/. Embryol. exp. Morph. Vol. 62, pp. 109-115, 1981
Printed in Great Britain © Company of Biologists Limited 1981
Foetal megakaryoblastic
abnormality associated with congenital limb
amputation in the br/br
rabbit
By MAUD EHRENSPERGER, 1 CLAUDE PETTER AND
LIM KEUKY
From the Laboratoire de Physiologie comparee,
Universite Pierre et Marie Curie, Paris
SUMMARY
Recent research on the br/br rabbit is summarized. In all homozygous individuals, limb
amputations result from in utero haemorrhages which develop in the extremities and lead to
necrosis. An inner-ear abnormality is observed in about 25% of the cases. By studying a large
number of gestations and birth, we observed that homozygous crossings result in about
100% amputated animals while crossings between heterozygous and homozygous rabbits
result in about 50% of the young being abnormal.
Observations of megakaryoblastic cells in 15- and 17-day foetal liver show a predominance
of immature cells in 15-day br/br foetuses. This abnormality disappears on day 17. The
possible role of megakaryoblastic cells in br/br rabbit amputation is discussed.
INTRODUCTION
Hereditary brachydactyly was described in rabbits by Greene & Saxton
(1939) and Jost, Roffi & Courtat (1969). This abnormality is characterized
by limb amputations of variable severity occuring during foetal life. These
result from haemorrhages which develop in the foetal extremities between
the sixteenth and twenty-first days and lead to necrosis (Greene & Saxton,
1939; Inman, 1941; Jost et al. 1969). This necrosis occurs generally in the
extremities of the phalanges, although the whole upper or hind limb may be
amputated. At birth, cicatrization is total and the limbs are stump-like
(Fig. 1).
Several treatments result in prevention of foetal limb amputations (hyperoxia and phenylhydrazine, Petter, Bourbon, Maltier & Jost, 1971, vitaminic
treatments: folic acid, vitamin B12, Petter et al. 1977). These experiments
1
Author's address: Laboratoire de Physiologie comparee, Universite Pierre et Marie
Curie, 4, Place Jussieu, 75230 Paris Cedex 05, France.
109
110
M. EHRENSPERGER, C. PETTER AND L. KEUKY
Fig. 1. Congenital limb amputations in a new-born br/br rabbit.
confirmed the role played by blood defects in the induction of foetal abnormalities in the br/br rabbit.
MATERIALS AND METHODS
The br/br rabbits were bred in our laboratory. In order to maintain fertility,
frequent outcrosses with 'Geants Normands' were necessary. The latter were
also used as controls. The animals were bred in individual cages and fed and
given water ad libitum. Females were introduced into the male's cage, and the
day of mating is considered day 0 of pregnancy since ovulation is induced by
mating in the rabbit. Two stages of gestation were chosen: day 15 and day 17
because they generally correspond to the appearance of limb haemorrhages in
the br/br rabbit.
The experiments were performed on: fourteen 15-day foetuses from three
control females 'Geant Normand' and twelve 15-day foetuses from three br/br
rabbit females. Six 17-day control foetuses from two females and seven 17-day
br/br foetuses from two females; in both cases, br/br females were mated with
br/br males.
Pregnant rabbits were anaesthetized with i.v. pentobarbital (24 mg/kg) and
laparatomized. The uterus was opened at each foetal site and the foetuses were
removed with their adnexa. Whole foetuses were fixed in Bouin's solution.
The liver was then separated, dehydrated with ethanol, placed in isoamylacetate,
embedded in paraffin, and cut in totality. Serial liver sections (5/tm) were
stained with Gabe and Martoja's trichrome.
