/ . Embryo!, exp. Morph. Vol. 40, pp. 285-289, 1977
Printed in Great Britain © Company of Biologists Limited 1977
285
Interdigital cell death during limb development
of the turtle and lizard with an interpretation of
evolutionary significance
By JOHN F. FALLON 1 AND JO ANN CAMERON 2
From the Department of Anatomy, University of Wisconsin
SUMMARY
Cell death accompanies the formation of free digits in birds and mammals. However, in
species with webbing between the adult digits, little or no cell death occurs in the prospectively webbed region of the developing interdigit. Cell death does not occur during the
formation of free digits in amphibians. In this paper we report that cell death accompanies
the formation of the digits in snapping and painted turtles and in the skink (a lizard). We
conclude that cell death accompanying the formation of free digits had its origin at the
point of amniote emergence during evolution.
INTRODUCTION
Massive cellular death accompanies the formation of free digits during limb
development of birds (Saunders & Fallon, 1967; Hinchliffe, 1974; Pautou,
1974) and mammals (Ballard & Holt, 1968). In animals that have webbed digits
cell death is sparse, if it occurs at all, in presumptive webbed areas. In limbs
that have partial webbing between the digits, only the interdigital area that
lacks webbing in the adult shows massive necrosis during embryonic development, e.g. the duck (Saunders & Fallon, 1967; Pautou, 1974) and the ferret
(Fallon, unpublished). Thus cell necrosis appears to be an integral part of free,
and partially free digit formation in birds and mammals.
In amphibian limb development cell death does not occur during the formation of free digits in several species of anurans and urodeles (Cameron & Fallon,
1977). Using tritiated thymidine injected into Xenopus laevis tadpoles, our
analysis demonstrated that initially the labeling index was comparable in the
prospective digital and interdigital regions of the free digited forelimb. However
as development proceeded, the labeling index decreased in the interdigit while
it was maintained at previous levels in the digit. We proposed that the observed
difference in proliferation rates accounts for the fact that the forelimb digits grow
1
Author's address; Department of Anatomy, 453 Bardeen Medical Laboratories, University of Wisconsin, Madison, Wisconsin 53706, U.S.A.
8
Author's address: Department of Biology, University of Virginia, Charlottesville,
Virginia 22901, U.S.A.
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J. F. FALLON AND JO ANN CAMERON
out free of webbing. Finally, in the webbed hindlimb of Xenopus the labeling
indexes remained comparable in digit and interdigit, and the two areas elongate
at essentially the same rate throughout development.
The question of when during evolution cell death became an integral part
of the formation of the digits may now be raised. In the present study we
examined digit formation in three reptiles and found that cell death accompanies
digit formation in the most primitive extant reptiles (turtles) and in a more
phylogenetically advanced lizard (skink) as well.
MATERIALS AND METHODS
Snapping turtle (Chelydra serpentina) and painted turtle {Chrysemys picta)
eggs were collected locally and incubated in a bed of moist vermiculite at 25 °C.
Development was monitored by candling the eggs, and the embryos were
staged according to the developmental stages of Yntema (1968). Two clutches
of skink eggs (Eumeces fasciatis) also collected locally, were allowed to develop
surrounded by moist moss. The limb stages of Mathur and Goel (personal
communication) were used.
Turtle embryos between stages 16 and 21 and skink embryos of stages 3638 were removed from the shell and placed in Hams FIO tissue culture medium
containing one part per 10000 (w/v) of either neutral red or brilliant cresyl blue.
These basic dyes vitally stained dying cells. Cell death patterns for each limb
were drawn with the aid of a camera lucida; the stippled areas in Figs. 1 and
5-7 indicate the intensity of vital dye staining. Embryos were also fixed for
light and electron microscopic observations.
RESULTS
Snapping and painted turtle limbs stained with brilliant cresyl blue during
stages 17 and 20 show similar patterns of degeneration between the digits at
each stage (Fig. 1). Cell death begins during stage 17 in both fore- and hindlimbs. While degeneration between the digits of the forelimb begins very early
during stage 17, a comparable degenerative pattern is not present in the hindlimb until stage 17 + . During stages 17, 18 and early 19, the outer rim of mesoderm of the prospective digits of fore- and hindlimb contains some dying cells.
