T-genes and limb bud development

� 2006 Wiley-Liss, Inc. American Journal of Medical Genetics Part A 140A:1407–1413 (2006)

Research Review

T-Genes and Limb Bud Development

Mary King,1 Jelena S. Arnold,2 Alan Shanske,3 and Bernice E. Morrow2,3*1Department of Obstetrics and Gynecology and Women’s Health, Albert Einstein College of Medicine, Bronx, New York

2Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York3Center for Craniofacial Disorders, Children’s Hospital at Montefiore, Albert Einstein College of Medicine, Bronx, New York

Received 27 January 2006; Accepted 9 March 2006

The T-box family of transcriptional factors is ancient andhighly conserved among most species of animals. Haploin-sufficiency of multiple T-box proteins results in severehuman congenital malformation syndromes, involving cra-niofacial, cardiovascular, and skeletal structures. Thesegenes have major roles in embryogenesis, including thedevelopment of the limbs. Formation of the limbs beginswith a limb bud and its morphogenesis requires complexepithelial–mesenchymal interactions. Recent studies haveshown that T, Tbx2, Tbx3, Tbx4, Tbx5, Tbx15, and Tbx18 are

all expressed in the limb buds, and many have develop-mental functions. The study of these genes is clinicallyrelevant as mutations in several of them cause humancongenital malformation syndromes. Furthermore, under-standing the function and biology of these genes is importantin understanding normal embryogenesis.� 2006 Wiley-Liss, Inc.

Key words: T-box genes; SHFM; limb bud development

How to cite this article: King M, Arnold JS, Shanske A, Morrow BE. 2006. T-genes and limb bud development.Am J Med Genet Part A 140A:1407–1413.

INTRODUCTION

The T-box family of transcriptional regulatoryfactors are required for embryonic development andwhen mutated are associated with human congenitalmalformation syndromes. Due to the association withhuman diseases, there has been much interest inunderstanding their molecular roles in development.Inactivationmutations inan important subset ofT-boxgenes are associated with limb defects, the subject ofthis review. The limb starts off as a set of lateral bulgesin the early embryo, referred to as limb buds.Recent research has focused upon understandingtheunique andcombinatorial roles of theT-boxgenesexpressed in the limb buds. These genes include T,Tbx2, Tbx3, Tbx4, Tbx5, Tbx15, and Tbx18. Theirgenetic roles have been delineated by gene targetingin the mouse andbyectopic overexpression studies inthe chick. The limb is particularly vulnerable toenvironmental and genetic factors leading to defects,and understanding the role of the T-box genes indevelopment will provide insight into the pathogen-esis of malformations in human disorders.

T-GENES AND HUMAN DISEASESWITH LIMB MALFORMATIONS

T-box genes were first described in 1927 with thediscovery of a heterozygous mutant, Brachyury or T

(for short tail), which caused truncated tails inmice [Dobrovoiskaia-Zavadskaia, 1927]. Homozy-gous mutations in Brachyury resulted in earlyembryonic lethality due to a complete lack of axialdevelopment [Wilkinson et al., 1990; Kispert andHerrmann, 1993]. Subsequent studies identified awhole family of related genes, sharing homologywith part of the protein, termed the T-box. Many ofthe T-box genes are associated with human geneticdisorders. They are frequently inherited in anautosomal dominant manner due to haploinsuffi-ciency. Haploinsufficiency of TBX1 is associatedwith the congenital malformation disorder calledvelo-cardiofacial syndrome (DiGeorge syndrome,22q11.2 deletion syndrome; MIM 188400) [Jeromeand Papaioannou, 2001; Lindsay et al., 2001;Merscher et al., 2001]. Haploinsufficiency of tworelated family members, TBX3 and TBX5, causesthe ulnar-mammary syndrome (MIM 181450) andHolt-Oram syndromes (MIM 142900), respectively,

*Correspondence to: Bernice E. Morrow, Department of MolecularGenetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue,Bronx, NY 10461. E-mail: [email protected]

DOI 10.1002/ajmg.a.31250

both associated with forelimb defects [Bamshadet al., 1997; Basson et al., 1997; Li et al., 1997].Hemizygous mutations in TBX4 cause small patellasyndrome (MIM 147891), an autosomal dominantskeletal dysplasia with defects in the skeletonincluding the feet [Bongers et al., 2004]. Thus, threeT-box genes, TBX3, TBX4, and TBX5, are associatedwith limb defects. Mutations in TBX22 have beenlinked to X-linked cleft palate and ankyloglossia(MIM 303400) [Braybrook et al., 2001, 2002]. T-boxgenes, therefore, represent a large family of tran-scription factors, essential for normal embryonicdevelopment and in many cases associated with limbmalformations.