Foetal megakaryoblastic abnormality in br/br rabbits
111
Sections of hepatic tissue were observed under a projection microscope. As
the diameter of megakaryoblastic cells is about 20-25 /«n, we studied one out of
five serial sections. All the cells of the megakaryoblastic line were observed and
counted in each section studied. Quantitative data were based on a 100-cell
count. These cells are visible among the hepatocytes and in the erythropoietic
tissue. They can be easily classified into two groups: (a) Mono and binucleated
cells, which are the youngest forms of megakaryoblastic cells. Their cytoplasm
is coloured yellow-green and their nucleus is diploid or tetraploid. (b) Giant
cells which represent the most mature forms of megakaryoblasts and the
promegakaryocytes. Their nucleus is polynucleated.
RESULTS
I. Recent data concerning the br/br rabbit stock
Considering only the visible lesions in the limb extremities on days 19 and 20
and at birth, we have obtained the following data. By mating homozygous
rabbits, we obtained 201 foetuses from 36 females and 199 presented visible
limb haemorrhages; 290 young were born from 73 females, only 2 had anomalies. By mating heterozygous females with homozygous males, we obtained
622 foetuses from 110 females, 314 were abnormal and 308 normal; 804 young
were born from 198 females, 338 were abnormal and 466 were normal. The
relatively low number of abnormal young was probably due to the high mortality rate of the most affected foetuses at the end of gestation.
Another abnormality, occuring spontaneously in about 25 % of the subjects
was also observed: an internal ear syndrome, generally unilateral, evolving
progressively only in homozygous individuals (Fig. 2). We have followed the
evolution of this syndrome throughout the life of 12 rabbits. We have observed
a lack of sensitivity of the external ear, often accompanied by ataxia. Preliminary results from histological sections of the brain indicate a lesion at the
level of the trigeminic nerve nucleus.
II. Study of the megakaryoblastic cell line in the br/br foetus
In the 15-day br/br foetuses, the mono or binucleated cells are more numerous than those of the control group. These young cells are often situated
close to the vessel lumen or even in the vessels (Fig. 3). However, in 17-day
br/br foetuses the frequencies of the different megakaryoblastic cells was
found to be the same as in controls. In both cases, the most mature polynucleated cells seem to be predominant, and the observed values are similar to
those in the 15-day control foetuses.
The observed abnormalities possibly result in blood platelet defects, and for
this reason, we have counted the blood platelets on day 16. Five foetuses from
normal 'New Zealand* rabbits were used as controls and compared with five
foetuses from homozygous br/br rabbits. After laparotomy and uterine
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M. EHRENSPERGER, C. PETTER AND L. KEUKY
Fig. 2. Abnormal posture in a br/br adult rabbit, probably linked with an inner-ear
abnormality.
Table 1. Megakaryoblastic cell count in the liver of control and br/br rabbit
foetuses on days 15 and 17
Subjects
On day 15
Control
br/br rabbits
On day 17
Control
br/br rabbits
% (± S.E.) mono or
binucleated megakaryoblasts
polynucleated megakaryoblasts
300 ±3-0(14)
73-5 ±3-1(12)
700 ± 3-0(14)
26-5 ±3-2(12)
20-5 ±0-5(6)
21-5 + 3-5(7)
79-5 ±4-5(6)
78-5 ±3-5(7)
%(±S.E.)
The numbers of foetuses examined are given in parentheses.
incision, the foetal splanchnopleura was carefully dried and a vitelline vein was
punctured. The blood was collected with a Potain's pipette. The dilution fluid
consists of 1/10 procaine chlorhydrate and 9/10 NaCl (0-15 % solution). An
hematimeter was used for the platelet count. The results are given in Table 2.
DISCUSSION
The br/br rabbit stock has been known for more than 40 years and the
genetic transmission of the abnormality has remained unchanged. The interest
of this stock is due to the total penetrance of the gene with respect to limb
Foetal megakaryoblastic abnormality in br/br rabbits
113
Fig. 3. Megakaryoblastic cells in a 15-day foetal rabbit liver. A, Megakaryoblastic
cell in a control (note the abundance of erythropoietic cells): L.c. Liver cell; Ex.,
erythropoietic cell; P.m., polynucleated megakaryoblastic cell. B, Predominance
of immature cells in the br/br rabbit. (Note the scarcity of erythropoietic cells):
L.c, liver cell; E.c, erythropoietic cell, i.b.m., immature binucleated megakaryoblastic cells.
defects. The observed lesions generally affect the limb extremities and their
severity is variable. We have also observed an inner ear syndrome in the br/br
rabbit, which has not been described previously.
