An inauspicious outpouching on the wall of an embryo undergoes a vastly complicated process to become a functional limb. While elongating, the limb bud develops along axes of proximal to distal, anterior to posterior, and ventral to dorsal in specific temporal sequences. Throughout the development process, cells are proliferating, migrating, and differentiating. Apoptosis cleaves paddles into digits and creates joints. The intricacies of this process involve a network of cues. Rapid advances in biomolecular research continually change the understanding of the control mechanisms. Visceral organs develop simultaneously with the musculoskeletal system. Causative factors of limb deficiency may relate spatially, temporally, or on a molecular basis to simultaneous organ development. Basic understanding of these complex interrelationships helps create more awareness of potentially associated syndromes and inheritance patterns.

The upper limb bud begins as an outpouching of the lateral plate of the embryo, known as the Wolff crest, at 4 weeks of embryonic growth. Formation progresses from cranial and proximal to caudal and distal. The lower limb bud develops 1 day later than the upper limb. Limb buds develop from the lateral plate mesoderm encased in thickened ectoderm. The inner layer of the thickened ectoderm at the apex of the limb bud is referred to as the apical ectodermal ridge (AER). The AER forms at the border of the future ventral and dorsal ectoderm. The AER influences the proximal to distal outgrowth and differentiation of the mesoderm of the limb bud. The longitudinal growth of the limb bud stems from proliferation of undifferentiated mesoderm immediately under the AER in the progress zone. A small area of mesoderm at the posterior portion of the limb bud, known as the zone of polarizing activity (ZPA), directs the anterior posterior/radioulnar or tibiofibular development including digit type and number (Figure 1). The limbs begin as continuous pentagonal paddles, which undergo fissuring through apoptosis. Initially fissures develop to separate the central rays and then the border digits.¹

Under the influence of the AER, the limb bud mesenchyme forms prechondrogenic condensations, the cells of which will undergo chondrogenic differentiation giving rise to cartilage anlage. The condensations form in a proximal to distal sequence. The cartilage anlage is the precursor to endochondral ossification. Capillary invasion from the trunk into the limb bud accompanies development.
However, the areas of mesenchymal condensation of the future cartilage anlage remain avascular. As the embryonic period proceeds to the fetal period, chondrocyte proliferation, maturation, and then hypertrophy stimulate vascular invasion and formation of primary centers of ossification. The elements of the physis are visible at this stage. The physis flattens into a platelike shape with the development of the secondary ossification center.

A single end artery is responsible for blood supply to the nascent limb prior to 10 weeks of gestation. The adult pattern of arterial arches forms by 11.5 weeks. The formation of arterial anastomosis provides an element of protection against vascular disruption.²

A host of interactive mechanisms creates the cascade of skeletal outgrowth, differentiation, and polarity. The signaling may occur via gradients, cell-to-cell communication, and matrix-to-cell communication.

Fibroblast growth factor (FGF) 8 expressed in the precursors to the ectodermal cells of the AER plays a role in the induction of this structure.³ Just under the AER lie the mesodermal cells of the progress zone. The continued outgrowth by these undifferentiated mesodermal cells is influenced by FGF 2, 4, and 8 from the AER. Control of the outgrowth requires a balance of FGF 2 upregulation offset by bone morphogenetic protein 2 downregulation.⁴

Factors found in the embryo prior to the formation of the AER determine the dorsoventral patterning. The AER formation occurs along this dorsoventral border. Wnt7a in the dorsal ectoderm induces formation of LMX1 in the mesoderm. The cells in the area of mesoderm with LMX1 undergo dorsalization.⁵ The dorsal ectoderm of the limb bud also expresses the gene radical fringe (r-fng), whereas the ventral ectoderm expresses Engrailed-1. Engrailed-1 represses the r-fng produced in the dorsal ectoderm, thus creating a border between cells with r-fng and those without.⁶

Anterior posterior development is directed by a gradient in Sonic hedgehog (SHH) and retinoic acid. SHH is a secreted protein that is predominant in the ZPA.⁷ Skeletal abnormalities may be associated with abnormalities in SHH or disruptions in the gradient that are stabilized by genes that enhance or suppress SHH. The ZPA regulatory sequence functions posteriorly to enhance SHH, whereas GLI3 repressor (GLI3R) functions anteriorly to suppress SHH.⁸

Mesenchymal cell prechondrogenic condensation is associated with cell-to-cell communication. The adhesion molecules N-cadherin and N-cell adhesion molecule are expressed in these areas during condensation and cell recruitment.⁹ Cell-to-cell communication is also responsible for differentiation into chondrocytes. Enhanced N-cadherin expression is noted in mesenchymal cell lines treated with transforming growth factor beta 1 or bone morphogenetic protein 2.¹⁰

Mesenchymal cells also communicate via the production of matrix molecules. Condensing mesenchyme expresses fibronectin, which may function to mediate pattern-specific condensation.¹¹ Type I collagen is a marker for mesenchymal cells. Chondrocyte progenitor cells begin producing cartilage matrix molecules including type II collagen and the cartilage-specific sulfated proteoglycan, aggrecan. Type II collagen undergoes splicing. Collagen type IIA is noted in the progenitor cells of chondrocytes versus type IIB in the differentiated cells. Type X is most prominent in the maturing hypertrophic chondrocytes. Mature hypertrophic chondrocytes stimulate vascular invasion by releasing an angiogenic factor.¹²

Initial evaluation of a child with a congenital limb deficiency always necessitates careful consideration of associated syndromes and abnormalities outside of the musculoskeletal system. These can range from major cardiac and life-threatening hematopoietic conditions to clinically minor alterations in the renal system. The associations correlate with the temporal or molecular insult.

Cardiac, craniofacial, and renal development occur simultaneously with limb development accounting for several of the common associations.

The terminology for limb deficiency generally stems from the area of most severe or most obvious involvement. The treatment team needs to keep in mind that the obvious deficiencies rarely occur in isolation. Potentially subtle proximal abnormalities often drive treatment plans, such as the presence or absence of functioning musculature, joint stability, and joint contractures. For instance, in deciding the best management strategy in children with longitudinal deficiency of the lower limb, not only the presence or absence of a foot but also the intrinsic stability and muscles controlling the knee need to be considered. Similarly, elbow motion and control dictate management of hand anomalies.

Preaxial deficiency of the upper extremity, also known as radial longitudinal deficiency and radial clubhand, may be seen with some of the severe comorbidities including life-threatening cardiac and hematopoietic deficiencies. Distal structures tend to be more involved than proximal structures.¹³