Different Regulation of T-Box Genes Tbx4 and Tbx5 During Limb

Developmental Biology 250, 383–392 (2002) doi:10.1006/dbio.2002.0801

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Paul Khan, Barbara Linkhart, and Hans-Georg Simon1 Developmental Systems Biology, Department of Pediatrics, Northwestern University, The Feinberg School of Medicine and Children’s Memorial Institute for Education and Research, Chicago, Illinois 60614

The T-domain transcription factors Tbx4 and Tbx5 have been implicated, by virtue of their limb-type speciﬁc expression, in controlling the identity of vertebrate legs and arms, respectively. To study the roles of these genes in developing and regenerating limbs, we cloned Tbx4 and Tbx5 cDNAs from the newt, and generated antisera that recognize Tbx4 or Tbx5 proteins. We show here that, in two urodele amphibians, newts and axolotls, the regulation of Tbx4 and Tbx5 differs from higher vertebrates. At the mRNA and protein level, both Tbx4 and Tbx5 are expressed in developing hindlimbs as well as in developing forelimbs. The coexpression of these genes argues that additional factors are involved in the control of limb type-speciﬁc patterns. In addition, newt and axolotl Tbx4 and Tbx5 expression is regulated differently during embryogenesis and regenerative morphogenesis. During regeneration, Tbx5 is exclusively upregulated in the forelimbs, whereas Tbx4 is exclusively upregulated in the hindlimbs. This indicates that, on a molecular level, different regulatory mechanisms control the shaping of identical limb structures and that regeneration is not simply a reiteration of developmental gene programs. © 2002 Elsevier Science (USA) Key Words: limb development; limb regeneration; pattern formation; Tbx4; Tbx5.

INTRODUCTION blastema resembles that of the embryonic limb bud, and regeneration is often viewed as a reiteration of embryogen- The vertebrate limb is a versatile model for studying the esis. However, the relationship between limb development cellular and molecular interactions that determine morpho- and limb regeneration is not yet understood. A fundamental logical pattern during development. Although shape and question is whether patterning mechanisms are identical in function can be very diverse, vertebrate limbs have a the developing and regenerating limb (Carlson et al., 2001). fundamentally similar design, consisting of a single proxi- Employing molecular techniques, signiﬁcant inroads mal element (stylopod), paired middle elements (zeugopod), have been made over the past years to elucidate underpin- and variable numbers of distal elements of the hand ning mechanisms in urodele limb regeneration. Many of and foot (autopod) (Johnson and Tabin, 1997). Unlike these studies, however, have been limited to adult newt other vertebrates, the urodele amphibians (newts and tissues (Savard et al., 1988; Ferretti et al., 1991; Simon and salamanders) can replace lost appendages through the for- Tabin, 1993; Savard and Tremblay, 1995; Brockes, 1997). mation of a regenerating blastema. The blastema cells are Little is known about embryonic and larval stages of newt derived by local dedifferentiation of adult mesenchymal development on the tissue, cellular, or molecular levels. tissue in the stump. They re-enter the cell cycle, proliferate, With the advent of breeding newts (Notophthalmus viride- and undergo differentiation and morphogenesis to com- scens) in the laboratory (Khan and Liversage, 1995b), we can pletely replace the lost structure (Brockes, 1997). Thus, now compare embryonic limb pattern formation and adult tissue regeneration holds the potential as an alternative regeneration in the same animal. option to embryonic stem cell use (Stocum, 1999; Pearson, By performing mRNA differential display screens of re- 2001). The growth and differentiation of the regenerating generating newt appendages, we previously identiﬁed Tbx5 to be exclusively expressed within forelimbs (Simon et al., 1 To whom correspondence should be addressed. Fax: (773) 880- 1997). Together with its family member Tbx4, which is 8266. E-mail: [email protected]. exclusively expressed in the hindlimbs, both genes have