T-box Genes in Transcriptional Regulation

T-box proteins regulate embryogenesis by activat-ing or repressing different target genes via theirpromoter or enhancer regions. The T-box is theregion conserved among T-gene family members,and it functions as the DNA binding domain. TheDNA binding properties were delineated first for theoriginal T-gene that was described, Brachyury or T[Kispert and Herrmann, 1993]. T binds a palindromicDNA binding site, termed the T-site, as a dimer,with each monomer binding to half of the sequence,or to theT-half site (50-AGGTGTGAAATT-30). Furtherstudies have demonstrated that all T-box proteinscan bind T-half sites as monomers. How canspecificity be determined? Binding specificity canbe achieved because there are variations in thepalindromic sequences. Different T-box genes haveslightly different affinities for the same T-site[reviewed in Naiche et al., 2005]. In vitro, it has beendemonstrated that different T-box proteins showpreference for different synthetic combinations of T-half sites, with varying orientations, numbers, andspacing, possibly to confer target specificity in vivo[Conlon et al., 2001].

T-box genes can act as transcriptional activators,thereby activating downstream genes or repressors,inhibiting transcription of target genes. Some haveboth activating and repressing functions, dependingon the particular transcriptional target. Specificprotein domains mediate transcriptional regulation.The activation domains of several T-box proteins,such asTbx5 andTbx1, havebeenmapped to their C-terminal domains [Plageman and Yutzey, 2004;Stoller and Epstein, 2005]. However, T-box proteinscan also repress transcription, as has been shown forTbx3 and Tbx2 [Sinha et al., 2000; Butz et al., 2004],which have been reported to act both as activatorsand/or repressors, depending on promoter context[reviewed in Naiche et al., 2005]. To understand thebasis for specificity in binding and in transcription,the crystal structures of a subset of T-box genes havebeen determined. The crystal structures of homo-dimers of bothT andTBX3, T-boxdomains, bound to

the canonical T-site have been solved [Muller andHerrmann, 1997; Coll et al., 2002]. Both proteinsmake contacts with the DNA via the same aminoacids, indicating strong conservation of DNA bindingfunctions. However, differences in ternary structuresof the two protein–DNA complexes have also beennoted, underlying the distinction in T-half sitepreferences among different T-box proteins, therebyproviding specificity.

Most T-box proteins require a co-factor to activate/repress target genes. Synergistic activation of targetgenes has been observed with T-box proteins andmembers of other major developmental proteins, thehomeodomain, GATA zinc finger, and LIM domainprotein family members [reviewed in Naiche et al.,2005]. The interactions between T-box proteins andtheir co-factors are thought to contribute to promoterspecificity as well. Thus, not only is the affinity ofbinding to the T-half sites important, but interactionwith additional transcription factors also is requiredfor conferring specificity to this major class oftranscription factors. Of interest, some of the T-boxproteins will interact with select members of oneparticular protein family. For example, Tbx20 hasbeen shown to interact with GATA5 but less so withGATA4 [Stennard et al., 2003]. In some cases, T-boxgenes may interact with different domains of thesame protein, allowing for different types of signal-ing. For example, Tbx4 and Tbx5 interact withdifferent LIM domain repeats of the same protein[Garg et al., 2003; Plageman and Yutzey, 2005].Several T-box genes are required for heart develop-ment and some have been shown to interact withthe mammalian homolog of the Drosophila home-odomain containing transcription factor, Tinman,referred to as Nkx2.5 [Plageman and Yutzey, 2004].The ability of T-box proteins to regulate transcriptionvia heterodimerization has biological relevance.Holt-Oram syndrome patients with point mutationsin TBX5 abolish its ability to bind to NKX2.5, a co-factor required for transcriptional activation [Garget al., 2003]. Thus, most T-box genes functionbiologically via protein–protein interactions andhuman disorders result when these interactions aredisrupted.

