The ingenious work of Riddle et al.1, Tickle2, and others in manipulating limbs in chick embryos provided the basic understanding of limb development. It was such work that defined the terms as well as the role of the apical ectodermal ridge, the progress zone, and the zone of polarizing activity in patterning early limb development. Embryology has moved beyond the primitive understanding of which structures form when, into the molecular realm of developmental biology and genetics. As the signaling pathways for limb differentiation become well understood at a molecular level, morphological anomalies in limbs are seen as patterning errors and offer clues to the role of both genetic and epigenetic effects.
Investigators now have more sophisticated tools of molecular genetics, such as microarray chips, which can simultaneously search for abnormalities in the expression of thousands of genes, and techniques that can snip and splice as well as amplify and analyze extremely small quantities of DNA. One of the most revolutionary tools is the "knockout" animal, bred specifically to answer the question "What happens if this particular gene is missing?"
With the availability of these molecular genetic tools, and the compilation of the human genome, we now have a good, but not perfect, understanding of what should happen in the normal processes of limb development. Before it can be determined whether there is a genetic cause of a congenital malformation, it is necessary to have a baseline understanding of normal limb development—what starts it, what regulates it, and what stops it.
From this understanding, we gain insights into uncontrolled growth, including congenital overgrowth conditions and the dysregulation of growth that causes the malignant tumors that occur throughout life. The interest of pediatric orthopaedic surgeons in these conditions stems from our limited control over the growth, ultimate size, and especially the function of limbs that have been affected by growth abnormalities.
Because the scientific process of ascertaining the effect, the interactions, the order of cascading steps, and the feedback mechanisms is so very meticulous, a single scientist or team must focus on only one part of the puzzle. Much has been learned in a very short time and it is impossible to stay abreast of all that is now accepted as scientific fact. This paper delves into the regulation and patterning of limb growth, and in particular, the osteocartilaginous elements in the limb.
The upper-limb bud appears in the human embryo approximately twenty-eight days after fertilization along the symmetrical lateral mesodermal plates known as the "Wolff crest." The early limb bud has two major components: a core of loose mesenchymal cells derived from the lateral plate mesoderm, and an outer layer of epithelial cells derived from the ectoderm. The skeletal elements and connective tissues (cartilage, bone, tendon, and vasculature) are derived from this mesenchyme. Limb muscles and nerves have separate lineages; musculature is formed from myogenic precursor cells that originate in mesodermal somites and migrate into the limb bud. The peripheral nerves, which arise from the neural crest, migrate and extend their axons later in response to trophic cues, such as ephrins, produced by the muscles and connective tissues3.
This rapid proliferation of an undifferentiated cellular substrate is orchestrated at the distal end of the limb bud in a region known as the progress zone. Proceeding from proximal to distal, this cellular matrix begins its differentiation by the condensation of cells that will form the cartilage templates of individual bones. The condensations that give rise to proximal limb elements form first. Cells at the tip of the limb bud remain undifferentiated. As the bud continues to enlarge, more distal skeletal elements differentiate sequentially until the complete set of condensations is laid down2. The formation of these condensations in a precise proximal (early) to distal (late) manner is controlled by the apical ectodermal ridge, a thickening in the limb ectoderm that forms at the distal tip of the growing limb bud. The apical ectodermal ridge produces fibroblast growth factors (FGFs) that promote proliferation and inhibit condensation and differentiation in mesenchymal cells nearest the apical ectodermal ridge. As the limb grows outward, the proximal mesenchymal cells that are farthest from the apical ectodermal ridge and destined to become skeletal elements undergo condensation and initiate chondrogenesis.