FIG. 1. Alignment of Tbx4 and Tbx5. Peptide sequences of the respective available Tbx proteins of newt (Nv), chick (Gg), mouse (Mm), and human (Hs) were compiled. The conserved T-domain is boxed. Identical amino acids and conservatively substituted residues in greater than 60% of the compared sequences are in white print on black background or in white print on gray background, respectively. Amino acids on colored background are speciﬁc for Tbx4 (green) and Tbx5 (yellow). Residues in Xenopus T-domain protein Brachyury that are important for DNA binding (red ﬁlled circles) and dimerization (blue open circles) are indicated (Mu¨ ller and Herrmann, 1997). Gaps were introduced to optimize alignment and indicated by dashes. Sequences currently not available are indicated as a dotted line. GenBank Accession Nos. for all sequences are as follows: NvTbx4 (AF537187), NvTbx5 (U64433), GgTbx4 (AF069395), GgTbx5 (AF069396), MmTbx4 (U57329), MmTbx5 (AF140427), HsTBX5 (Y09445).

been shown to have key functions in controlling limb for a role in controlling limb pattern comes from TBX5 type-specific patterns (Gibson-Brown et al., 1998; Isaac et mutations in humans, which cause malformations of the al., 1998; Logan et al., 1998; Ohuchi et al., 1998). In upper limbs as well as defects of heart septation (Basson et developing chick and mouse limbs, Tbx4 and Tbx5 are al., 1997; Li et al., 1997). expressed early in the hindlimb or forelimb fields, respec- Recent misexpression studies have shown that Tbx4 and tively, and then throughout the respective limb mesen- Tbx5 play crucial roles in determining limb identity and the chyme during limb bud outgrowth. Limb-specific expres- regulation of limb outgrowth. However, the partial trans- sion of Tbx4 and Tbx5 is shared from humans to fish, determination of the limbs also argues that limb-type making these genes candidates that control the specific specification is more complex and additional factors are shaping of hindlimbs and forelimbs in virtually all verte- needed (Logan and Tabin, 1999; Rodriguez-Esteban et al., brates (Gibson-Brown et al., 1996; Basson et al., 1997; Li et 1999; Takeuchi et al., 1999). A correlation between limb al., 1997; Logan et al., 1998; Tamura et al., 1999; Begemann phenotype and respective Tbx message has been demon- and Ingham, 2000; Takabatake et al., 2000). Direct evidence strated, yet the actual distribution of the proteins and their

© 2002 Elsevier Science (USA). All rights reserved. T-Box Genes in Limb Development and Regeneration 385 activity remain unknown. To gain a more complete under- were cloned into the pET21b expression vector (Novagen). To test standing of Tbx4 and Tbx5 function, we have generated the speciﬁcity of the Tbx4 or Tbx5 antisera, Western blots with antibodies to detect Tbx4 and Tbx5 proteins in vitro and in Escherichia coli lysates of the recombinant Tbx proteins were vivo. Using both the developing and regenerating newt limb processed with the respective primary antibodies [T7-tag (Nova- as a model, we investigate here the timing and spatial gen), Tbx5ab1, or Tbx4ab1], secondary antibody (goat anti-rabbit conjugated to HRP; Jackson ImmunoResearch), and visualized by distribution of Tbx4 and Tbx5 mRNA and protein during enhanced chemiluminescence (Pierce). limb pattern formation.

RNase Protections MATERIALS AND METHODS RNA preparation and RNase protections were done as described (Simon and Tabin, 1993). To avoid cross-hybridization, a 237-bp Unless otherwise noted, all standard cloning techniques were Tbx4 probe covering part of the carboxy-terminal coding region performed according to Sambrook et al. (1989). All enzymes and without conserved T-box was used (Fig. 1, pos. 508–744). The molecular biology reagents were obtained from Roche Molecular signals were normalized with reference to the signals of the EF-1␣ Biochemicals, or as otherwise indicated. probe.