Evolution of T-box Gene Family MembersExpressed in the Limb Buds

T-box genes are highly conserved among speciesand are required for generating the metazoanembryonic body plan. This family of genes predatesthe divergence of arthropods and chordates, arisingapproximately 600 million years ago. T-box familymembers have been identified in most animalsstudied to date. The discovery of multiple membersof the T-gene family occurred after the original ‘‘T’’gene was identified and when it was realized thatboth a Drosophila optomoter-blind (DmOmb) gene

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American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

and theTgene shared a conserved region, termed theT-box, which is a novel DNA binding domain[Pflugfelder et al., 1992;Kispert andHerrmann, 1993].

T-box genes evolved as a result of gene duplicationand subsequent cluster dispersion. In most organ-isms, they are dispersed throughout the genome.The sharing of this common tool kit by suchorganisms as diverse as mammals and Drosophilapresents an obvious paradox. How does the use ofthese common tools coincidewith the apparent greatmorphologic diversity between phyla? The solutionto this paradox lies within changes in the timing andlocation of expression of the members of this genefamily, as well as changes in protein structure,dictated by alteration in the DNA coding sequence.The alterations in coding sequence occurred primar-ily outside the T-box. Sequence variations upstreamor downstream of the exons, also had significantbiological effects because some of these comprisetranscriptional regulatory elements. Genes that sharesimilar regulatory elements show regions of co-expression in embryonic tissues. Changes in tran-scriptional regulatory elements over time resulted indifferential gene expression, particularly relevantwith respect to limb development.

Regarding limb development, there are seven T-box genes of interest that are highly expressed in thelimb buds T, Tbx2, Tbx3, Tbx4, Tbx5, Tbx15, andTbx18. In vertebrates, the loci for Tbx2 and Tbx4 arelinked on chromosome 11 in the mouse, chromo-some 17 in the human, and those of Tbx3 and Tbx5on chromosome 5 in the mouse (human chromo-some 12) (Fig. 1). Tbx2 and Tbx3 compose onecognate pair and Tbx4 and Tbx5 another, evolvingfrom tandem duplication and cluster dispersion[Agulnik et al., 1996; Wattler et al., 1998]. Two otherT-box genes, Tbx15 and Tbx18 are also highlyrelated in sequence (Fig. 1) and share overlappingexpression patterns. The original T gene is on adifferent branch on the phylogenic tree than the sub-branch shown in Figure 1. Thus, the T-genesrequired for limb development comprise tandem

duplicated genes that have evolved to serve uniqueand redundant functions.

FORMATION OF THE LIMB BUDS

In order to understand the role of T-box genes inlimb development, it is important to describe thesteps in forming the limb bud. Following gastrula-tion, the mesoderm is divided spatially into threesegments, paraxial, intermediate, and lateral plate,based upon its proximity to the closing neural tube.The vertebrate limb bud develops from the lateralplate mesoderm. Its early formation is a complexprocess requiring organization of the growing limbbud along three axes: proximal-distal (PD), anterior-posterior (AP), and dorsal-ventral (DV). The devel-opment of many organs and structures in the earlyembryo depends on tissue interactions between theepithelia and the underlying mesoderm, looselyreferred to as mesenchyme. Different epithelial-mesenchymal signaling mechanisms are thought tocontrol these axes of development [reviewed byMartin, 2001]. The initial events in limb developmentinvolve proliferation of the lateral plate mesodermand induction of the apical ectodermal ridge (AER).The diffusible growth factor, fibroblast growthfactor ligand, Fgf10 is expressed in the lateral platemesoderm, which then induces the expression ofanother Fgf family member, Fgf8 in the ectoderm.Fgf8 induces continued high levels of expressionof Fgf10 in the mesoderm and allows continuedcell proliferation and expansion of the limb bud.Inactivation of Fgf10 results in a complete loss oflimbs [Min et al., 1998; Sekine et al., 1999]. The limbbud contains an area of rapidly proliferating cells,which form the progress zone, and these cells thendifferentiate to form limb structures. The AER directsPD outgrowth of the limb. In the posterior area ofthe limb bud, the zone of polarizing activity (ZPA)forms in response to signals from the AER. TheZPA expresses another diffusible morphogen, Sonichedgehog (Shh), and this signal is the principalorganizer for the AP axis of the limb. Signaling from athird family of morphogens, the bone morphogenicproteins (BMPs), belonging to the transforminggrowth factor superfamily, is thought to regulatethese processes, both by stimulating AER inductionand by inducing expression of dorsal and ventralectodermal genes, which establish DV polarity of thelimb. BMP signaling causes expression of othergenes required for patterning the limb, such as En1(Engrailed 1) in the ventral ectoderm and Wnt7a inthe dorsal ectoderm which then signal to the under-lying mesenchyme [Liu et al., 2003].