Except for the earliest stage the prospective digit has a very small amount of
necrosis when compared to the numbers of cells dying in the interdigits. At
all stages the zones of degenerating cells are most intense in the peripheral
part of the interdigit and, except during stage 18, these zones are not found
deeper than the terminal sinus. Stage 18 is the time of peak interdigital degeneration, when massive cell death extends from the apical ectoderm to just
beneath the terminal sinus. A few dying cells may also be found deeper in the
interdigit (small arrows, Figs. 2 and 4) at this stage. Cell death ceases between
Cell death in reptile interdigits
1 Interdigital cell death in the turtle
Forelimb
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J. F. FALLON AND JO ANN CAMERON
the forelimb digits during early stage 21, whereas necrosis ceases between the
hindlimb digits late in stage 21. Sections through the interdigit of painted and
snapping turtle forelimb (Figs. 2 and 4) at stage 18 show many pycnotic nuclei
that appear to be phagocytized. This observation was confirmed by electron
microscopy of one of these zones (Fig. 3).
In contrast to the limited interdigital zones of necrosis described for the
turtle, stage-36 skink limbs stained with brilliant cresyl blue (Fig. 5) show substantial numbers of degenerating cells throughout the interdigit. For the most
part the dying cells were within macrophages.
DISCUSSION
In this paper we describe the patterns of cell death which accompany the
formation of the digits in three species of reptiles. In snapping and painted
turtles, the zones of cell death are limited to the distal parts of the interdigits
with very little degeneration in the proximal parts of the interdigits. In the
skink, cells die in great number?, throughout the interdigits. The corollaries to
these observations are that the adult turtle digits are webbed for most of their
length and the adult skink digits are completely free of webbing. Similar parallels
have been observed in avian and mammalian species. For example, the adult
duck foot is partially webbed and the massive interdigital zones of cell
death (see Fig. 6) are limited to the distal, prospectively free interdigit, while
fewer cells in the prospectively webbed regions die (cf. Saunders & Fallon,
1967; Pautou, 1974). Conversely, the digits of the adult chicken or quail foot
are essentially free of webbing and massive cell death occurs throughout the
embryonic interdigit (see Fig. 7) as the free digits are forming (cf. Saunders &
Fallon, 1967). In the reptiles, birds, and mammals examined to date, the
amount of cell death in the interdigit portends how much of the digit will be
FIGURES
1-7
Fig. 1. A drawing of the cell death patterns in the turtle fore- and hindlimb interdigits between stages 17 and 20. The zones of necrosis are limited compared to the
skink (cf. Fig. 5). Adult turtle digits are webbed for most of their length.
Figs. 2-4. Frontal sections through interdigit 3-4 of the forelimb of the snapping
turtle (Fig. 2) and painted turtle (Fig. 4). The zone of necrosis lies primarily distal
to the terminal sinus (TS). Pycnotic nuclei are indicated by large arrows. Small
arrows indicate degenerating cells deeper in the interdigit. Figure 3 is an electron
micrograph from the area of large arrows in Fig. 2. This demonstrates that the
majority of the dead cells are within phagocytic cells. The arrows point to a single
phagocytic cell.
Fig. 5. A drawing depicting the degeneration of cells throughout the interdigit of the
skink (lizard). The zones of necrosis are extensive (cf. Fig. 1). The adult skink has
free digits.
Figs. 6 and 7. A further comparison of the degenerative pattern in an animal with
partial webbing (limited massive degeneration) between the digits (duck, Fig. 6)
and an animal with free (extensive massive degeneration) digits (quail, Fig. 7).
Cell death in reptile interdigits
289
free in the adult. However, where necrosis occurs between the digits it is
important to note that cell death may not be the only mechanism accounting
for the formation of free digits. In this regard, Kelley (1970) has suggested
that cells may migrate from the interdigit to the digit as the digits become free.
Further, we have noted that differential growth may also play a significant role.
Clearly the latter has a significant, if not the primary role in free digit formation
in Xenopus (Cameron & Fallon, 1977).
The selection of snapping and painted turtle for these studies took into
account the facts that turtles were the first forms to branch off of the modern
reptilian line and that snapping and painted turtles are among the most ancient
of extant reptiles (Romer, 1966). In this context it is reasonable to conclude
that cell death became an integral part of the formation of free or partially
free digits with the emergence of the Amniota, while cell death probably was
never a part of free digit formation among the Amphibia.
Supported by NIH Grant HDO7402, NIH Training Grant GM00723 and NSF Grant Nos.
GB24704 and GB40506. We are grateful to Drs A. W. Clark, R. O. Kelley, B. H. Lipton,
H. W. Mossman, and D. B. Slautterback for their critical reading of this manuscript, to
Dr E. F. Allin for helpful discussions and to Dr S. C. Goel for permitting us to see his reptile
limb stages before their publication. We thank Mrs B. K. Kirk for technical assistance,
Ms L. Taylor for the drawings, and Mr R. Vogt for supplying the reptile eggs.
REFERENCES
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{Received 3 March 1977)