Animals and Treatment In Situ Hybridization Mature red-spotted newts (N. viridescens) were purchased from Whole-mount in situ hybridizations were carried out as reported M. Tolley (Donelson, TN), and axolotl (Ambystoma mexicanum) by Gardiner et al. (1995), and Simon et al. (1997). A newt Tbx4- larvae were obtained from the Indiana Axolotl Colony. Newts were specific (Fig. 1, pos. 744-1704) and a Tbx5-specific (Simon et al., spawned and the resulting progeny reared in our lab as described 1997) probe covering the carboxy-terminal protein domain and 3Ј (Khan and Liversage, 1995a,b). Newt and axolotl larvae were grown untranslated region of the cDNA were employed. to desired developmental stages (Gluecksohn, 1931), covering developing fore- and hindlimbs. Regenerating limbs of adult newts were staged (Iten and Bryant, 1973) and tissues harvested as Immunohistochemistry described (Simon and Tabin, 1993). Newt or axolotl larvae were fixed in Dents (80% MeOH, 20%

DMSO) and bleached in 10% H2O2, 90% MeOH. For whole-mount Isolation of Newt T-Box cDNA Clones, Sequence immunohistochemistry, rehydrated larvae were blocked in 1ϫ Analysis, and Alignment TBS, 5% DMSO, and 20% calf serum and then incubated with either Tbx4ab1 (1:200) or Tbx5ab1 (1:200) primary antibody in T-domain-containing PCR fragments (Simon et al., 1997) were blocking solution. After washing, secondary antibodies (goat anti- used as probes to screen newt forelimb or hindlimb midbud-stage rabbit conjugated to biotin) were applied, and embryos were then blastema cDNA libraries (Simon and Tabin, 1993). Tbx5- and reacted with an avidin-biotin complex (Vector Labs). Color reaction Tbx4-speciﬁc clones were isolated, and full-length cDNAs were was with a Diamino-benizidene (DAB) peroxidase reaction accord- Ј Ј obtained by 5 or 3 RACE (Marathon Kit; Clontech) and veriﬁed by ing to the manufacturer (Roche Molecular Biochemicals). Speci- sequencing. Overlapping cDNA sequences were assembled into mens were embedded in gelatin and cryosectioned. continuous contigs. Peptide alignments were created by using MacDNASIS (Hitachi), SEQPUP (D. Gilbert), and MacBOXSHADE (M.D. Baron) software. RESULTS

Peptide Design and Generation of Tbx4- and Tbx5- Evolutionarily Conserved Tbx4 and Tbx5 Specific Antibodies Sequences In order to identify evolutionarily conserved amino acids that are Using mRNA differential display with newt regenerating specific for either Tbx4 or Tbx5, deduced peptide sequences of the fore- and hindlimbs, we previously identified cDNAs ho- respective orthologous genes of newt, chick, mouse, and human mologous to Tbx5 exclusively expressed in regenerating were compiled (Fig. 1). Peptide motifs in the carboxy-terminal forelimbs (Simon et al., 1997). Employing 5Ј RACE, we domains were tested for their likelihood to be exposed on the extended the partial Tbx5 sequence (NvTbox1) beyond the surface of the proteins, and Tbx4- and Tbx5-specific peptides (Fig. T-domain to its ATG initiation codon, yielding a composite 1, peptide A for Tbx4: RERVPPSSFPRERVHPSLCERK; and peptide B for Tbx5: TKRKDEECSTTEHPYKKPYM) were used for immuni- Tbx5 cDNA of 2219 bp, which codes for the complete zations of rabbits and the production of polyclonal antisera (Re- 518-amino-acid protein (Fig. 1). From a pool of other newt search Genetics, Inc). T-box genes (Simon et al., 1997, and this report), we isolated 874 bp of a cDNA with homology to mouse and chick Tbx4. In order to produce Tbx4 gene-specific probes, we extended Expression of Recombinant Tbx Proteins and the cDNA by RACE beyond its coding domain into the 3Ј Western Blots untranslated region. The assembled composite newt Tbx4 For the production of recombinant Tbx proteins, the C-terminal cDNA of 2054 bp comprises a partial Tbx4 peptide 423 domains of chick Tbx2, Tbx3, Tbx4, and Tbx5 (Logan et al., 1998) amino acids long (Fig. 1).

reactivity was demonstrated by immunohistochemistry (data not shown).