Role of T in the Function of the AER

Much has been learned about early limb develop-ment from vertebrate animal models by expression

FIG. 1. Partial phylogenetic tree of T-box gene family members [adaptedfrom Naiche et al., 2005]. Tbx2, 3, 4, 5, 15, and 18 are expressed in the limbbuds. Related family members, Tbx15 and Tbx18, mapping to mousechromosomes 3 and 9 (human chromosomes 1 and 6), respectively. Tbx2and Tbx4 map adjacent to each other on mouse chromosome 11 (humanchromosome 17). Tbx3 and Tbx5 map adjacent to each other on mousechromosome 5 (human chromosome 12). Tbx5 is expressed exclusively in theforelimb (blue) and the Tbx4 is expressed exclusively in the hindlimb (red).

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studies, gene inactivation, andmisexpression experi-ments. Naturally occurring mutants of T wereidentified in 1927, as described above. In thesemutants, it was shown that T is required for theformation of the mesoderm during gastrulation.Inactivation of T results in catastrophic defectsleading to lethality by E10.5 days in development(normal mouse gestation is 20 days). However, theformation of the AER can still be studied as it occursearlier in development [Liu et al., 2003]. Furthermore,misexpression studies can be performed in the chickin which expression is driven by injection of retro-viral vectors. This can provide additional clues as tothe function of genes when inactivation is notforthcoming. Tmisexpression experiments in a chickmodel showed an anterior expansion of the AERand phenotypic changes in the skeletal patternconsistent with expanded AER function, such asanterior digit duplications, posterior transformationof anterior digits, and thickening of the most anteriormetatarsal [Liu et al., 2003]. Milder phenotypes werealso observed, consistent with a role in the functionin the AER. Fgf10 expression was increased in the Tmisexpressing limb buds suggesting a disruption inepithelial–mesenchymal interactions. Shh expres-sion, which is dependent on AER signals, is alsoabsent [Liu et al., 2003]. Both the timing and locationof T expression as well as the results of theirexperiments support a role for mesodermal Texpression in directing the formation of a mature,organized AER [Liu et al., 2003].

Tbx2 and Tbx3 Are Requiredfor Digit Development

Tbx2 and Tbx3 are closely related T-box familymembers with similar patterns of expression inseveral tissues including the limb buds. Tbx2 isexpressed in a stripe along the anterior and posterioredges of the limb buds (Fig. 2). Its pattern ofexpression is similar in both forelimb and hindlimbbuds and a role in specification of the identity of the

posterior digits have been suggested for both Tbx2and Tbx3 [Suzuki et al., 2004]. Tbx3 is also expressedalong the anterior and posterior edges of the limbbud, but in addition, it is expressed in the mouse AER[Suzuki et al., 2004]. Experiments in chick embryoshave shown that Tbx2 and Tbx3 expression corre-lates with digits IV and III, respectively, and thatthese genes direct BMP and Shh signaling pathwaysto allow normal development of the digits. Chickmissense expression experiments with Tbx2 andTbx3 have shown posterior transformation of digitidentity [Suzuki et al., 2004]. Both of these genes,Tbx2 and Tbx3, seem to be important in patterningthe posterior digits. Mice were generated harboring aTbx2 null mutation; these null mutants die betweenE10.5 and E14.5 due to cardiac malformations[Harrelson et al., 2004]. Those embryos that survivedto E13.5 had bilateral duplications of digit IV inthe hindlimbs only [Harrelson et al., 2004; Suzukiet al., 2004].

An inactivating mutation of Tbx3 was generated[Davenport et al., 2003]. Tbx3 homozygous nullmutant mouse embryos die by mid gestation due toyolk sac defects [Davenport et al., 2003]. The nullmutant embryos have mild forelimb and hindlimbabnormalities including abnormal hand and footplates as well as major defects in the formation ofmammary glands [Davenport et al., 2003; Suzukiet al., 2004]. Hemizygous deletion of TBX3 isresponsible for ulnar-mammary syndrome, in whichpatients have posterior forelimb deficiencies invol-ving the ulna and fifth digit as well as apocrine andmammary gland defects, including abnormal nipples[Davenport et al., 2003]. In addition, split hand/footmalformation (SHFM) can occur in this syndrome.TheTbx3heterozygousmouse embryos donot showanydefects, but the homozygous nullmutants clearlyresemble the human syndrome.

