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letters to nature
NATURE
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VOL 390
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Received 5 June; accepted 18 August 1997.
1. Lebacq-Verheyden, A.-M., Trepel, J., Sausville, E. A. & Battey, J. in Handbook of Experimental
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Acknowledgements. Wethank J.-I. Miyazaki for ES cells and training regarding ES cells; A. F. Parlowfor
mouse growth hormone and polyclonal monkey anti-rat GH serum; K. Wakabayashi for goat anti-
monkey IgG serum; H. Ohno for rat UCP1 probe; T. Nishikawa for providing Animex Auto; N. M. Le
Douarin, K. Mikoshiba, R. S. Petralia and J. Smith for critical reading of the manuscript. This work was
supported in part by research grants from the Ministry of Education, Science, Sports and Culture, the
Ministry of Health and Welfare, the Science and Technology Agency of Japan, the Japan Health Science
Foundation.
Correspondence and requests for materials should be addressed to H.O.-H. (e-mail: hamazaki@prit.go.jp)
or K.W. (e-mail: wada@ncnaxp.ncnp.go.jp).
Math1 is essential for genesis
of cerebellar granule neurons
Nissim Ben-Arie*†, Hugo J. Bellen*‡k, Dawna L. Armstrong§,
Alanna E. McCall†, Polina R. Gordadze†, Qiuxia Guo§,
Martin M. Matzuk*‡§& Huda Y. Zoghbi*†k
Departments of *Molecular and Human Genetics, †Pediatrics, ‡Cell Biology,
§Pathology, and kHoward Hughes Medical Institute, Baylor College of Medicine,
Houston, Texas 77030, USA
.........................................................................................................................
The cerebellum is essential for fine motor control of movement
and posture, and its dysfunction disrupts balance and impairs
control of speech, limb and eye movements. The developing
cerebellum consists mainly of three types of neuronal cells:
granule cells in the external germinal layer, Purkinje cells, and
neurons of the deep nuclei1. The molecular mechanisms that
underlie the specific determination and the differentiation of
each of these neuronal subtypes are unknown. Math1 (refs 2, 3),
the mouse homologue of the Drosophila gene atonal 4, encodes a
basic helix– loop–helix transcription factor that is specifically
expressed in the precursors of the external germinal layer and
their derivatives. Here we report that mice lacking Math1 fail to
form granule cells and are born with a cerebellum that is devoid of
an external germinal layer. To our knowledge, Math1 is the first
gene to be shown to be required in vivo for the genesis of granule
cells, and hence the predominant neuronal population in the
cerebellum.
The mammalian cerebellum consists of deep centrally located
neurons, referred to as the deep nuclei, and a peripheral cortex. The
cortex contains two principal neuronal subtypes, the Purkinje cells
and the cells of the internal granular layer (IGL). The IGL neurons,
which eventually constitute most of the neurons in the cerebellum,
are derived from peripherally located cells in the external germinal
layer (EGL) which migrate postnatally along radial glia1,5. The
mouse cerebellar anlage is specified at embryonic-day 9 (E9) just
anterior to the area where closure of the neural tube is incomplete.
The neuroepithelium of the ventricular zone in the metencephalon
gives rise to the precursors of the neurons of the deep nuclei and
Purkinje cells6. The precursors of the EGL are derived at E13–E15
from a structure named the rhombic lip (also known as the germinal
trigone), which is located at the posterior edge of the cerebellar
anlage1,5,7 (Fig. 1). The proliferating cells of the rhombic lip, which
are committed to become EGL neurons, disperse rostrally over the
surface of the cerebellar anlage, where they establish the EGL.
Proliferation of these granule cell precursors continues in the EGL
until postnatal day 15 (P15). The EGL cells migrate inwardly to give
rise to the IGL, starting at birth until day P20, when maturation of
the cerebellum is complete1,5,7.