Tissue Distribution and Expression Patterns of Tbx4 during Limb Regeneration We have demonstrated forelimb-specific expression of Tbx5 during newt limb regeneration (Simon et al., 1997). Using RNase protection, we analyzed here the expression pattern of Tbx4 in normal and regenerating appendages as well as in different organs, and found mRNA expression exclusively in the hindlimb territory. As shown previously for Tbx5 in forelimbs, we detected a protected Tbx4 band in normal hindlimb RNA, indicating basal activity (Fig. 3B). After amputation at a proximal level (mid femur), we observed a strong induction of Tbx4 mRNA in midbud stage regenerating blastemas. In early digit-stage regener- ates, Tbx4 expression was lower, and in fully regenerated hindlimbs, the expression was further reduced to the level detected in normal, unamputated hindlimbs. Thus, Tbx4 expression was highest in the blastema, when limb patterning is reprogrammed. Because limb pattern is specified by the mesenchymal “blastema” cells (Stocum and Dearlove, 1972), it was important to ascertain whether Tbx4 is expressed by these cells. Hindlimb midbud blastemas were dissected into epithelial and mesenchymal fractions, and FIG. 2. Specificity of Tbx protein antisera. Western blots with E. the Tbx4 message was found exclusively in the mesen- coli protein lysates were processed with antibodies against: T7-tag, chyme (Fig. 3C). We did not detect Tbx4 mRNA in fore- Tbx5, and Tbx4. Lanes: (1) T7-tag/Tbx5, uninduced; (2) T7-tag/ limbs, forelimb blastemas, tail, or tail blastemas, nor in a Tbx5 induced; (3) T7-tag/Tbx4, induced; (4) T7-tag/Tbx3, induced; variety of organs and tissues (Fig. 3D, and data not shown). (5) T7-tag/Tbx2 induced. Molecular weight standards in kDa (New England Biolabs) are indicated on the left. Thus, Tbx4 expression in adult tissues is limited to normal and regenerating hindlimbs.

Tbx4 and Tbx5 mRNA and Protein Expression in There is substantial identity between T-box family mem- Limb Development bers across the DNA-binding domain (T-domain); however, When newt larvae became available for experimentation, no extended homologies have been observed outside this we extended our studies and compared Tbx gene regulation region (Agulnik et al., 1996; Papaioannou and Silver, 1998). in regenerating and developing limbs. To our surprise, in A comparison of the T-domains among orthologous Tbx whole-mount mRNA in situ hybridizations, we detected proteins in newt, chick, mouse, and human revealed dis- both Tbx5 and Tbx4 mRNA in developing forelimbs and tinct amino acids unique to each Tbx protein within the hindlimbs (see Figs. 4 and 5). This result contrasted the Tbx2/3/4/5 subfamily (Simon, 1999). When this compari- mouse and chick data, but also those obtained with newt son is extended downstream to the potential Tbx4 or Tbx5 regenerating limbs. transactivation domain, we identified stretches of identities In embryogenesis, Tbx5 mRNA was first detected when separated by divergent sequences. Importantly, amino acid the forelimbs bud out from flank mesoderm (Figs. 4A and motifs characteristic for either Tbx4 or Tbx5 could be 4B). During limb bud formation and subsequent growth, recognized as a common feature of the two subfamily Tbx5 mRNA is uniformly distributed throughout the limb members (shown in Fig. 1 with Tbx4 highlighted in yellow; mesenchyme (Figs. 4C–4E). Tbx5 forelimb expression in Tbx5 highlighted in green). We selected two amino acid the mesenchyme persisted through palette and digit forma- motifs that are evolutionarily conserved and specific for tion. In late digit development, Tbx5 mRNA is downregu- Tbx4 (peptide A) or Tbx5 (peptide B), and used those to lated from distal to proximal (Figs. 4E and 4F). In contrast to generate specific antisera. In in vitro (Fig. 2) and in vivo (see most vertebrates, newt forelimbs and hindlimbs develop Figs. 4 and 5) experiments, both antibodies proved highly sequentially; only after the forelimbs are completely specific for their respective proteins. In addition, when the formed do the hindlimbs start developing (Fig. 4G). At Tbx4 and Tbx5 antibodies were titrated with their respec- closer inspection, a clearly visible Tbx5 signal was also tive antigen, specific inhibition of Tbx4 or Tbx5 protein detectable in the emerging hindlimb buds (Fig. 4H). Tbx5

