/. Embryol. exp. Morph. Vol. 68, pp. 87-98, 1982
Printed in Great Britain © Company of Biologists Limited 1982
Interaction of granule, Purkinje and inferior
olivary neurons in Lurcher chimaeric mice
I. Qualitative studies
By RICHARD WETTS 1 AND KARL HERRUP
From the Department of Human Genetics,
Yale University School of Medicine
SUMMARY
Heterozygous lurcher (+/Lc) mutant mice lose 100% of their Purkinje cells (PCs), 90%
of their granule cells, and 75 % of their inferior olivary neurons. In order to determine the
primary site of Lc gene action, lurcher <-» wild-type aggregation chimaeras were produced.
The cerebella of the three chimaeras examined were intermediate or normal in size compared
to + /Lc and wild-type cerebella. The PCs were reduced in number. Using the /?-glucuronid&se
locus (Gus) as a cell marker, all of the PCs present were identified as having descended from
the wild-type embryo. It appears that all of the + /Lc PCs degenerated. Hence, the Lc gene
acts directly on PCs to cause their degeneration.
The inferior olivary nuclei of the chimaeras seemed to have fewer neurons than wild-type
but more than + /Lc animals. As revealed by /?-glucuronidase histochemistry, both + / +
and + [Lc cells were present, and the ratio of genotypes was similar to the ratio seen in other
regions of the brain. The evidence suggests that the death of olivary neurons in lurcher is
secondary to another defect, probably the loss of PCs. /?-glucuronidase is not an accutate
cell marker for granule cells, and so no conclusion concerning the action of the Lc gene on
granule cells could be made with these chimaeras.
INTRODUCTION
For the past twenty years, neurological mutant mice have added major insights to our understanding of the mammalian nervous system. During development of the nervous system, numerous events occur simultaneously, and genetic
mutations are a useful tool for analysing these events individually. The importance of cell-cell interactions and the specificity of synaptic connections have
been revealed through the use of these mutant mice (see reviews by Sidman,
1972 and Rakic, 1976). The heterozygous lurcher (gene symbol: Lc) is one such
mutant. Affected animals possess a rocking gait and impaired balance (Phillips,
1960). These behavioural abnormalities are associated with the loss of certain
neurons in the central nervous system. Virtually all of the cerebellar Purkinje
cells (PCs) degenerate between the second and twelfth weeks of postnatal life
1
Author's address: Department of Human Genetics, Yale University School of Medicine,
333, Cedar Street, New Haven, CT 06510. U.S.A.
88
R. WETTS AND K. HERRUP
(Caddy & Biscoe, 1979). The major afferents to the PCs are also affected. The
number of granule cells in the cerebellar cortex is decreased by 90 % (Swisher &
Wilson, 1977). In the ventral brain stem, 75 % of the neurons of the inferior
olivary nucleus degenerate by the twelfth postnatal week (Caddy & Biscoe,
1979). The role of the Lc gene in this pathology remains elusive. All genes are
physically present in all cells, but only a specific subset of genes are expressed
in any given cell type. The cell or cells in which the mutant gene is expressed is
referred to as the primary site(s) of gene action (Mullen, 1977 b). These cells may
or may not be morphologically affected by the intrinsic action of the mutation.
Other cells, which possess the mutant gene but do not express it, can be affected
indirectly as a consequence of a critical interaction with those cells which are
the primary site of gene action. Although in lurcher we know that the PCs,
granule cells, and olivary neurons degenerate, this does not reveal which, if any,
of these cells is the primary site of gene action. For example, the Lc gene could
act intrinsically within all three cell types to cause their death. It is also possible,
however, that these neurons die as a secondary consequence of the Lc gene
being expressed in some other cell type on which they all depend. A third
hypothesis is that the Lc gene acts intrinsically within only one cell type, the
PC, whereas the granule and olivary neurons are affected indirectly by the gene.
This explanation was suggested by Caddy & Biscoe (1979) on the basis of their
morphological observations of the mutant. In the present study, we have
examined this hypothesis by producing lurcher *-+ wild-type chimaeric mice.
