Vertebrate Limb Formation

Elucidating the mechanisms that control morphogenesis (the origin of form) is one of the most exciting frontiers in modern biology. Vertebrate limb placement is one system in which the mechanisms of morphogenesis can be investigated. The long-term objectives of the research conducted in my laboratory are to determine what parameters establish the location and size of a limb on the vertebrate body, and to determine how those parameters disfunction in birth defects involving the limbs.

To evaluate the influence of various embryonic tissues on the development of the chick limb, we inhibited wing development by implanting foil barriers between the body axis and the future wing territory. In another study, the neck, wing, flank, or leg regions of prelimb-stage embryos, with or without adjacent medial tissues, were grafted into the coelom of host embryos.

As a result of nearly 300 barrier implants and over 7400 coelomic grafts, we have demonstrated that very early in development the central axis of the embryo exhibits limb-stimulating activity. This activity is not confined to only the limb regions, but the neck and flank axial regions also exhibit limb-stimulating activity. In the wing and leg regions, there is a cascade through time of limb-forming potential from the central axial tissue, to the somitic mesoderm, and to the intermediate mesoderm to the lateral plate (where the limbs develop). In the neck and flank, the cascade of limb stimulation begins in the central axis and moves to the somitic mesoderm but is not transferred to the intermediate mesoderm or lateral plate.

We have also been able to restore limb-forming ability in a wing region isolated by a foil barrier from the embryo axis by implanting axial tissue lateral to the barrier. We are currently analyzing limb initiation at the chemical level by implanting foil barriers to inhibit wing development and then implanting gel beads soaked in various chemicals, such as growth factors, lateral to the barriers, in an attempt to learn what chemical signals may be involved in restoring limbness in the wing territory.

The number of grafts we have accomplished has been possible, in part, because of the number of people involved in the study: two faculty, one postdoctoral fellow, and forty-one students, including eight graduate students, one dental student, thirty-one undergraduates, and two high school students. We are interested in determining whether the involvement of undergraduates in our research projects would affect the results of the study. Therefore, in addition to evaluating the grafts, we also evaluated the students participating in this research. Survival of the embryos following surgery was nearly the same for undergraduates (62%) and graduate students (63%). Growth of the grafts was quite a bit lower for undergraduates (55%) than for graduate students (66%), but the percent of limbs developing in the growths was lower for the graduate students (12%) than for the undergraduates (15%).