FIG. 3. Expression of Tbx4 in normal and regenerating tissues. RNase protection analysis with RNA of the respective tissue samples employing probes speciﬁc for newt Tbx4 and EF-1␣, respectively, to normalize expression levels. (A) Schematic diagram representing tissue (shaded) used for RNA preparation. (B) Expression in normal and regenerating hindlimbs. Lanes: (1) input probe; (2) yeast tRNA; (3) normal hindlimb (NHL); (4) proximal blastema hindlimb (BHL(P)); (5) proximal digit-stage hindlimb (DHL(P)); (6) proximal regenerated hindlimb (RHL(P)). (C) Epidermal/mesenchymal expression. (7) proximal blastema hindlimb - epidermis (BHL(P)Epi); (8) proximal blastema hindlimb - mesenchyme (BHL(P)Mes). (D) Expression in normal and regenerating forelimbs. (9) normal forelimb (NFL); (10) proximal blastema forelimb (BFL(P)). The size of the input probes and the respective protected fragments are indicated. No Tbx4 mRNA was detected in the forelimb territory, nor in any other tissue or organ tested (data not shown).

expression became evenly distributed throughout the hindlimb buds are discernible (Fig. 5F). During hindlimb bud limb mesenchyme as the limb elongated (Fig. 4I). However, formation and subsequent growth, Tbx4 mRNA was evenly Tbx5 mRNA in the developing hindlimbs appears down- distributed throughout the mesenchyme (Figs. 5G–5I). regulated earlier than in forelimbs and was undetectable When digits have formed, Tbx4 message is gradually local- beyond the three-digit stages (Figs. 4J and 4K). ized to the more posterior and proximal portion of the Surprisingly, Tbx4 mRNA distribution was very similar growing footplate. Tbx4 mRNA decreased ﬁrst in the more to Tbx5 in developing limbs (Fig. 5). Early in forelimb differentiated anterior digits I and II (Fig. 5J). At this stage, development, the Tbx4 message was expressed in limb bud the actively growing digits III and IV strongly expressed mesenchyme (Figs. 5A and 5B). However, Tbx4 mRNA is Tbx4 mRNA. Soon afterwards, the expression domain downregulated rapidly after the forelimb digits form (Figs. moved posterior and proximal but still included digits IV 5C–5E). In the hindlimb territory, Tbx4 mRNA accumu- and V (Fig. 5K). Thus, it appears that during digit formation, lated in the lateral plate mesoderm well before the hind- Tbx4 expression decreases in a wave from anterior to

FIG. 4. Tbx5 mRNA and protein expression in developing newt limbs. In all images, anterior is toward the right. Scale bars, approximately 0.6 mm. Digits are indicated by roman numerals. Tbx5 mRNA whole-mount in situ hybridization (purple color), and Tbx5 protein immunohistochemistry (insets, brown color) in successive stages of development of larval newts. (A) Dorsal view of a larva just prior to the visualization of forelimb buds (fb). (B) Dorsal view of a larva with established forelimb buds (fb). (C) Lateral view of a rod-shaped forelimb bud (fb), external gills (g). (D) Lateral view of a digit-forming forelimb bud. (E) Ventral–lateral view of a developing three digit forelimb. (F) Dorsal–lateral view of a four-digit larval forelimb, proximal stylopodial region (x). (G) Ventral–lateral view of a larva possessing four-digit forelimbs (ﬂ) and hindlimb buds (hb), external gills (g). (H) Lateral view of an emerging hindlimb bud (hb). (I) Lateral view of a rod-shaped hindlimb bud (hb). (J) Lateral view of a digit-forming hindlimb bud. (K) Dorsal view of a larval hindlimb possessing three digits and an emerging fourth digit. (L) Transverse section through a hindlimb bud probed for Tbx5 protein. Note brownish pigmentation at back and side of the animals.

