of combinations with other level-median tissues (1, 3, or 4+5 in Fig. 5C). The relative amount of 2+5 was more than twice that of 4+5 if the amount of Tbx5 in the median tissues themselves is considered (2 and 4 in Fig. 5C). In contrast, median tissue for the presumptive flank region (limbless space between the wing and leg, 3 in Fig. 5A) inhibited Tbx5 expression (compare 3+5 with 4+5, with considering lanes 3 and 5 in Fig. 5C). The amount of Tbx4 expression in the combination 2+5 is appreciably smaller than in any other combinations (Fig. 5D; P<0.05). We obtained similar results in samples cultured for 24 hours, although each amount was relatively low (data not shown). The results described above suggest that the presumptive wing level-median tissue has the ability not only to accelerate Tbx5 expression but also to repress Tbx4 expression. Although we tested the presumptive wing region with the same combinations, no significant differences were seen in the amounts of Tbx5/Tbx4 transcripts in all samples (data not shown).
Finally, we investigated whether the presumptive leg level-median tissue has effects on the presumptive leg region. We compared the amount of Tbx5/Tbx4 expression in cultured un-separated explants (experiment 3 in Fig. 6A-D) that contained the presumptive leg region and leg level-median tissues with that in cultured separated explants (experiment 2 in Fig. 6A-D. The presumptive leg region and leg level-median tissues were cultured separately and then, assayed together). As a control, we determined the amount of Tbx5/Tbx4 expression in the same region before incubation (experiment 1 in Fig. 6A-D). When they were cultured for 24 hours, expression of both Tbx5 and Tbx4 were considerably stimulated in both conditions. The amounts of Tbx5 expression in experiment 2 and 3 were significantly different (Fig. 6B,C; P<0.05). The relative amount of Tbx5 expression in experiment 2 was about 1.9-times greater than that in experiment 3. These data suggest that the presumptive leg-level median tissues repress Tbx5 expression.
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DISCUSSION |
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Limb-type specification, determination and Tbx5/Tbx4 expression
Tbx5 and Tbx4 in the presumptive limb regions and limb buds are responsible for the distinct identities of limbs (Rodriguez-Esteban et al., 1999; Takeuchi et al., 1999), and hence these two genes can be viewed as selector genes of limb-type identity in vertebrates [see review by Weatherbee and Carroll (Weatherbee and Carroll, 1999)]. In the present study, we have demonstrated that the expression of both Tbx5 and Tbx4 began to be up-regulated in each presumptive forelimb/hindlimb region at stage 13 (Fig. 1), soon after limb identity had been determined (see below). Taken together, the results suggest that Tbx5/Tbx4 expression in each presumptive limb region is one of the earliest phenotypes representing limb-type identity. In other words, the process of commitment to limb-type is possibly complete at around stage 13. This possibility is supported by the results of transplantation experiments, showing the timing of determination of each region. It is likely that the presumptive wing region is determined before stage 9, since wing-type identity (Tbx5 expression and wing-type cartilage pattern) was irreversible after stage 9 (Fig. 3, Fig. 4). However, the presumptive leg region is thought to be determined at a later stage, stages 12/13, because supernumerary limbs derived from the presumptive leg region between stages 9 and 12/13 were capable of ectopically expressing Tbx5 and making a wing-type cartilage pattern, but the leg-type phenotype became irreversible after stages 12/13 (Fig. 3, Fig. 4 and Table 1, Table 2). In terms of limb-type specification, the fate of both regions seems to be specified before stage 9, because all presumptive limb regions isolated from stage 9-14 embryos and placed in culture dishes were capable of expressing innate Tbx5/Tbx4 genes autonomously (Fig. 2). The timings of specification, determination, and Tbx gene expression for the commitment process of limb identity are shown in Fig. 7. The stage at which we suggest the presumptive leg region is determined only approximately corresponds with previous study (Stephens et al., 1989). They estimated the limb-forming potential of the presumptive neck-, forelimb-, flank- and hindlimb-level lateral plate mesoderm and suggested that stages 12-14 are characterized by the determination of the specific limb in the future wing and leg. However, they used a different transplantation system; they transplanted each presumptive wing/leg region into the coelomic cavity, where the effects of other tissues would be much less, and they did not estimate the stage by expressions of specific marker genes. Moreover, they did not clearly distinguish between specification and determination. These are thought to be the reasons why estimated stages in their study and those in the present study are somewhat different.