The Drosophila atonal gene is essential for the generation of the
chordotonal organs, the proprioreceptors, and the R8 photorecep-
tor precursors in the eye4,8. Adult flies that lack atonal function do
not have photoreceptors, are uncoordinated and tend not to fly8.
Expression of Math1, the mouse atonal homologue, is highly
restricted to a few neurons in the hindbrain and dorsal neural
tube2,3.Math1 is expressed in the precursors of the EGL in the
rhombic lip starting at E13, and later in the EGL of the developing
cerebellum (Fig. 1). Math1 expression ceases during the inward
migration phase of the granule cells to the IGL. To investigate the
role of Math1 in cerebellar development, a targeted deletion of the
Math1 gene (Math1
m1
) was generated by using embryonic stem (ES)
cell technology (Fig. 2a). Heterozygous mice were intercrossed to
obtain mice homozygous for the Math1 deletion (Fig. 2b). Absence
of a hybridization signal with DNA fromhomozygous mutant mice,
when probed with an internal probe spanning the basic-helix–
IO
IV
URL/GT
MC
MO
IST
Cb
NE
EGL
LRL
Cb
EGL
NE
IV
SC
PO
URL/GT
CP
AQ
IC
CP
ab
Figure 1 The developing cerebellum at E16.5. The sagittal (a) and coronal (b)
views show the localization of the cerebellum (Cb) between the inferior colliculus
(IC), pons (PO), mesencephalon (MC) and medulla oblongata (MO). The germinal
matrix, which gives rise to EGL precursors, is the rhombic lip, with an upper and a
lower component (URL, LRL, respectively). The proliferative zone at the URL, the
germinal trigone (GT), has three prongs: EGL, neuroepithelium (NE), and choroid
plexus (CP). Granule cell precursors originate at the URL/GTand migrate (arrow)
to form the EGL. The LRL will contribute to the medullar precerebellar nuclei,
including the inferior olive (IO). Math1 is expressed in granule precursors in GT
and in emerging EGL (shaded area). AQ, aqueduct of Sylvius; IST, isthmus; IV,
fourth ventricle; SC, superior colliculus. Adapted from ref. 1.
Nature © Macmillan Publishers Ltd 1997
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170 NATURE
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loop– helix (bHLH) domain, confirmed that the mutation is a null
allele (data not shown).
Mice heterozygous for the Math1 deletion are viable, fertile and
appear normal. Genotyping of newborn mice from heterozygous
intercrosses demonstrated that the targeted allele did not result in
embryonic lethality. The observed ratio of genotypes fitted that
expected for a fully penetrant, mendelian recessive inheritance
pattern, with 28 (25.2%) wild types, 55 heterozygote (49.5%) and
28 (25.2%) homozygote (null) mice. Math1-null mice are born
alive, but fail to breathe, become cyanotic, and die a few minutes
after birth. We observed no morphological abnormalities in the
lung (data not shown). Furthermore, the lack of expression of
Math1 in the lungs, combined with the expression pattern in the
brainstem2, suggest a central mechanism for this respiratory failure.
The structure and histology of the cerebellum of Math1-null mice
is abnormal compared to their littermates. The most peripheral cell
layer in the cerebellum, the EGL, is normally about eight cells thick
at E18.5. These rapidly proliferating cells are the granule cell
precursors and express Math1 abundantly2.Math1-null mice com-
pletely lack the EGL, as shown by haematoxylin-and-eosin staining
(Fig. 3a, b), and cresyl violet staining (data not shown), and have a
cerebellum that is reduced in size. To analyse the changes in the
cytoarchitecture of the cerebellum, we stained the two principal
neuronal populations in the cerebellum, the Purkinje cells (Fig. 3c,
d) and the granule cells (Fig. 3e, f ). RNA in situ hybridization using
RU49, a zinc-finger transcription factor that labels the EGL, the
migrating granule cells and the IGL9, demonstrates the absence of
the EGL and migrating granule cells in the cerebella of Math1-null
mice (Fig. 3e, f ). In contrast to the lack of granule neurons, the
Purkinje cells and the neurons of the deep nuclei are still present in
mutant mice, as demonstrated by staining with anti-calbindin
antibody (Fig. 3c, d) and Nissl staining (data not shown). However,
because of the absence of the EGL, the Purkinje cells, which are
normally located beneath the EGL, are now localized at the
periphery of the cerebellum and do not form a distinct and
organized layer. In addition, a significant subpopulation of Purkinje
cells fails to migrate from the central area of the cerebellum into the
periphery (Fig. 3c, d). Staining with glial fibrillary acidic protein10
(GFAP) and nestin11, which both label glial cells, demonstrates that
there is not excessive gliosis, and that the radial glia are localized
properly (data not shown). These results show that Math1 is
essential for the formation of the EGL but not the Purkinje cells,
the neurons of the deep nuclei, or the glial cells. In Math1-null mice,
the absence of the EGL also leads to the lack of foliation of the
cerebellum typically observed in normal embryos starting from E18
(Fig. 3a, b).