posterior, correlating with the differentiation of individual cant reduction in protein within differentiated digits (Fig. digits. 5J, inset, and data not shown). For both limbs, Tbx4 and These unexpected expression patterns could have been Tbx5 expression was exclusively mesenchymal, but not explained by an anomaly on the message level, since epidermal (Figs. 4L and 5L, and data not shown). mRNA is not necessarily translated into protein. To address The spatiotemporal expression of Tbx5 and Tbx4 proteins this possibility, we produced Tbx4- and Tbx5-specific anti- during forelimb and hindlimb development revealed a pro- sera (Figs. 1 and 2) and performed immunohistochemistry tein distribution consistent with our mRNA findings; Tbx4 on a range of developing newt limbs. Consistent with and Tbx5 were transcribed into mRNA and translated into mRNA, we found that Tbx5 protein is expressed by devel- protein in both developing forelimbs and hindlimbs. In oping forelimbs (Figs. 4C and 4D, inset). Moreover, and in addition, our studies demonstrated that the pattern of agreement with the mRNA data, Tbx5 protein was also protein expression correlated with the distribution of expressed in the developing hindlimbs, from early to late mRNA. The concomitant expression of message and pro- stages (Figs. 4I and 4J, inset). Comparable to Tbx5 mRNA, tein indicates a regulation of these Tbx genes on the protein expression becomes reduced from anterior to poste- transcriptional level. rior during digit formation, before it rapidly decreased after the completion of digit formation (data not shown). Re- Tbx4 and Tbx5 Coexpression in Forelimbs and markably, Tbx4 protein is also expressed in both developing Hindlimbs during Urodele Embryogenesis forelimbs and hindlimbs (Figs. 5B, 5C, 5I and 5J, inset). Comparable to Tbx4 mRNA expression, we detected Tbx4 To exclude the possibility that the different regulation of protein in proliferating cells in growing digits and a signifi- Tbx4 and Tbx5 was a newt-specific finding, we turned to

FIG. 5. Tbx4 mRNA and protein expression in developing newt limbs. In all images, anterior is toward the right. Scale bars, approximately 0.6 mm. Digits are indicated by roman numerals. Tbx4 mRNA whole-mount in situ hybridization (purple color), and Tbx4 protein immunohistochemistry (insets, brown color) in successive stages of development of larval newts. (A) Lateral view of a larva with evident forelimb buds (fb). (B) Lateral view of a rod-shaped forelimb bud (fb). (C) Lateral view of a digit-forming forelimb bud. (D) Dorsal–lateral view of a four digit forelimb, zeugopod (z). (E) Lateral view of a larva with a four digit forelimb (ﬂ) and an early stage hindlimb bud (hb). Pharyngeal–lower mandibular region (x). (F) Higher magniﬁcation of early-stage hindlimb bud (hb). (G) Lateral view of an elongating hindlimb bud (hb). (H) Lateral view of a rod-shaped hindlimb bud (hb). (I) Lateral view of an early-stage-digit forming hindlimb bud (hb). (J) Lateral view of a more advanced, three digit hindlimb. (K) Lateral view of a four-digit larval hindlimb. (L) Transverse section through a hindlimb bud probed for Tbx4 protein. Note brownish pigmentation at back and side of the animals.

