Cellular Contribution to Supernumerary Limbs in the Axolotl - PDF document

Cellular Contribution to Supernumerary Limbs in the Axolotl, Ambystoma mex&mm KENMUNEOKAANDSUSAN V. BRYANT Developmental Biology Center and Department of Developmental and Cell Biology, University of California, Irvine, California 9.2717 Received January 6, 1984; accepted in revised fwm April 30, 1984 Using the triploid cell marker, the cellular contribution from graft and stump to the supernumerary limbs which result from contralateral grafts of limb bu

ds and regeneration blastemas In urodeles, supernumerary limbs can result from a number of different experimental grafting manipula- tions of the regeneration blastema and the developing limb bud (regeneration-reviewed in Tank and Holder, 1981; development-Maden and Goodwin, 1980; Thorns and Fallon, 1980; Muneoka and Bryant, 1982). It is from such experimental manipulations that our current un- derstanding of how the limb pattern is established or reestablished

during limb outgrowth has evolved. The polar coordinate model (French 0012-1606/84 $3.00 Copyright 0 by Academic Press, Inc. All rights of reproduction in any form reserved. 166 BRYANT Cell Chtribution to Axolotl Limbs 167 Xenopus larvae, Cameron and Fallon (1977) also con- cluded that the cellular contribution (based on the l- nu cell marker) to supernumerary limbs resulting from 180” rotation of the hindlimb bud is solely from anterior tissue. In th

e developing axolotl, however, Thorns and Fallon (1980) using the triploid cell marker, showed a cellular contribution from both graft and stump to su- pernumerary limbs which were formed after various axial misalignments of the forelimb bud. Studies of the relative contribution from A. mexicanum, A. maculatum, and A. texanum (Stocum, 1982). Taken as a whole, these blas- tema studies have indicated that supernumerary limbs arise from graft tissue only, stump

tissue only, or from a combination of both stump and graft tissue. However, both markers are less than ideal, and cannot be followed on a cell by cell basis in the supernumerary outgrowth (see Discussion). MATERIALS AND METHODS All experiments were performed on axolotls (A. mex- icanum) spawned at the University of California, Irvine. Animals were maintained at 20 f 1°C. Prior to grafting, young larvae were kept in pans of 20% Steinberg’s so- lution.

They were changed and fed newly hatched brine shrimp daily. Experimental animals were maintained individually in 100 X 25-mm plastic petri dishes in 25% Holtfreters solution until they reached a body length of 5 cm. They were changed and fed tubifex worms daily. Animals larger than 5 et al. (1984). Triploid larvae possess cells with three nucleoli while diploid larvae possess cells with two (Fankhauser and Humphrey, 1943). Triploid and diploid larvae were ma

intained under identical conditions but were kept separately. Grafting The grafting procedures used in this study are shown in Fig. 1. As shown in Fig. la, hindlimb buds were et al, 1976) and were from forelimbs of animals ranging in body so as 2 100 in digits bud and or less 1, 2, 3, 2, 3, 1, 2, 3, 1, 2, 1, 2, 4, 3, 2, 1, 3, 4. but not M~JNEOKA AND BRYANT Cell Cimtributim to Axolotl Limbs 171 a + d e FIG. 4. (a) Whole-mount dermal preparation. The dermi

s was man- ually isolated from the epidermis and analyzed in bismuth-stained whole mounts for cellular contribution to supernumerary limbs. An example of a dorsal dermal preparation is shown here. Epidermis is left at the digit tips to prevent the dermal preparation from curling. The limb from which this preparation was derived formed super- numerary limbs anterior (left 4 digits) and posterior (right 4 digits) to the graft (central 3 digits). X7. (b, c) A highe

r magnification of bismuth-stained triploid (b) and diploid (c) regions in the dermis of a supernumerary limb. (d, e) Examples of trinucleolate (d) and bin- ucleolate (e) cartilage cells in paraffin sections stained with bismuth. FIG. 5. Summary diagram of the cellular contribution boundaries in the dorsal dermis, cartilage, and ventral dermis of 10 supernumerary limbs resulting from contralateral limb bud grafts. Five supernumerary limbs formed anterior to the

graft (left column) and five formed pos- terior to the graft (right column). The experimental limbs which gave rise to these supernumerary limbs are numbered l-5. The boundary position in the dorsal and ventral dermis and in the cartilage of each supernumerary limb was derived from data similar to those shown in Fig. 2. These data are presented for each individual supernumerary limb in Table 1. Complete supernumerary limbs are those with five digits and are show

n diagrammatically as full ellipses. Incomplete supernumerary limbs possess less than five digits and are shown as partial ellipses. Cross hatching indicates triploid tissue. In the remaining 10 cases derived from limb bud grafts, both the stump and the graft contributed to the dermis of the supernumerary limbs. The amount of con- tribution from the graft versus that from the stump, and the position of the boundary again varied from limb to limb as was the case

for the 10 limbs discussed above. Considering all 20 supernumerary limbs, the dorsal and ventral boundaries within each individual limb, for the most part, did not fall at the same position on the an- terior-posterior axis of the supernumerary limb. There was a tendency for the dorsal boundary to be closer to the anterior edge of the limb, and for the ventral bound- ary to be closer to the posterior edge. This indicates that cells derived from the posterior edge

of the stump or graft tend to contribute more to the dorsal than to the ventral part of the supernumerary limb while cells from the anterior edge of the stump or graft tend to contribute more to the ventral than to the dorsal part of the supernumerary limb. The nucleoli stain dark brown and are easily distinguished with this staining procedure. In diploid tissue cells have nuclei with two nucleoli while in triploid tissue, cells have nuclei with three nucleoli.