Chimaeras are formed by aggregation of pairs of embryos to produce a single
mouse. If one of the embryos is genotypically lurcher and the other wild-type,
then mutant and normal cells contribute to and interact during the formation
of a single organism. The nervous system of a chimaera is a fine-grained mosaic
of cells each of which is descended from one of the original embryos. If the
Lc gene acts intrinsically on a given cell type, then all and only those cells with
the mutant gene will have the mutant phenotype. To make this correlation, we
must be able to determine a cell's genotype (mutant or wild-type) independently
from its phenotype (i.e. whether the cell is present or absent). This is done by
making chimaeras from two embryos each of which is homozygous for a different
allele atthe/?-glucuronidase locus. This difference can be detected histochemically,
and, thus, can be used to identify the genotype of each cell (Condamine, Custer
& Mintz, 1971; Mullen & Herrup, 1979). The accuracy of using ^-glucuronidase
activity as an independent cell marker has been amply demonstrated. The
staining pattern of PCs using /#-glucuronidase (Mullen, 1977 a) is similar to the
pattern seen using either differences in /?-galactosidase activity (Dewey, Gervais
& Mintz, 1976) or antigenic differences between isozymes of glucosephosphate
isomerase (Oster-Granite & Gearhart, 1981). In addition, /?-glucuronidase
activity correlated exactly with the PC phenotype in Purkinje cell degeneration
(Mullen, 1977a) and staggerer chimaeras (Herrup & Mullen, 1979). Using
/ft-glucuronidase histochemistry, we report here that the degeneration of the
Site of Lc gene action using chimaeric mice
89
PCs in lurcher is an intrinsic defect while the loss of inferior olivary neurons is
an extrinsic event.
MATERIALS AND METHODS
Chimaeras were produced by a modification (Mullen & Whitten, 1971) of the
8-cell embryo aggregation method of Mintz (1962, 1965) and Tarkowski (1961).
Heterozygous lurcher mice were obtained from the Children's Hospital Medical
Center (Boston) where they had been outcrossed. As a result some of these
animals carried the agouti gene (gene symbol: A). Males from this colony were
mated to C57BL/6J females. The embryos obtained from this mating were
homozygous for the high-activity, Gusb, allele at the /?-glucuronidase locus, wildtype at the retinal degeneration (gene symbol: rd) locus, and were either + / +
or + /Lc. Inbred C3H/HeJ mice were obtained from the Jackson Laboratories
(Bar Harbor). These embryos were A /A, rd/rd, homozygous for the lowactivity, Gush, allele of /?-glucuronidase, and wild-type at the Lc locus. Pairs of
these embryos were cultured together overnight and then transplanted into the
uteri of pseudopregnant females. The chimaeras finished development and were
born normal in size.
All animals were prepared for histology as described (Mullen, \911a). Briefly
they were perfused transcardially with cold 4 % paraformaldehyde in phosphate
buffer, embedded in polyester wax, and serially sectioned in the sagittal plane.
Certain sections were stained for /?-glucuronidase activity by the method of
Hayashi, Nakajima & Fishman (1964) with the modifications of Feder (1976).
This procedure stains Gusb/Gusb cells with a red chromophore which appears
black in the figures. The amount of background staining can vary depending on
the time of incubation, the quality of the substrate, etc. Unstained Gush/Gush cells
appear green due to the methyl green counterstain. Although these cells are
easy to identify in colour, they contrast poorly with the background in the
black-and-white photographs.
RESULTS
After reaching maturity, the chimaeras were each mated to a C57BL/6J
mouse. Four chimaeras (designated #11, xl, ^13, and ^12) produced progeny
which exhibited the lurcher behavioural phenotype. This confirmed that the
embryos from the + /Lc x + / + mating carried the Lc gene. Hence, the geflotype of three of the four mice was + / L c a/a +Gusb/ + Gusb *-»+/+ A/A rd
Gush/rd Gush. The genotype of the fourth chimaera is slightly different, as
explained below. Despite the presence of the Lc gene, all of the chimaefas
behaved normally. The age of the chimaeras at the time of sacrifice is presented
in Table 1. Note that all of the chimaeras were sacrificed after the twelfth postnatal week, which is when the neuronal degeneration is complete in +/Lc
animals (Caddy & Biscoe, 1979). Yet the chimaeras never displayed any behavioural abnormalities. One chimaera (#12) is now 16 months old and has not
90
R WETTS AND K. HERRUP
Table 1. Percentage of wild-type cells in the chimaeras
X13
Sex
M
Age at sacrifice (postnatal
P87
days)
Coat
100%*
0%
Liver hepatocytes
Posterior choroid plexus and
10%
brain stem nuclei
10%
Purkinje Cells
22%
Inferior olivary nucleus
* Both embryos carried an
M
P261
F
P186
F
—
80%
30%
30%
98%
80%
50%
60%
—
—
60%
61%
—
—
30%
18%
agouti gene.
