Julie Lander and Dr. Jeffery Barrow, Physiology and Developmental Biology
Limb abnormalities are among the more common birth defects, occurring in up to 1 in 1,000 live births. Although limb development has been studied for decades, many mechanisms that regulate limb outgrowth remain currently unknown. The object of this study was to further elucidate some of the means by which the limb grows outward from the body and forms its distinctive shape.
Morphology is among the most complex concepts in human development, and the limb represents an accessible and useful model for studying outgrowth. This project has helped to ascertain the particular mechanism whereby a structure in the distal ectoderm, the Apical Ectodermal Ridge (AER), regulates limb outgrowth and patterning.
The AER was found many decades ago to play a critical role in limb outgrowth as well as proximal-distal patterning. If the AER is removed, limb development is halted. This project tested the hypothesis that the AER plays a critical role in recruiting cells towards it, and that this leads to limb outgrowth and patterning. Dr. Barrow’s lab had previously observed that removing only a portion of the AER allowed for continued limb growth, but created depressions in the limb bud in the area where the AER had been removed. There were three possible reasons for the depressions:
1. The AER promotes cell division. These depressions result from a loss of cell division in regions of AER removal.
2. The AER is necessary to prevent apoptosis (programmed cell death). Areas of AER removal show increased cell death and thus form depressions.
3. The AER recruits cells towards itself. Thus the regions of AER removal create depressions because flanking areas where AER is still present are recruiting cells preferentially in their direction leaving a depression in their wake.
To test these hypotheses, we used the chick embryo as a model organism. We could manipulate the chick in ovo, and then allow it to continue to develop for several more hours. Briefly, we took embryos that had developed for 85 hours. We cut a window in the egg, and carefully removed the middle one-third of the AER of one of the forelimbs. We then placed Scotch tape to cover the window, and placed the eggs back into the incubator. We collected the embryos at intervals of 5, 7, 9, 12, 16, 20, and 24 hours following the AER removal procedure. The limbs were removed and embedded in gelatin. They were then sectioned into slices 12 micrometers thick to allow for further analysis.
To ascertain the role of cell proliferation in limb outgrowth, we detected proliferating cells in the limb sections using fluorescently tagged antibodies directed against phosphohistone 3, a marker of cell proliferation. In addition, we counterstained the tissue using the fluorescent nuclear stain, ToPro3. The limb sections were divided into control (regions of limb mesenchyme adjacent to the AER) and experimental (areas adjacent to regions where the AER has been surgically removed) regions, and we calculated the number of proliferating cells per 100 nuclei in each region. The initial data do not indicate that there is any decrease in cell division in areas of AER removal. In fact, it may even be slightly higher at some stages. This is shown in the graphs below. Therefore, the Apical Ectodermal Ridge does not appear to regulate limb outgrowth by merely increasing cell division.
The next hypothesis, regarding apoptosis, or cell death, was assessed using a similar technique as above. Instead of an antibody for proliferating nuclei, we used an Apop-Tag kit that labels apoptotic cells. The data regarding cell death are somewhat inconsistent; most of the time little or no apoptosis could be seen, even in modified regions of the limb. There were, however, a few isolated cases of very clear apoptosis in areas where the AER had been removed. It should be noted that when the entire AER is removed there is about 200 micrometers of apoptosis about 7 hours following removal. It is possible in the cases in which we saw apoptosis that too much of the AER had been removed to prevent cell death. Further investigation is necessary to validate a conclusion regarding the role of apoptosis, but the majority of data do not support cell death as a major cause for lack of limb growth.
As neither cell division nor cell death seem to play conclusive roles in determining limb outgrowth, the final hypothesis, that the AER recruits cells towards it, seems quite likely. Time did not permit the experiments to test this directly, suffice it to say that preliminary data produced in our lab previously show that cells labeled in regions where the AER has been removed divide preferentially towards the peripheral areas where the AER is still present.
The data produced in this study have helped to determine the precise role of the Apical Ectodermal Ridge in limb outgrowth. The preliminary results of this study argue that the AER regulates outgrowth by promoting directional growth of the adjacent mesenchyme rather than by cell proliferation or by preventing apoptosis. Further studies will be performed to confirm this conclusion. The knowledge gained from studying the limb can also be applied to the growth of other structures of the body, and contributes to an increased comprehension of morphogenesis in general.