the axolotl. These urodeles are also used as a model in limb however, they do form morphologically different forelimbs development and limb regeneration. In performing immu- and hindlimbs. In addition, while Tbx4 and Tbx5 are nohistochemistry on axolotl larvae, the antibodies demon- coexpressed in the limbs during development, they display strated reactivity across species in detecting orthologous a different expression during regenerative pattern forma- Tbx4 or Tbx5 proteins. During axolotl embryogenesis, we tion: Tbx4 is exclusively reactivated in hindlimb blastemas, detected Tbx4 and Tbx5 protein expression in both devel- and Tbx5 is exclusively reactivated in forelimb blastemas. oping forelimbs and hindlimbs from early bud to late digit Regeneration of the lost limb portion rebuilds the ap- stages (Figs. 6A–6F, and data not shown), consistent with pendage both structurally and functionally (Wallace, 1981; and confirming the newt data. These results demonstrate Brockes, 1997). The basic paradigm of epimorphic regenera- that coexpression of Tbx4 and Tbx5 in developing fore- and tion in the adult newt implies that, following the formation hindlimbs is a common feature in urodele amphibians. of the blastema, the amputated limb retraces its embryonic development. In support of this notion, a number of devel- opmentally important homeobox genes are known to play DISCUSSION similar roles in development and regeneration. This suggests that the mechanisms controlling limb outgrowth and The limb type-specific expression of Tbx4 or Tbx5 is patterning are both utilized during development and regen- thought to be critical for controlling the identity of verte- eration (Simon and Tabin, 1993; Gardiner et al., 1995; brate legs and arms. Our data with newts and axolotls Savard and Tremblay, 1995; Simon et al., 1995). Impor- demonstrate that Tbx4 and Tbx5 are distinctly regulated in tantly, and unlike other vertebrates, genes with a restricted different vertebrates. These two amphibians coexpress limb type-specific expression, including Tbx4 and Tbx5, are Tbx4 and Tbx5 in both limb types during embryogenesis; constitutively expressed at low levels in the respective

ably the same cells. It is possible that a combination of two or more gene activities determines limb identity. In the hindlimbs, the homeobox transcription factor Pitx1 has been demonstrated to act upstream of Tbx4, and its hindlimb-specific expression is critical for hindlimb patterning (Logan et al., 1998; Lanctot et al., 1999; Logan and Tabin, 1999; Szeto et al., 1999). Different early genes expressed in presumptive limb regions have been described, but their roles in limb induction and/or specificity remain elusive (Isaac et al., 2000). In addition, Wnt-2b and Wnt-8c signaling molecules are involved in chick limb bud initiation in a forelimb/hindlimb-specific manner (Kawakami et al., 2001). However, it is not known whether Wnt-2b/Tbx5 in the forelimbs and Wnt-8c/Tbx4 in the hindlimbs work together in a pathway. Other important factors that either operate in a regulatory hierarchy or act as regulators by binding to Tbx proteins may not yet have been identified. In larval salamanders, such as newts and axolotls, developing digits form with individual buds (Wake and Shubin, 1994). The successive budding of digits one to five is accompanied by a dynamic high-level expression of Tbx4 and Tbx5 within the proliferating cells of the growing digits. However, their expression becomes downregulated FIG. 6. Tbx4 protein expression in axolotl limb development. In in an anterior to posterior sequential manner and correlates all images, anterior is toward the right. Scale bars, approximately to the differentiation stage of the individual digit being 0.6 mm. (A–F) Tbx4 protein immunohistochemistry with different formed. This successive development of individual digits is stages of axolotl larvae. (A) Overview of developing fore- and in contrast to that of amniotes, where all digits form hindlimbs. (B) Control experiment with comparable stage embryo simultaneously from a paddle-like structure as a result of as in (A) processed without primary antibody. (C) Detail of an early interdigital apoptosis, and gradual distal to proximal down- forelimb bud (fb) and (D) of a three-digit forming forelimb (fl). (E) regulation of Tbx4 or Tbx5, respectively (Gibson-Brown et Detail of an early hindlimb bud (hb) and (F) of a three-digit larval al., 1996; Logan et al., 1998). Developing limbs as well as hindlimb (hl). regenerating limbs are characterized by high uniform levels of Tbx4 and/or Tbx5 message in their proliferating mesenchymal cells. The correlation of growth and high levels of Tbx expression is particularly striking during the shaping of adult newt limbs (Simon et al., 1997). This may suggest that digits in urodele limbs, and would suggest a role for Tbx4 residual gene activity is needed to define limb identity in and Tbx5 in the regulation of cell proliferation. A feedback adulthood and is therefore critical for regeneration to occur. loop of Tbx and FGF expression has been demonstrated in During development of mouse and chick (Gibson-Brown Xenopus for the T-box family member brachyury (Schulte- et al., 1996, 1998; Isaac et al., 1998; Logan et al., 1998), Merker and Smith, 1995). Similarly, Rodriguez-Esteban et anuran amphibians such as Xenopus (Takabatake et al., al. (1999) have argued that Tbx4 and Tbx5 are linked to the 2000), and zebrafish (Tamura et al., 1999; Begemann and activity of signaling proteins such as FGF, BMP, and Wnt. Ingham, 2000), Tbx5 is exclusively expressed in the While Tbx4 and Tbx5 seem to regulate outgrowth in the forelimb/wing/pectoral fin, whereas Tbx4 is exclusively limb, it has been recently reported that, in the developing expressed in the hindlimb/leg/pelvic fin. Recent work in heart, Tbx5 is inversely related to cellular proliferation chick limb development has identified a reciprocal negative (Hatcher et al., 2001). These opposing observations could be feedback loop where Tbx5 in the wing field represses Tbx4, reconciled by proposing that Tbx4 and Tbx5 have different and Tbx4 in the leg field represses Tbx5 (Saito et al., 2002). regulatory activities by associating with diverse proteins. Despite very low Tbx5 background levels in chick leg buds, The distinct and conserved amino acid motifs within either the repression appears very tight, arguing that this inhibi- Tbx4 or Tbx5 are likely to have functional significance, and tory regulation insures limb type-specific Tbx expression it is conceivable that such amino acid residues serve as and is hence important for the determination of limb binding sites for accessory proteins. Evidence for this hy- identity. Such an inhibitory regulation pathway between pothesis comes from the finding that Tbx4 and Tbx5 Tbx4 and Tbx5 is apparently not active in developing proteins can bind to different cellular proteins in the heart urodele limbs. It is not clear how newts and axolotls (Bruneau et al., 2001; Hiroi et al., 2001) and in the limbs manage to develop discrete forelimbs and hindlimbs with (H-G.S. et al., manuscript in preparation). It is therefore Tbx4 and Tbx5 expressed at comparable levels in presum- possible that the specific interaction with diverse accessory