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Fig. 7 shows a model of limb development at various stages. In the presumptive wing region, wing-type identity is specified and determined before stage 9. This irreversible determination is maintained until stage 13, and this region begins to exhibit a wing phenotype as Tbx5 expression at stage 13. In this process, the abilities to induce Tbx5 and to repress Tbx4 in the presumptive wing level-median tissues contribute to determination and maintenance of wing-type identity. In the presumptive leg region, leg-type identity is specified before stage 9 but is not yet determined at this stage. Determination occurs at stages 12/13, and subsequently, this region begins to differentiate into a leg bud with Tbx4 up-regulation at stage 13. Tbx5-repressing ability in the presumptive leg level-median tissues contributes to the determination of leg-type identity. The period from determination of leg-type identity to Tbx4 up-regulation in the presumptive leg region is considerably shorter than the period in the presumptive wing region. It should be noted that the inhibitory regulation on the limb-type-specific expression of Tbx genes is the major factor in the determination of limb identity.
Possible molecular mechanisms concerned with regulation of limb-type identity
Although little is known about upstream regulators in the lateral plate mesoderm that control the specific expression of Tbx5/Tbx4, it is possible that the expression of Tbx5/Tbx4 is regulated by some transcription regulatory factors in the presumptive limb region. Pitx1, a bicoid-related transcription factor, is possibly an upstream regulator of Tbx4, because Pitx1 expression precedes Tbx4 expression in leg-region lateral plate mesoderm (Takeuchi et al., 1999), and misexpression of Pitx1 in the chick wing bud induces ectopic Tbx4 expression (Logan et al., 1998; Takeuchi et al., 1999). The other candidates for upstream regulators are Hox genes, which represent positional identity and determine the position-dependent morphologies of axial structures along the antero-posterior (AP) body axis. It is thought that each combination of Hox genes (Hox code) acts upstream of Tbx5/Tbx4 in the determination of limb-type identity for the following reasons. A specific Hox code exists in each presumptive limb region at early stages (Gaunt and Strachan, 1994; Gaunt and Strachan, 1996), limb-type-specific expression patterns of Hox genes in ectopic limbs induced at the flank of the chick embryo mimic the endogenous expression patterns in the limb field (Cohn et al., 1997), and a loss-of-function mutation of a Hox gene, Hoxb5, in the mouse shifts the axial position of the limb bud (Rancourt et al., 1995). A recent study on Wnt signaling (Kawakami et al., 2001) demonstrated that new members of the Wnt family, Wnt2b and Wnt8c, are expressed in the presumptive forelimb and hindlimb regions of the lateral plate, respectively, and that they also have limb-forming ability, suggesting that these factors might control Tbx expression in a particular region of the lateral plate.
It should be questioned whether specific expressions of all these molecules for limb-type identification, Hox genes, Pitx1 and Tbx5/Tbx4 are regulated autonomously in the lateral plate mesoderm or whether some regulation from other tissues are involved in this process. The results of the present study are the first to suggest that median tissues can regulate Tbx5/Tbx4 expression and that this regulation includes repressive factor(s) as well as inductive ones. These factors could be diffusible since they can act between discontinuous tissues in a culture condition, although the molecular nature of these regulators remains unknown. It is expected that these factors are specifically or differently expressed at the level of the wing or leg region in the median tissue and possibly regulated by another combination of Hox genes that acts to demarcate functional domains of the median tissues such as neural tissue, muscle structure, and skeletal elements along the primary body axis. The imaginary molecules may regulate Pitx1 and a specific Hox code in the presumptive limb region, resulting in the specific expression of Tbx5/Tbx4 and limb-type identity. It is possible that combinations of the above molecules may regulate the specific expression of Tbx genes and limb identity, and the possibility that unknown new molecules are also involved in this determination cannot be excluded. Based on the findings presented here, it is concluded that inhibitory regulation from the surrounding tissues is conducting the limb-type-specific Tbx expression.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Chaube, S. (1959). On axiation and symmetry in transplanted wing of the chick. J. Exp. Zool. 140, 29-77.