yet been sacrificed. We plan to sacrifice this animal at an advanced age in order
to study long-term changes.
Three chimaeras displayed coat mosaicism, and the percentages are given in
Table 1. The fourth (%11) was 100% agouti and was not noticeably ataxic, yet
he produced agouti, + /Lc offspring when mated to a C57BL/6J female. This
animal was one of our first chimaeras, and he was produced before the A gene
was eliminated from our lurcher colony. His genotype, therefore, differs from
the other chimaeras in that he is A/a instead of a/a in the + /Lc component.
Thus both cell genotypes carried the A gene, and so there was no mosaicism in
the coat of x l l . In addition to coat mosaicism, the percentage of chimaerism
was also estimated (to the nearest 10%) by a visual examination of the retina
and the liver. The photoreceptor degeneration seen in rd/rd mice is expressed
in chimaeric mice and is probably due to the intrinsic action of the rd gene
(Wegmann, Lavail & Sidman, 1971). Little or no degeneration was observed in
the retina of #11 suggesting the retina was largely + / + at the rd locus hence
+ /Lc. Liver sections were stained for /?-glucuronidase activity, but no Gush/Gush
cells were seen in the %11 liver. Approximately 80 % and 30 % of the hepatocytes
of #13 and ;\7, respectively, were Gush/Gush (Table 1) and, hence, were descended
from the C3H/HeJ embryo. Despite the lack of mosaicism in the rest of the
body of #11, wild-type cells were present in the central nervous system. Approximately 10 % of the cells of the posterior choroid plexus and various brainstem
nuclei were Gush/Gush. The percentages for the other chimaeras are given in
Table 1.
Upon dissection of the brains, the cerebella of the chimaeras were examined
and found to be intermediate or normal in size compared to + /Lc and wildtype cerebella. This is illustrated by the sagittal sections in Fig. 1. The cortices
of the chimaeric cerebella were trilaminar structures as were the + /Lc and wildtype cerebella. However, the granule cell layers and the molecular layers of #11
and xi w e r e intermediate in cross-sectional area. In ,^13, these layers had
approximately the same area as the wild type. It is readily apparent from the
Site of Lc gene action using chimaeric mice .
B
•'•*5: \
i*«;
•v'7
r.i>
Fig. 1. Cresyl-violet stained, sagittal sections of lurcher, chimaeric and wild-type
cerebella. The cerebella of xl1 (Q and ^7 (D) are intermediate in size between the
wild-type (A) and lurcher (B) cerebella. The cerebellum of ^13 (E) is similar in size
to the wild-type (A). Scale bar = 500 /*m.
R. WETTS AND K. HERRUP
Fig. 2. The cerebellar cortices of four chimaeric mice. The sections were stained for
ytf-glucuronidase activity; the red reaction product appears black in these photographs. The Gush/Gush cells are not stained by the /?-glucuronidase reaction but are
stained light green by the methyl green counter stain. These cells are hard to distinguish from the background staining. In the control chimaera which does not
possess the Lc gene (A), Gush/Gush PCs (arrows) are interspersed with the stained
Gusb/Gusb PCs. In the lurcher chimaeras (#11, B; #7, C; #13, D), the unstained
Gush/Gush +/+ PCs (arrows) are separated by gaps; no Gusb/Gusb + Lc PCs are
present. Scale bar = 50 ftm.
Site of Lc gene action using chimaeric mice
93
reduced cerebellar size that the presence of + /Lc cells affects cerebellar development in the chimaeras.
