A review of: Takeuchi JK, Koshiba-Takeuchi K, Matsumoto K, et al. 1999 Tbx5 and Tbx4 genes determine the wing/leg identity of limb buds. Nature 398:810–814. Rodriguez-Esteban C, Tsukui T, Yonei S, et al. 1999 The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity. Nature 398:814–818. Logan M, Tabin CJ 1999 Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity. Science 283:1736–1739. Szeto DP, Rodriguez-Esteban C, Ryan AK, et al. 1999 Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development. Genes Dev 13:484–494. Lanctot C, Moreau A, Chamberland M, et al. 1999 Hindlimb patterning and mandible development require the Ptx1 gene. Development 126:1805–1810.

EVER WONDER WHAT determines the characteristic differences between arms and legs? There is now evidence that certain genes control the “identity” of a developing limb bud, that differential expression of those genes is the basis for morphologic and functional variation, and that these pathways are conserved among vertebrates.

It was the initial observation that the expression patterns of the T-box transcription factor genes Tbx5 and Tbx4 were restricted to the forelimb and hindlimb (1), respectively, that led investigators to consider these genes as important components of the genetic hierarchy determining limb identity. Forcing expression of Tbx5 in the developing hindlimb bud, where it is normally not expressed, was shown to induce a partial transformation of leg structures into wing-like structures. It has also been reported that when Tbx4 was misexpressed in developing wing buds, the resulting limb had features similar to those of a leg (2, 3).

In other work, investigators studying the pituitary transcription factor Pitx1 found that this gene was also expressed exclusively in the hindlimb (4). Suspecting that Pitx1 was involved in specifying hindlimb identity, recent studies demonstrated that misexpression of Pitx1 in the wing bud of chicken embryos induced the hindlimb-specific genes Tbx4, Hoxc10, and Hoxc11, and resulted in a forelimb with leg-like morphology (5, 6). Further evidence for the involvement of Pitx1 in hindlimb specification came from the analysis of mice lacking the Pitx1 gene (6, 7). Hindlimb development in these animals was characterized by reduced Tbx4 expression, supporting the conclusion that Pitx1 regulates Tbx4. Furthermore, the fully developed “hindlimbs” of these mutant mice resembled forelimbs. Therefore, in addition to its role in pituitary development, Pitx1 is likely to be a key regulator in the determination of hindlimb identity.

These papers have made important contributions toward building molecular models relevant to determination of limb identity. However, many questions are unresolved. For example, Pitx1, Tbx4, and Tbx5 are all transcription factors, and the identity of their downstream target genes and upstream regulators is incomplete. Second, the misexpression studies described above resulted in partial transformations from one limb type to another, suggesting that other factors yet to be identified influence limb specification.

How does this new information relate to pediatric research or human limb malformations in general? There are approximately 40 reported inherited human limb malformation syndromes that predominantly or exclusively involve either the upper or the lower limbs (8). One of these, Holt-Oram syndrome, which is associated with mutations in TBX5 (9, 10), primarily affects the upper limbs and the heart, both regions of TBX5 expression. Do the genes mutated in these other syndromes also have restricted expression to the upper or lower limbs? Are these genes important for sculpting limb bone length and width, or in influencing muscle attachment points, nerve growth or vascular anatomy characteristic of a forelimb or hindlimb?

We now have a window into the genetic hierarchy regulating features of limb identity. Interestingly, this insight originated with the opportune discovery of differentially expressed genes. A prospective, comprehensive determination of gene expression at several stages of limb development might uncover new genes whose differential expression may be involved in regulating the specific morphogenetic events unique to the forelimb and hindlimb. The relationship of those newly discovered differentially expressed genes to Pitx1, Tbx4, and Tbx5 would be valuable in understanding how these molecular cues influence morphology and could provide candidate genes for inherited limb malformation syndromes.