/ . Embryo!, exp. Morph. Vol. 49, pp. 13-16, 1979
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Printed in Great Britain © Company of Biologists Limited 1979
Ventral horn cell counts in a Xenopus with
naturally occurring supernumerary hind limbs
By ALAN H. LAMB 1
From the Department of Neuropathology, Royal Perth Hospital
SUMMARY
In a Xenopus toad, two partially functioning supernumerary hind limbs developed naturally
on the right side and were innervated by the ipsilateral side of the spinal cord. The number of
ventral horn cells on the right was 18% higher than on the left while the combined mass of
limb muscle on the right was estimated to be a 100 % greater. This result corroborates studies
with transplanted supernumerary limbs.
INTRODUCTION
The effect of transplanted supernumerary limb-buds in reducing the large
number of dying ventral horn cells (lateral motor column cells) during normal
development has now been well documented in chicks (Hollyday & Hamburger,
1976) and toads {Xenopus laevis; Hollyday & Mendell, 1976). The resulting
increase in number of surviving cells is small, usually less than 20 % (in Xenopus
this represents only a 5 % fall in death rate). It is important to corroborate this
figure because it would imply that the number of surviving cells cannot be
dependent solely on the mass of limb tissue. In that case increases of 100 %
would be expected. The survival rates obtained from the transplant studies may
have been depressed by the operation itself, for example by affecting the blood
supply, development, or innervation of the transplanted, or normal limb. In
addition, future ventral horn cell survival might be partly dependent on the
presence of limb precursor cells before the time of the operation. For these
reasons comparison with cases of naturally occurring supernumerary limbs are
valuable. However only one such case {Rana fused) has previously been reported
in which ventral horn cells were counted (Bueker, 1945). A 22-74% increase
was found on the side bearing two extra functioning hind limbs.
I report a case of a Xenopus toad with two extra partially functioning hind
limbs on the right side in addition to the normal right and left limbs, in which
the ventral horn cells counts are comparable to the figures obtained in Bueker's
case and in the transplant studies.
1
Author's address: Department of Neuropathology, Royal Perth Hospital, Perth, Western
Australia.
2
EMB 49
14
Fig. 1. Silhouette of the juvenile Xenopus toad drawn freehand from the live animal
and post-mortem photographs (dorsal view) with the layout of the nerve trunks to
the hind limbs superimposed. The graph shows the numbers of ventral horn cells
per 15 /tm of the rostrocaudal length of the right (dashed) and the left (continuous)
ventral horns. The total numbers of cells on the right and left were 1857 and 1578
respectively. F, Femoral (crural nerve); S, sciatic nerve.
MATERIALS AND METHODS
The tadpole was first noticed at stage 51 (Nieuwkoop & Faber, 1967) in a
normal batch obtained by induced ovulation (Nieuwkoop & Faber, 1967), and
raised under standard conditions (tap water 23 °C, fed with soya flour and
spinach powder). At the completion of metamorphosis (stage 66) it was fixed in
10% formal saline. Serial transverse paraffin sections (10 jtim) of the limbs and
the trunk were prepared using standard methods and stained with haematoxylin
and eosin. Ventral horn cells (excluding cells of the medial motor column) were
counted in every third section using the criteria of Prestige (1967). The pathways
of the nerve trunks were plotted by recording their positions from the serial
sections onto graph paper.
OBSERVATIONS
The extra limbs were partially fused and grew on the right side just caudal to
the normal limb. Their combined mass was 1-5 times a normal limb. Movements
were normal at the ankles and toes, but diminished at the hip and knees,
Ventral horn cell counts
15
probably because of joint abnormalities. Homologous and independent movements were observed in all four hind limbs.
The origins and distributions of the nerve trunks to the hind limbs are given in
Fig. 1. The nerves to the extra limbs came from the right side of the cord and
were similar in appearance to the normal sciatic nerve and its branches. Because
of the plexus, it was impossible to say which segments of the spinal cord
innvervated the extra limbs.
The number of lumbar ventral horn cells on the right (1857) was 18 % higher
than on the left (1578), the increase being confined to the caudal two thirds of
the lumbar ventral horn (Fig. 1). Brachial ventral horn cell counts on the right
(837) and left (825) differed by only 1 %. Cell counts were made in normal
animals of the same maturity and the highest difference between sides was 3 %.
The combined volume of the 8th, 9th and 10th dorsal root ganglia on the
right was 1-7 times larger than on the left.
DISCUSSION
Despite there being at least twice the normal mass of limb muscle on the
right side because of the extra hind limbs, only an 18% increase in surviving
ventral horn cells was found on that side compared to the left. This is comparable
to figures from earlier studies with natural and transplanted extra limbs
(Bueker, 1945; Hollyday & Hamburger, 1976; Hollyday & Mendell, 1976) and
strengthens the conclusion that the majority of ventral horn cell deaths cannot
be prevented by extra limbs. It therefore seems unlikely that the number of
surviving ventral horn cells is regulated solely by the amount of limb tissue.
Other factors independent of the limb may also affect ventral horn cell survival
including the possibility that some cells are not endowed with the capacity to
survive. Another explanation may be that ventral horn cells are specified for
particular limb regions with which they must connect in order to survive. Once
axons had grown to a limb the task of reaching their appropriate regions would
be unaffected by additional limbs and the survival chances of their parent cells
would not be increased. In that case the failure of extra limbs to significantly
increase the survival rate would be expected.
I am grateful to Dr L. D. Beazley for her help with the manuscript. Support was provided
by a Saw Medical Fellowship from the University of Western Australia.
REFERENCES
BUEKER, E. D. (1945). Hyperplastic changes in the nervous system of a frog (Rana) as
associated with multiple functional limbs. Anat. Rec. 93, 323-331.
HOLLYDAY, M. & HAMBURGER, V. (1976). Reduction of naturally occurring motor neuron
loss by enlargement of the periphery. /. comp. Neurol. 170, 311-320.
16
A. H. LAMB
M. & MENDELL, L. (1976). Analysis of moving supernumerary limbs of Xenopus
laevis. Expl Neurol. 51, 316-324.
NIEUWKOOP, P. D. & FABER, J. (1967). Normal Table o/Xenopus laevis (Daudin). Amsterdam:
North Holland.
PRESTIGE, M. C. (1967). The control of cell number in the lumbar ventral horns during the
development of Xenopus laevis tadpoles. /. Embryol. exp. Morph. 32, 819-833.
HOLLYDAY,
(Received 9 May 1978, revised 1 September 1978)