© 2002 Elsevier Science (USA). All rights reserved. T-Box Genes in Limb Development and Regeneration 391 proteins in different tissues provides Tbx transcription and the specification of forelimb/hindlimb identity. Mech. Dev. factors with multiple regulatory activities, such as promo- 56, 93–101. tion or repression of proliferation, regulation of differentia- Gibson-Brown, J. J., Agulnik, S. I., Silver, L. M., Niswander, L., and tion, and formation of pattern. Papaioannou, V. E. (1998). Involvement of T-box genes Tbx2– Growth and differentiation of the regeneration blastema Tbx5 in vertebrate limb specification and development. Devel- opment 125, 2499–2509. resemble that of the embryonic bud; however, here we Gluecksohn, S. (1931). Aeussere Entwicklung der Extremitaeten present clear molecular evidence that the patterning und Stadieneinteilung der Larvenperiode von Triton taeniatus mechanisms in these two structures are controlled by Leyd. und Triton cristatus Laur. Roux’s Arch. Dev. Biol. 125, different gene activities. Our data suggest that additional 341–406. cofactors are required, which may interact with Tbx4 and Hatcher, C. J., Kim, M. S., Mah, C. S., Goldstein, M. M., Wong, B., Tbx5 proteins to regulate downstream genes in a limb Mikawa, T., and Basson, C. T. (2001). TBX5 transcription factor type-specific fashion. Future experiments that elucidate regulates cell proliferation during cardiogenesis. Dev. Biol. 230, such accessory factors will shed light on the molecular 177–188. network of proteins that ultimately controls the distinct Hiroi, Y., Kudoh, S., Monzen, K., Ikeda, Y., Yazaki, Y., Nagai, R., morphological patterns of an arm or a leg. and Komuro, I. (2001). Tbx5 associates with Nkx2–5 and syner- gistically promotes cardiomyocyte differentiation. Nat. Genet. 28, 276–280. ACKNOWLEDGMENTS Isaac, A., Cohn, M. J., Ashby, P., Ataliotis, P., Spicer, D. B., Cooke, J., and Tickle, C. (2000). FGF and genes encoding transcription We thank R. 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