Chapman, D. L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S. I., Gibson-Brown, J. J., Cebra-Thomas, J., Bollag, R. J., Silver, L. M. and Papaioannou, V. E. (1996). Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev. Dyn. 206, 379-390.[Medline]
Cohn, M. J., Patel, K., Krumlauf, R., Wilkinson, D. G., Clarke, J. D. and Tickle, C. (1997). Hox9 genes and vertebrate limb specification. Nature 387, 97-101.[Medline]
Gaunt, S. T. and Strachan, L. (1994). Forward spreading in the establishment of a vertebrate Hox expression boundary: The expression domain separates into anterior and posterior zones, and the spread occurs across implanted glass barriers. Dev. Dyn. 199, 229-240.[Medline]
Gaunt, S. T. and Strachan, L. (1996). Temporal colinearity in expression of anterior Hox genes in developing chick embryos. Dev. Dyn. 207, 270-280.[Medline]
Gibson-Brown, J. J., Agulnik, S. I., Chapman, D. L., Alexiou, M., Garvey, N., Silver, L. M. and Papaioannou, V. E. (1996). Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity. Mech. Dev. 56, 93-101.[Medline]
Gibson-Brown, J. J., Agulnik, S. I., Silver, L. M., Niswander, L. and Papaioannou, V. E. (1998). Involvement of T-box genes Tbx2-Tbx5 in vertebrate limb specification and development. Development 125, 2499-2509.
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stage in the development of the chick embryo. J. Morphol. 88, 49-92.
Isaac, A., Rodriguez-Esteban, C., Ryan, A., Altabef, M., Tsukui, T., Patel, K., Tickle, C. and Izpisua-Belmonte, J. C. (1998). Tbx genes and limb identity in chick embryo development. Development 125, 1867-1875.
Kawakami, Y., Capdevila, J., Buscher, D., Itoh, T., Rodriguez-Esteban, C. and Izpisua-Belmonte, J. C. (2001). WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo. Cell 104, 891-900.[Medline]
Kimura, J. and Ide, H. (1998). Shh, Bmp-2 and Hoxd-13 gene expression in chick limb bud cells in culture. Dev. Growth Differ. 40, 457-464.[Medline]
Li, Q. Y., Newbury-Ecob, R. A., Terrett, J. A., Wilson, D. I., Curtis, A. R., Yi, C. H., Gebuhr, T., Bullen, P. J., Robson, S. C., Strachan, T., Bonnet, D., Lyonnet, S., Young, I. D., Raeburn, J. A., Buckler, A. J., Law, D. J. and Brook, J. D. (1997). Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. Nat. Genet. 15, 21-29.[Medline]
Logan, M., Simon, H. G. and Tabin, C. (1998). Differential regulation of T-box and homeobox transcription factors suggests roles in controlling chick limb-type identity. Development 125, 2825-2835.
Ohuchi, H., Takeuchi, J., Yoshioka, H., Ishimaru, Y., Ogura, K., Takahashi, N., Ogura, T. and Noji, S. (1998). Correlation of wing-leg identity in ectopic FGF-induced chimeric limbs with the differential expression of chick Tbx5 and Tbx4. Development 125, 51-60.
