• Ultrastructure of Developing Human Limb
• Structure of the Limb Bud
• Cells that Contribute to Mesoderm of the Limb Bud
• SEM of Human Limb Bud
• The Structure of the Human Fore-Limb
• The Organization and Polarity of the Developing Limb Bud
• Early Limb Development
• Epithelial-Mesenchymal Interactions during Limb Development
• Mesoderm Defines Type of Structure Formed
• ZPA & Retinoic Acid
• Is Retinoic Acid the Morphogen?
• SEM of "Paddle Stage" of Human Limb Development--40 day old (10mm)
• Signal Transduction & Limb Formation
• Understanding Thalidomide and Limb Development
• Fibroblast Growth Factor (FGF): a family of factors
• Events of Signal Transduction & Limb Formation

• 34 day old human embryo (5mm)
• 34 pairs of somites
• Forelimb (lower left) started to develop
• Hindlimb just beginning (right side)

• Limb bud: Mesoderm & Epithelial Ectoderm
• Ectoderm over mesoderm
• Ectoderm thickened as Apical Ectodermal Ridge (AER)

• Limb mesoderm (mesenchyme) comes from the somite and lateral plate mesoderm
• The Lateral Plate Mesoderm contributes to the skeleton, blood vessels & connective tissue
• The Somite Mesoderm contributes to the Musculature
• Nerve cells & Neural crest cells migrate in as well
• Motor Axons from spinal cord will innervate limb
• Neural Crest gives rise to sensory nerves, schwann cells, pigment cells

Scanning electron microscope picture (SEM) reveal details about the developing limb bud. A thickened ridge called the apical ectodermal ridge (AER) is clear in the picture on the left which has been coloured yellow on the right to indicate where you should look.

• The limb is a complex structure
• Large number of different cell & tissue types
• Precise pattern & polarity of differentiated tissues

The limb bud has a strict pattern and polarity. Development is organized around the A-P, D-V and P-V axes. The tissues of the limb will differentiate in a specific pattern that is defined in part by the existing embryonic regions: the Apical Ectodermal Ridge (AER), the Zone of Polarizing Activity (ZPA) and the Progress Zone (PZ). The following figure demonstrates these relationships in the developing limb. The AER acts as the organizing region for the proximodistal axis of the limb. The ZPA organizes the limb along the A-P axis. It does this in part through the expression of Sonic hedgehog resulting in the production of the soluble sonic hedgehog protein. Sonic hedgehog mediates many developmental events. In the limb it not only meditates A-P Axis formation but also the maintenance of the AER. Some of the experiments that delineated the roles of the AER and ZPA are detailed below.

• Limb grows & develops proximo-distally
• Zone of Cell Division: Region of actively dividing cells
• Zone of Differentiation: Region of cell specialization

• Experiments originally done in chickens
• Modified here to show how results might apply to human limb
• Removal of AER stops limb development
• Addition of AER causes formation of 2nd limb
• Splitting AER leads to 2nd limb
• Thus, AER controls limb development

• Limb mesoderm dictates limb development; almost any epithelial ectoderm can replace normal limb epithelium
• Type of limb depends on type of mesoderm
• Not species specific: Inter-specific grafts show same induction
• Inducer may be universal

• Transplantation exps. reveal ZPA
• Transplant 2nd ZPA to Host Limb: Extra Limb Parts Induced
• Retinoic Acid Induces Same Effects

• RA: Exists in limb, Receptors present
• The ZPA likely controls the A/P polarity of the limb through the secretion of sonic hedgehog
• To learn more about the ZPA and the role of bone morphogenetic proteins in the control of limb development see: Dahn & Fallon, 1999. BioEssays 21: 721-725.

During limb development the limb bud grows away from the body in a proximo- (close) distal (away from) fashion. Developmentally, it is important to realize that as the limb bud lengthens and limb components are specified and start to differentiate, what were once distal regions become proximal as new distal regions form. Thus in early embryogenesis, the humerus as it forms is initially the most distal component but once the radius and ulna and subsequent components form, it becomes proximal to them. This continues until the limb is fully developed and the final relationship of limb components is defined.

By the progress-zone model of limb development, the AER secretes FGF that influences the closest cells (those in the progress zone) to develop into distal structures. FGF is a distalizing factor in limb development. Those cells that are not within range of the AERs influence remain proximal in nature. As the AER extends out due to the continued division of cells in the progress zone, it continues to affect the closest cells by causing them to be specified as distal structure cells. Those that fall out of the range of influence of the AER are no longer influenced by the effects of FGF and retain their previously defined status (i.e., are now proximal components not influenced by the distalizing effect of FGF).

In the lecture on Critical Periods in Development we mentioned the effect of the teratogen thalidomide as an inducer of congenital limb malformations. The mode of action of thalidomide in limb development is still a controversial issue. However, it is generally believed that exposure to thalidomide leads to an inhibition of the growth of the limb bud mesenchyme. The end product is a limb with distal structures (hand/fingers) attached directly to the shoulders (i.e., the long bones are lost) resembling the flippers of a seal. The problem is fitting the effects of thalidomide into current models of limb development. By the progress-zone model one interpretation is that the lack of growth of the limb mesenchyme means that all of those cells are continually influenced by FGF released by the AER (Tabin, 1996. Nature 396: 322-323). Thus these cells never fall out of the sphere of influence of the AER to become proximal cells. Instead they only receive the distalinzing signals ultimately resulting in their differentiation only into distal components. This is shown in the following diagram. Clearly there is a range of limb shortening (phocomelia) syndromes reflected in thalidomide syndromes but this model begins to elucidate the possible underlying mechanisms and may lead to a better understanding of normal and defective limb development. A proposed mechanism of thalidomide teratogenesis at the molecular level has been recently reviewed (Stephens et al, 2000. Biochemical Pharmacology 59: 1489-1499).

• FGF is linked to the initiation of bud formation, maintaining bud outgrowth, and the induction of a regeneration
• FGFs are secreted primarily by AER
• Tyrosine kinase FGF receptor is expressed on the surface mesenchyme cells

• FGF Released by AER binds to FGF Receptor (a receptor tyosine kinase or RTK) & activates It
• RTK then phosphorylates critical proteins
• This causes the mesenchyme cells to release retinoic acid (RA)
• RA induces Hox Gene Expression in target cells