The development of this proximal-distal axis is integrated with the development of the anterior-posterior axis (in human development this corresponds to the radial-ulnar axis in the upper limb, and tibial-fibular axis in the lower limb). This axis is controlled by the zone of polarizing activity, a region of limb mesenchyme located on the posterior side of the limb bud near its junction with the flank mesenchyme. The zone of polarizing activity is the source of the secreted protein Sonic hedgehog (Shh)1, which controls the radial-ulnar or tibial-fibular patterning of limbs and specifies both digit number and digit identity2. A key mediator of these effects of Shh signaling in the limb is the conversion of the intracellular transcription factor Gli3 from a repressor to an activator. In the absence of Shh, Gli3 represses digit formation. Mutations that lead to ectopic Shh expression, or mutations in Gli3 that convert it to an activator, have been associated with some forms of polydactyly. This finding demonstrates the ability of the zone of polarizing activity and Shh to control digit number. In accordance with the fact that Shh acts through Gli3, mutations in Gli3 itself that cause syndromic polydactyly have been described in Greig cephalopolysyndactyly syndrome and Pallister-Hall syndrome in humans4; these syndromes are listed in the human genetic database Online Mendelian Inheritance in Man (OMIM) as entry numbers 175700 and 146510, respectively. (For conditions referenced in this paper, the reader is encouraged to go to and enter the OMIM identifier number in the search field.) Mutations may also occur in distal regulatory regions upstream of the Shh gene that lead to ectopic Shh expression; these have been associated with other forms of preaxial polydactyly in humans5,6. In addition to the control of digit identity by graded expression of Shh along the anterior-posterior or the radial-ulnar axis of the limb bud, there is evidence that locally acting bone morphogenetic proteins (BMPs), produced in the interdigital mesenchyme, are required for normal differentiation7.
Beyond its role in specifying digit number and identity, Shh is required for proximal-distal outgrowth. The requirement for Shh in regulating outgrowth is seen in Shh-deficient mice, in which the limbs are truncated at the most proximal skeletal element. This aspect of Shh function is a reflection of the reciprocal requirement for Shh to maintain the expression of FGFs in the apical ectodermal ridge. Thus, loss of either Shh or FGFs in the limb bud leads to limb truncation and therefore loss of differentiation distally. BMPs and Gremlin, a secreted inhibitor of BMPs, also participate in this pathway; the BMPs produced throughout the limb mesenchyme and in the apical ectodermal ridge inhibit production of FGFs by the apical ectodermal ridge. A key role of Shh is to activate expression of the BMP antagonist Gremlin, thereby preventing this inhibition of FGF expression. Hence, overexpression or underexpression of FGFs, Shh, or BMPs in the early limb bud leads to phenotypes associated with limb truncations or patterning defects (alterations in digit identity and number), but not to limb overgrowth (Fig. 1).
Control of growth of individual cells is an important process during embryogenesis and throughout life. A serine-threonine protein kinase, involved with growth, replication, and motility through regulation of DNA transcription and protein synthesis, is known as FK506-binding protein 12-rapamycin complex-associated protein 1 (FRAP1), or mammalian target of the immunosuppressive drug rapamycin (mTOR).
mTOR plays a central role in the regulation of cell growth23, receiving input from multiple signaling pathways to stimulate protein synthesis. Dysregulated mTOR activity caused by mutations in genes that normally repress mTOR activity is associated with several hamartoma syndromes, including Cowden disease, Proteus syndrome and Proteus-like syndrome, and tuberous sclerosis complex (TSC). For example, phosphatase and tensin homolog (PTEN), a tumor suppressor gene involved in the regulation of cell-cycle length and in the initiation of apoptosis, negatively regulates mTOR activity. Homozygous loss of PTEN leads to embryonic lethality, whereas heterozygous loss in epithelial tissues leads to neoplasia24. Germline mutations in PTEN (OMIM 601728) have been found in multiple overgrowth syndromes, including 85% of Cowden disease cases (OMIM 158350), 65% of Bannayan-Riley-Ruvalcaba syndrome cases (OMIM 153480), and 20% of Proteus syndrome cases (OMIM 176920)25. Although there is controversy regarding the relevance of PTEN mutations as a specific cause of Proteus syndrome, the finding of mutations in this gene in multiple overgrowth syndromes is compelling, and it is likely that mutations in genes other than PTEN that also negatively regulate mTOR activity may underlie at least some cases of Proteus syndrome24. If this turns out to be the case, then treatment with rapamycin, a negative regulator of mTOR activity, may be of benefit. Initial results indicate that this treatment ameliorates at least some aspects of overgrowth in Proteus syndrome26. 