The T-gene, Tbx3, typically functions as a tran-scriptional repressor during normal limb bud devel-opment [Carlson et al., 2001]. However, Tbx3 canalso act as an activator. Recently, it was shown thatTbx3 helps determine the axial positioning of thelimb, functioning as a transcriptional activator,causing the limb to shift to a more caudal locationand as a repressor, causing a shift to a more rostrallocation in the chick [Rallis et al., 2005].

Tbx4 and Tbx5 in Hindlimb and ForelimbDevelopment, Respectively

Tbx4 andTbx5 are another pair of closely relatedT-genes that have two main functions, initiation of limbbud outgrowth and specification of forelimb andhindlimb identities along with additional genes(Figs. 1 and 2). Most genes involved in limbdevelopment are expressed similarly in forelimband hindlimb, but Tbx4 is expressed only in thehindlimb and Tbx5 is expressed only in the forelimb

FIG. 2. Diagrams of the forelimb (blue) and hindlimb (red) showing theoverlapping patterns of T-box gene expression in the limb buds in the mousefrom E10.5-12.5. Note that Tbx2 and Tbx3 are expressed in the anterior andposterior margins of the limb buds. Tbx5 is forelimb specific and Tbx4 ishindlimb specific.

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(Fig. 2) [Chapman et al., 1996; Gibson-Brown et al.,1996]. Vertebrate limb development has been arecent subject of interest as researchers try to clarifythe mechanisms that allow forelimb to developdifferently from hindlimb. Tbx4 and Tbx5 arethought to be involved in specifying the structuraldifferences between fore- and hindlimb since theyare differentially expressed early in the limb bud(Fig. 2). In complex mouse genetics crosses withtransgenic expression of Tbx4 in Tbx5 null mutantmice, Tbx4was able to replaceTbx5 in rescue of limboutgrowth, suggesting that these genes do notdetermine limb-specific morphologies [Minguillonet al., 2005]. Pitx1, a bicoid homeodomain contain-ing transcription factor also plays a role in hindlimb-specific development, but again, it alone is notnecessary and sufficient for hindlimb specification[Minguillon et al., 2005].

Tbx4 and Signaling in Hindlimb Development

A null mouse model for Tbx4 mutation has beengenerated and shows embryonic lethality at E10.5due to its function in the allantois [Naiche andPapaioannou, 2003]. Hindlimb budding can bestudied by this developmental stage, and theseembryos initiate normal budding of the hindlimbwith normal activation of Fgf receptors and normalFgf8 expression in the AER. However, they then failto maintain Fgf10 expression in the mesenchymeand limb bud outgrowth ceases [Naiche andPapaioannou, 2003]. As mentioned above, Fgf10in the lateral plate mesenchyme is required viaepithelial–mesenchymal interactions in a positivefeedback loop with Fgf8 for limbbudoutgrowth [Minet al., 1998; Sekine et al., 1999]. This model isinteresting in that signal transduction has beenuncoupled. Mice with mutations in Twist, a regulatorof the fibroblast growth factor receptor 1 gene, Fgfr1,also begin normal limb development as for the Tbx4null mutant, and then fail to maintain Fgf10 expres-sion and halt limb outgrowth. In addition, AER-specific ablation of Fgf8 and Fgf4 produces a similareffect, suggesting thatTbx4 is required in relaying Fgfsignaling from the AER to the mesenchyme and thatFgf10may be a direct target of Tbx4 [Sun et al., 2002].The Tbx4 homozygous null embryos also have somedisruption of AP organization with an expansion ofthe posterior domain, which is presumably due toanother later function of Tbx4 [Naiche and Papaioan-nou, 2003]. It was also suggested that Tbx4 mayregulate expression of Tbx2 [Naiche and Papaioan-nou, 2003]. Both wingless and legless phenotypeswere generated by an early injection of dominant-negative forms of Tbx5 and Tbx4, respectively,supporting a critical role for these genes in initiationof limb bud outgrowth [Takeuchi et al., 2003]. Theyfound truncation defects with later injection andtheorized that there is a critical period during which

Tbx4 and Tbx5 are required to interact with Wnt andFgf signaling pathways in order to have normal limboutgrowth. Furthermore, it was found that laterinjection of the dominant-negative forms interferedwith a less important role in maintaining limboutgrowth [Takeuchi et al., 2003].