Possible causes for the missing EGL in mutant mice include
failure in fate determination of granule precursors at the rhombic
lip, aberrant migration of precursors to form the EGL, or degenera-
tion of the neurons already localized at the EGL. The granule neuron
precursors originate at E13–E15 in the proliferation zone of the
rhombic lip1,7. At E14, the EGL precursors initiate migration from
the rhombic lip over the surface of the cerebellar primordia. At this
stage, Math1 is expressed in the granule precursors localized at the
rhombic lip and in the emerging EGL2. As shown in Fig. 4a– d,
Math1-deficient mice contain fewer cells in the rhombic lip than do
control embryos. In addition, there is no migration of cells out of
the rhombic lip to form the EGL. Bromodeoxyuridine (BrdU)
labelling demonstrates the absence of proliferating cells in both
sites that normally expand the pool of granule cells: the rhombic lip
and the cells that have already migrated to the EGL (Fig. 4e, f ).
Hence, the lack of the cerebellar EGL in Math1-null mice is evident
early on and is caused either by the absence of the granule cell
precursors or by the inability of these cells to proliferate at the
rhombic lip. Thus, the agenesis of granule cell precursors in Math1-
null mice is a much earlier event than the postnatal apoptotic
degeneration of the EGL seen in weaver mice12,13.
8.5 kb (WT)
6.4 kb (mutant)
Math1
locus
AXRI RV RIHA H AA
X ARVRI
B
PGKhprt
HRI
BN N
1 kb
X ARVRIHA
Targeting
vector
Targeted
Math1
locus
3' probe5' probe B
PGKhprt
HRI
RI
MC1-TK
a
b
Figure 2 Targeted disruption of the Math1 locus and generation of Math1-null
mice. a, Homologous recombination between the 3.7-kb and 3.1-kb fragments in
the targeting vector and the genomic locus, resulted in the replacement of 3.2 kb
(black box) which include the entire coding region (hatched box).b, Tail DNA from
newborn littermates obtained by heterozygous intercrosses was digested with
EcoRI and HindIII and hybridized with the 39diagnostic probe. The three pups
homozygous for the deletion died a few minutes after delivery, whereas the four
wild-type (WT) and five mice heterozygous for the deletion were viable.
Figure 3 Cerebellar abnormalities in E18.5 Math1-null embryos. a,b, Midsagittal
sections through the cerebellum stained with haematoxylin and eosin. The
cerebellum in wild-type (a) mice is well developed and foliated (asterisks), but is
smooth in Math1 null mice (b). The external germinal layer (EGL) is absent and the
Purkinjecell layer (PCL)less organized,with somePC locateddeep in thecerebellum,
as revealedby anti-calbindin staining (c,d). Parasagittalsections ofthe cerebellum of
wild-type (e) and mutant (f) embryos were hybridized with the granule-cell marker
RU49. Wild-type EGL is strongly positive for RU49, whereas the cerebellum of
Math1-null mice is negative. Original magnifications: a–d,×100; e,f,×40.
