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I suggested that phocomelia (missing proximal limb elements) results from lack of progress-zone proliferation in the context of continued distalization by fibroblast growth factor (FGF) signalling1. Neubert et al. consider this suggestion implausible because the established period of thalidomide susceptibility in marmosets (starting at stage 11, equivalent to stage 16/17 in the chick) is before the formation of the apical ectodermal ridge (AER), the structure at the distal tip of the limb bud that is the source of FGFs. But what matters is not when a morphological AER forms, but when FGFs are first produced at the distal tip.

In all species examined, including mice, chicks and frogs, FGF-8 is first expressed in a stripe of ectoderm corresponding to the future AER just before the initial outgrowth of the nascent limb bud2,3, at stage 16 in the chicken or what would be early stage 11 in the marmoset. The AER forms later, and at different relative times in different species, arising later in the mouse than in the chick, and not at all in amphibians. The stages of limb development at which marmosets are susceptible to thalidomide, as previously defined by Neubert and co-workers4, correspond extremely well to the stages of limb development when proximal limb elements are specified under the influence of the distal ectoderm5, lending explicit support to my proposal.

Neubert et al. also criticize my model for failing to explain other aspects of their criteria. But I never intended to address the pharmacological basis of thalidomide's effects, and indeed, as I pointed out, the pharmacology of this compound remains controversial. Whatever the mechanism, it undoubtedly has similar cellular effects in different regions of the embryo. However, in the context of the limb bud, the consequence of this is an ultimate decrease in growth of the proximal limb elements, and the point of my hypothesis was to explain in developmental terms specifically why proximal elements should be missing. This is a distinct question from the other important aspects of Neubert et al. 's list of criteria, such as the specificity of species susceptible to the drug or the specific set of organs affected, which probably involves factors such as differential metabolism of thalidomide.

Neubert et al. propose an alternative hypothesis to mine. They suggest that thalidomide could cause its characteristic limb defects by blocking cell migration. However, consideration of limb development suggests that this is unlikely because limb-bud initiation does not involve significant migration of lateral plate mesoderm and, even if it did, blocking such migration would not lead to phocomelia.

They cite results6 suggesting that limb-bud formation depends on cell migration from the somatopleura and from the somites. But the work cited actually shows that there are two distinct sets of limb precursors (myogenic cells from the somites, and connective tissue or chondrogenic cells from the somatopleura) and that the myogenic cells migrate. It does not provide any evidence that somatopleural cells migrate. Indeed, subsequent work has shown that they do not7. Rather, the limb bud forms by local proliferation of the somatopleural mesoderm. Only the myoblasts migrate.

However, even if thalidomide were to block myoblast migration completely, it would not affect the proximodistal pattern of the limb, as removal of all myogenic precursors results in a limb with a normal skeletal pattern but no muscle8. Moreover, even if, despite data to the contrary, a migratory block were to decrease the number of somatopleurally derived cells as well as myogenic cells in the early limb bud, this would still not result in phocomelia. As has been shown surgically9 and by various methods including drug treatment, decreasing the number of cells in the early limb bud results in a narrower limb bud and subsequent loss of elements along the anteroposterior axis, but does not affect proximodistal patterning. This contrasts with the result of experimentally preventing proliferation within the progress zone during limb development, which does result in phocomelia10.

Neubert et al. cite the downregulation of integrin expression11 to support their model. However, integrins are also important mediators of growth control and are key regulators of angiogenesis12, so these data are compatible with other explanations for the teratogenicity of thalidomide.

See also Neubert et al.