The qualitative appearance of the PC layers was similar in all three chimaeras,
and the PCs which were present appeared morphologically normal. However,
there were large gaps in the PC layer, indicating a decreased number of PCs as
compared to wild type. ^11 possessed very few PCs, while ^13 had the greatest
number of PCs (approximately 60% as many as wild type). For each chimaera,
the number of PCs corresponded approximately with the percentage of Gush/
Gush cells in the rest of the central nervous system (Table 1). All of the PCs
present were unstained by the /?-glucuronidase reaction (Fig. 2). Hence, they
were all Gush/Gush cells, descended from the wild-type embryo. Since the number
of PCs was reduced in all chimaeras and since there were no Gusb/Gusb PCs
observed in any of the chimaeras, we conclude that all + /Lc PCs degenerated.
Thus, the Lc gene acts intrinsically on the PCs to cause their degeneration.
The inferior olivary nuclei of the chimaeras are shown in Fig. 3. The nuclei
of x l l and ^13 appeared to be approximately normal in volume, although the
density of cells was lower than in wild type. The nucleus of xl was the most
severely affected compared to the other chimaeras. The cross-sectional area
appeared somewhat smaller than wild type, and the density of cells was much
lower than in the other two chimaeras. The decreased density of neurons suggests
that there may have been some loss of olivary neurons in the chimaeras, but
the amount of loss did not seem to correlate with the percentage of + /Lc
neurons in the rest of the central nervous system. It is important to point out,
however, that considering both density and volume, the number of neurons was
clearly greater in all of the chimaeras than in the + /Lc animal (Fig. 3A). When
stained for /?-glucuronidase activity, both Gusb/Gusb and Gush/Gush cells were
seen (Fig. 4). If the degeneration of the +/Lc olivary neurons was intrinsic, one
would expect to see an increased proportion of + / + Gush/Gush cells. For
example, if initially 90 % of the olivary neurons in %11 were + /Lc, then after
75 % of these cells degenerate, the percentage of + / + Gusn/Gush cells would
have increased from 10 to 31 %. Preliminary counts1 indicated that the actual
percentage was 22%. Similarly, assuming again that the initial percentage of
wild type olivary neurons was the same as the observed percentage of wild type
PCs, one would expect 63 and 86% of the olivary neurons in x7 and #13,
respectively, to have been + / + Gush/Gush. In fact, 18 and 6 1 % of the cells
were observed to be + / + Gush/Gush. For each of the three chimaeras, the
actual proportion of wild-type cells was lower than expected if the degeneration
1
At least 150 olivary neurons were counted for each chimaera. In control Gusb/Gusb
sections, the staining of the olivary neurons ranged from heavy to light, but olivary neurons
of Gush/Gush animals were always unstained. In the chimaeras, the genotypes of $ome
neurons were ambiguous due to intercellular enzyme transfer, variability in background
staining, etc. The number of ambiguous neurons was less than 5 % of our counts, and so these
factors did not prevent us from accurately determining the genotypes of the vast majority of
the olivary neurons.
4
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94
R. WETTS AND K. HERRUP
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Site of Lc gene action using chimaeric mice
95
of + /Lc olivary neurons were intrinsic. Further, the ratio of + /Lc to + / +
olivary neurons was similar to the ratio seen in the rest of the central nervous
system (Table 1, lines 5-7). This suggests that if there was any cell loss, both
+ /Lc and + / + cells were equally affected. Furthermore, a substantial majority
of the olivary neurons in %11 were Gusb/Gusb, hence + /Lc in genotype. Since
the number of olivary neurons was much greater in the chimaera than in the
+ /Lc animal, many +/Lc neurons survived in ^11 which would not have
survived in a + /Lc animal. Therefore, we conclude that the death of the
inferior olivary neurons in the + /Lc mouse is secondary to some other defect.
