A reevaluation of X-irradiation induced phocomelia and proximodistal limb patterning

Abstract

The congenital disorder phocomelia is a rare limb malformation that became more familiar in the 1960s as a side effect of the use thalidomide in pregnancy. Phocomelia is mimicked in developing chick limb buds exposed to X-irradiation, and studies of the chick model provided important evidence for the long-established progress zone model of limb development, in which fibroblast growth factor produced by the apical ectoderm ridge directs cell fate. New work, involving molecular analysis and lineage tracing, shows that X-irradiation-induced phocomelia is not a patterning defect as was thought, but results from a time-dependent loss of skeletal progenitors. This finding challenges the current model of phocomelia aetiology as well as the predictions of the progress zone model. The condition of phocomelia, a human birth defect in which the long bones are shorter than normal, is mimicked in developing chick limb buds exposed to X-rays. Studies of X-irradiation-induced phocomelia have served as evidence supporting the 'progress zone' model of limb patterning. Here, X-irradiation-induced phocomelia is shown not to be a patterning defect at all; rather, it results from a time-dependent loss of skeletal progenitors. Phocomelia is a devastating, rare congenital limb malformation in which the long bones are shorter than normal, with the upper portion of the limb being most severely affected. In extreme cases, the hands or fingers are attached directly to the shoulder and the most proximal elements (those closest to the shoulder) are entirely missing. This disorder, previously known in both autosomal recessive and sporadic forms, showed a marked increase in incidence in the early 1960s due to the tragic toxicological effects of the drug thalidomide, which had been prescribed as a mild sedative1,2. This human birth defect is mimicked in developing chick limb buds exposed to X-irradiation3,4,5. Both X-irradiation5 and thalidomide-induced phocomelia5,6 have been interpreted as patterning defects in the context of the progress zone model, which states that a cell’s proximodistal identity is determined by the length of time spent in a distal limb region termed the ‘progress zone’7. Indeed, studies of X-irradiation-induced phocomelia have served as one of the two major experimental lines of evidence supporting the validity of the progress zone model. Here, using a combination of molecular analysis and lineage tracing in chick, we show that X-irradiation-induced phocomelia is fundamentally not a patterning defect, but rather results from a time-dependent loss of skeletal progenitors. Because skeletal condensation proceeds from the shoulder to fingers (in a proximal to distal direction), the proximal elements are differentially affected in limb buds exposed to radiation at early stages. This conclusion changes the framework for considering the effect of thalidomide and other forms of phocomelia, suggesting the possibility that the aetiology lies not in a defect in the patterning process, but rather in progenitor cell survival and differentiation. Moreover, molecular evidence that proximodistal patterning is unaffected after X-irradiation does not support the predictions of the progress zone model.