Rancourt, D. E., Tsuzuki, T. and Capecchi, M. R. (1995). Genetic interaction between hoxb-5 and hoxb-6 is revealed by nonallelic noncomplementation. Genes. Dev. 9, 108-122.[Abstract]
Rodriguez-Esteban, C., Tsukui, T., Yonei, S., Magallon, J., Tamura, K. and Izpisua-Belmonte, J. C. (1999). The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity. Nature 398, 814-818.[Medline]
Ruvinsky, I., Oates, A. C., Silver, L. M. and Ho, R. K. (2000). The evolution of paired appendages in vertebrates: T-box genes in the zebrafish. Dev. Genes. Evol. 210, 82-91.[Medline]
Saunders, J. W. J. (1948). The proximo-distal sequence of the origin of the parts of the chick wing and the role of the ectoderm. J. Exp. Zool. 108, 363-404.
Saunders, J. W., Jr, Cairns, J. M. and Gasseling, M. T. (1957). The role of the apical ectodermal ridge of ectoderm in the differentiation of the morphological structure and inductive specificity of limb parts in the chick. J. Morph. 101, 57-88.
Saunders, J. W., Jr, Gasseling, M. T. and Cairns, J. M. (1959). The differentiation of prospective thigh mesoderm grafted beneath the apical ectodermal ridge of the wing bud in the chick embryo. Dev. Biol. 1, 281-301.
Saunders, J. W., Jr and Gasseling, M. T. (1968). Ectodermal-mesenchymal interactions in the origin of limb symmetry. In Epithelial-Mesenchymal Interactions (ed. R. Fleishmajer and R. E. Billingham), pp. 78-97. Baltimore: Williams and Wilkins.
Saunders, J. W., Jr, Gasseling, M. T. and Errick, J. E. (1976). Inductive activity and enduring cellular constitution of a supernumerary apical ectodermal ridge grafted to the limb bud of the chick embryo. Dev. Biol. 50, 16-25.[Medline]
Sharma, K. and Izpisua-Belmonte, J. C. (2001). Development of limb neuromuscular system. Curr. Opin. Cell Biol. 13, 204-210.[Medline]
Simon, H. G., Kittappa, R., Khan, P. A., Tsilfidis, C., Liversage, R. A. and Oppenheimer, S. (1997). A novel family of T-box genes in urodele amphibian limb development and regeneration: candidate genes involved in vertebrate forelimb/hindlimb patterning. Development 124, 1355-1366.
Slack, J. M. W. (1991). From Egg to Embryo: Regional Specification in Early Development. New York: Cambridge University Press.
Stephens, T. D., Beier, R. L., Bringhurst, D. C., Hiatt, 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.[Medline]
Takabatake, Y., Takabatake, T. and Takeshima, K. (2000). Conserved and divergent expression of T-box genes Tbx2-Tbx5 in Xenopus. Mech. Dev. 91, 433-437.[Medline]
Takeuchi, J. K., Koshiba-Takeuchi, K., Matsumoto, K., Vogel-Hopker, A., Naitoh-Matsuo, M., Ogura, K., Takahashi, N., Yasuda, K. and Ogura, T. (1999). Tbx5 and Tbx4 genes determine the wing/leg identity of limb buds. Nature 398, 810-814.[Medline]
Tamura, K., Yonei-Tamura, S. and Izpisua-Belmonte, J. C. (1999). Differential expression of Tbx4 and Tbx5 in zebrafish fin buds. Mech. Dev. 87, 181-184.[Medline]
Weatherbee, S. D. and Carroll, S. B. (1999). Selector genes and limb identity in arthropods and vertebrates. Cell 97, 283-286.[Medline]
Wolpert, L., Beddington, R., Brockes, J., Jessell, T., Lawrence, P. and Meyerowitz, E. (1998). Principles of Development. Oxford University press.
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.[Medline]
Yoshida, N., Urase, K., Takahashi, J., Ishii, Y. and Yasugi, S. (1996). Mucus-associated antigen in epithelial cells of the chicken digestive tract: Developmental change in expression and implications for morphogenesis-function relationships. Dev. Growth Differ. 38, 185-192.