The study of megakaryoblastic cells in the br/br rabbit foetus liver clearly
114
M. EHRENSPERGER, C. PETTER AND L. KEUKY
Table 2. Number of blood platelets in control and bx/bv rabbit foetuses on day 16
Controls
Nl
N2
N3
N4
N5
358000/mm3
470000/mm3
366000/mm3
442000/mm3
418000/mm3
br /br rabbits
AN1
AN2
AN3
AN4
AN5
235400/mm3
242500/mm3
243800/mm3
310000/mm3
243800/mm3
The difference between controls and br/br animals is significant at the 0 05 level. (Student's
t test.)
shows an abnormality in their development which is especially apparent on day
15. At this stage, there is an abnormally high frequency of young cells and these
lie close to the vessel lumen. It has already been shown that the br/br liver is
especially poor in erythropoietic tissue at this stage and progressively becomes
normal towards day 17 (Petter et ah 1977). It seems possible that a developmental defect involving all the haemopoietic cells is linked with the induction
of the abnormalities. Treatment with vitamins, which increases the erythropoietic tissue and at the same time prevents limb lesions (Petter et al. 1977)
may also play a preventive role in abnormal megakaryoblastic cell formation.
Although unpublished studies have shown that the platelet count is approximately the same in adult br/br rabbits and in controls, this does not exclude the
possibility of temporary platelet deficiency during the critical stages in br/br
foetuses. The preliminary experiments (on day 16) shown in Table 2 indicate that
this indeed occurs. This deficiency could be responsible for both limb and liver
haemorrhages. Haemorrhages may also destroy nervous tissue and thereby
induce an inner-ear syndrome. Due to the exceptional fragility of 15-day-old
foetal membranes, it was impossible to count the platelets at this stage.
This megakaryoblastic abnormality, associated with congenital limb amputations in the rabbit resembles the TAR syndrome in man discussed in detail in
the survey of Hall et al. (1969). The characteristics of this human disease are
usually radius aplasia and platelet deficiency at birth. A hypomegakaryocytic
thrombocytopenia was observed in the children studied.
At the present time, further research is being carried out in order to better
understand the haemopoietic defects observed in the br/br stock.
REFERENCES
H. S. N. & SAXTON, J. A. Jr. (1939). Hereditary brachydactylya and allied
abnormalities in the rabbit. /. exp. Med. 69, 301-314.
GREENE,
HALL, J. G., LEVIN, J., KUHN, J. P., OTTENHEIMER, E. J., VAN BERKUM, K. A. P. & MCKUSICK,
V. A. (1969). Thrombocytopenia with absent radius (TAR). Medicine 48, 411-439.
O. R. (1941). Embryology of hereditary brachydactyly in the rabbit. Anat. Rec. 79,
483-505.
INMAN,
Foetal megakaryoblastic abnormality in br/br rabbits
115
A., ROFFI, J. & COURTAT, M. (1969). Congenital amputation determined by the br
gene and those induced by adrenalin injection in the rabbit foetus. In Limb Development
and Deformity: Problems of Evaluation and Rehabilitation. Charles C. Thomas, Publisher.
PETTER, C., BOURBON, J., MALTIER, J. P. & JOST, A. (1971). Prevention des amputations
cong&iitales hereditaires du lapin par une hyperoxie maternelle. C.r. hebd. Seanc Acad.
ScL, Paris, 273, 2639-2642.
PETTER, C , BOURBON, J., MALTTER, J. P. & JOST, A. (1977). Simultaneous prevention of blood
abnormalities and hereditary congenital amputations in a brachydactylous rabbit stock.
Teratology, 15, 149-158.
JOST,
(Received 20 February 1980, revised 24 October 1980)