Tbx5 and Forelimb Development

Tbx5 has also been shown to be essential for theformation of the forelimb in several vertebratespecies including zebrafish, chick, and mouse. [Ahnet al., 2002; Agarwal et al., 2003; Rallis et al., 2003;Takeuchi et al., 2003]. In the absence of Tbx5, noforelimb bud is formed. Inactivation of Tbx5 has noeffect on hindlimb formation. Several groups haveconsistently found, working in chick and mousemodels, that Tbx5 is necessary and sufficient forforelimb outgrowth. Molecular pathways directingforelimb growth involve establishment of the fore-limb field, followed by Tbx5 direct activation ofFgf10, via binding to its promoter [Agarwal et al.,2003]. This leads to expression of Fgf8 in the AER andlimb bud outgrowth. Wnt signaling from theectoderm also activates Fgf10 and is required forexpression of normal levels of Fgf10.

At later stages of forelimb outgrowth, Tbx5 seemsto play another role in contributing to the main-tenance of the AER, especially the anterior portion ofthe AER. Misexpression experiments using a laterstage injection of a dominant-negative form of Tbx5in chick wing bud show immaturity of the AER anddecreasedexpressionofFgf8 in the anteriorAERonly[Rallis et al., 2003]. Results suggest that Tbx5 isrequired for the induction and maintenance of theAER, and this is consistent with the observation thatTbx5 is required to maintain adequate Fgf10 expres-sion in the mesenchyme [Agarwal et al., 2003; Ralliset al., 2003]. This disruption in the anterior limb isconsistent with the defects seen in Holt-Oramsyndrome, which is due to haploinsufficiency ofhuman TBX5.

Tbx15 and Tbx18 may Serve RedundantFunctions in Limb Development

The T-box gene, Tbx15, is expressed in the murinelimb mesenchyme (Fig. 2), pharyngeal apparatus,and the craniofacial region [Agulnik et al., 1998;Candille et al., 2004; Singh et al., 2005]. To determinethe function of Tbx15, it was inactivated in themouse. Mice carrying one inactivated allele of Tbx15are normal and many survive to adulthood [Singhet al., 2005]. The Tbx15 homozygous null mutantembryos have skeletal defects that are mild com-pared to that of complete inactivation of other T-boxgenes. They have a mild reduction in bone size andalterations in bone shape due to delays in endochon-dral bone development, including those of the limbs



[Singh et al., 2005]. It is surprising that the defects inthe limb were quite mild in light of its strongexpression in the limb buds during embryogenesis.Tbx18 is expressed in the segmented somites and

resulting sclerotome, as well as in the limb budmesenchyme (Fig. 2) [Kraus et al., 2001]. Inactivationof one allele of Tbx18 in the mouse does not result ina detectable phenotype. Mice with inactivation ofboth alleles of Tbx18 die soon after birth with severeskeletal malformations of the vertebral column,defects in the pedicles and ribs, all deriving fromthe lateral sclerotome [Bussen et al., 2004]. Ectopicexpression of Tbx18 in somites also affects scler-otome development. Interestingly, no limb defectswere noted. It is possible that none were detectedbecause the expression of its closest related T-boxfamily member, Tbx15, with 93% sequence con-servation, is co-expressed and might serve redun-dant functions [Singh et al., 2005]. This is especiallyrelevant as the limb defects in Tbx15 homozygousnull mutant mice were mild [Singh et al., 2005]. ThusTbx15 and Tbx18 are co-expressed in the limbmesenchyme and when inactivated separately donot result in major limb malformations.

CONCLUSIONS

The seven T-box genes, T, Tbx2, Tbx3, Tbx4, Tbx5,Tbx15, and Tbx18, are important genes for thedevelopment of the limb bud and resulting forelimbsand hindlimbs. Most of the T-box genes listed hereare clustered into pairs based upon sequenceconservation, overlapping expression, and similaror complementary function. The pairs of genesinclude Tbx2-Tbx3, Tbx4-Tbx5, and Tbx15-Tbx18.A significant proportion of the T-box genes havebeen associated with human congenital malforma-tion syndromes, including an important subsetwith limb malformations. Thus, understanding themolecular biology of these genes is important forknowledge of normal limb development andalso for understanding the pathogenesis of limbmalformations.

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T-genes and limb bud development

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