DISCUSSION
Our results indicate that the Lc gene acts intrinsically on the PCs to cause
their degeneration. In chimaeras which do not possess the Lc gene, both
Gush/Gush and Gusb/Gusb PCs are present (Fig. 2A). In contrast, the + /Lc
Gusb/Gusb PCs of the lurcher chimaeras were totally absent. None was rescued
by the presence of up to 60% of the wild-type number of PCs. The degeneration
of PCs was observed in the chimaeras to correlate with the presence of the Lc
gene. Thus, the PC is a primary site of Lc gene action. We cannot be certain
whether any of the PCs which did not possess the Lc gene (the Gush/Gush cells)
also degenerated. The number present corresponded roughly with the proportion of wild-type cells in the rest of the central nervous system, however,
and this strongly suggests that there was no degeneration of wild-type PCs. The
intrinsic action of the Lc mutation on the PCs is analogous to the conclusion
of Mullen (1977a), who studied chimaeras of another PC degeneration mutant,
pcd.
Gush/Gush«-» Gusb/Gusb chimaeras display mosaicism in the inferior olivary
nucleus (Figure 4A). The presence of the Lc gene does not alter this mosaicism.
If the degeneration of olivary neurons was intrinsic to + /Lc cells, then these Cells
would have also degenerated in the chimaera and the ratio of + / + to + /Lc
cells would have been greatly increased. This was not observed. Hence, the
results suggest that the degeneration of olivary neurons is secondary to some
other defect. The loss of the PCs may be responsible, since they are the major
post-synaptic target of the olivary neurons. Since the chimaeras possessed more
olivary neurons than did + /Lc animals (Fig. 3), the question arises how many
olivary neurons are rescued by a given number of PCs? This issue of plasticity
Fig. 3. Cresyl-violet stained, sagittal sections of inferior olivary nuclei. The nuclei
are outlined by arrowheads. The inferior olive of the lurcher (A) is greatly reduced in
cross-sectional area, while the inferior olives of the chimaeras (#11, B; #7, C;
#13, D) are similar in size to the wild type (E). However, note that the cell density
in the chimaeras is somewhat reduced compared to wild type. Anterior is to the
right in A, D, and E and to the left in B and C. Scale bar = 200 fim.
4-*
96
R. WETTS AND K. H E R R U P
Fig. 4. Sections of the inferior olivary nuclei of four chimaeras stained for /?glucuronidase activity. As in Fig. 2 the arrows point to the unstained Gush/Gush
+ / + neurons. Both stained (Gusb/Gusb +/Lc) and unstained (Gush/Gush +/ + )
neurons are present in the lurcher chimaeras (xll, B; ^7, C; #13, D) just as in
the control chimaera (A) which does not possess the Lc gene. Scale bar = 25 fim.
Site of Lc gene action using chimaeric mice
97
is now being investigated by counting the number of PCs and olivary neurons
in each of the chimaeras.
/?-Glucuronidase is a cytoplasmic enzyme, and granule cells have very little
cytoplasm, so it cannot be used to accurately identify the genotype of the granule
cells (Mullen & Herrup, 1979). Since the identification of the PC as a primary
site of gene action does not rule out the possibility that the Lc gene also acts
intrinsically within other cell types, no conclusion concerning the granule cells
can be drawn at this time. That the granule cell death is possibly an extrinsic
phenomenon can be inferred from the findings in another cerebellar mutant,
staggerer. In this animal the death of the granule cells is believed to be due to
the lack of spines on the PC dendrites, which prevents the developing granule
cells from synapsing with their major post-synaptic target (Sotelo & Changeux,
1974; Landis & Sidman, 1978). In lurcher, the granule cells may not be able to
form synapses with the PCs because the latter begin to degenerate during the
period of synaptogenesis. So although the extrinsic nature of the granule cell
death has not yet been demonstrated in lurcher, a similar mechanism may be
involved.
The survival of neurons in many systems has been shown to depend on the
presence of a target (Cowan, 1973; Purves & Lichtman, 1980). Which neurons
survive may be determined by the stabilization of their synapses with the target
organ (Changeux & Danchin, 1976). In a similar way, PCs seem to be impottant
to the normal development of the granule cells and the inferior olivary neurons.
In contrast, the PCs may be less important to the development of their postsynaptic target, since the number of neurons in the deep cerebellar nuclei are
unaffected in lurcher (Caddy & Briscoe, 1979).
We would like to thank Mr Thomas Diglio and Ms Sandra Wilczynski for their technical
assistance. This work was supported by a Basil O'Connor Starter Grant from the March of
Dimes and by grant HD 12213 from the NIH.
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(Received 5 June 1981, revised 23 October 1981)