Nature © Macmillan Publishers Ltd 1997
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It has been suggested that inferior olive neurons are derived
from the rhombic lip14, which consists of an upper and a lower
component (Fig. 1) and that it is the lower lip that gives rise to the
neurons of the inferior olive1.Math1 is only expressed in the
germinal trigone that is part of the upper lip and not the lower
lip2(Fig. 1). The inferior olive neurons are present in Math1-null
mice and appear normal (Fig. 5), consistent with Math1 expression
in the upper rhombic lip. This finding supports the histological and
functional division of the rhombic lip into an upper and a lower lip,
each contributing to the generation of different types of neurons
that migrate to separate destinations.
As Math1-null mice die shortly after birth from respiratory
failure and Math1 is expressed in the hindbrain, we wanted to
establish whether there is selective loss of neuronsimplicated in the
control of respiration, including those of the nucleus tractus
solitarius, nucleus ambiguus, dorsal vagus nucleus and trigeminal
ganglion. These brainstem nuclei were identified and analyzed for
neuronal density and morphology. No differences between Math1-
null mice and littermate controls were noted (Fig. 5). The lack of
morphological defects suggests that Math1 has different roles in the
brainstem and cerebellum, or that Math1 is required in other
unidentified neuronal cells involved in respiratory control.
We have shown that the Math1 gene is required for generation of
the EGL cells and their derivatives. Although ,30 mutations in
mice cause cerebellar defects and ataxia15 –17, none of these muta-
tions causes a phenotype like that of Math1-null mice. For example,
the developmental mutant reeler causes a defect in cell migration in
the cerebellum, cerebral cortex and hippocampus, rather than a
defect in cell specification or proliferation18. In the cerebellum of
weaver mutant mice, proliferation of EGL precursor cells is normal,
but failure of granule cell migration from the EGL to the IGL causes
degeneration of this neuronal population19.
The cerebellum is derived from the posterior end of the mesen-
cephalon and from the metencephalon. The expression of wnt-1 at
the mesencephalon–metencephalon junction stabilizes the expres-
sion of the engrailed homologues En-1 and En-2, thereby specifying
the cerebellar anlage20. Absence of Wnt-1 or of En-1 and En-2 causes
a loss of cerebellar components, including the deep nuclei, Purkinje
cells and IGL21–26. In the absence of Math1, the cerebellum develops
but there is selective loss of only the EGL precursors and granule
neurons. Other defects, like the ectopic localization of the Purkinje
cells and the lack of foliation are most likely to be secondary to the
absence of EGL. Mice lacking Mash1, the murine homologue of
the Drosophila proneural gene complex achaete-scute, suffer from
loss of olfactory neuronal progenitors and are arrested in the
differentiation of sympathetic neuronal precursors27. Therefore,
Mash1 and Math1 are both essential for the generation of neurons
through progression of a differentiation programme, possibly in
selected progenitors. On the basis of our results, we propose that
Math1 plays a role in cerebellar granule fate specification or
proliferation. This role is functionally similar, although not iden-
tical, to the role of its homologue, the Drosophila atonal gene4,8. In
both mice and fruitflies, loss of function leads to the absence of
specific neuronal cell types early in neurogenesis. However, Math1 is
expressed in cells that are already committed to be neural tissue,
whereas atonal plays a role in the decision of epidermal rather than
neuronal cell fate. M
.........................................................................................................................
Methods
Targeted deletion of Math1.The genomic locus was targeted by homologous
recombination using two isogenic flanking fragments of 3.7 kb (EcoRI–EcoRV)
and 3.1 kb (XbaI–ApaI), which were isolated from a mouse 129/SvEv genomic
DNA library (Stratagene). A human hypoxanthine–guanine phosphoribosyl-
transferase (hprt) and the herpes simplex virus thymidine kinase gene were
used as positive and negative selection markers, respectively. The linearized
targeting construct was electroporated into hprt-negative AB2.1 embryonic
stem (ES) cells and selection was achieved using HAT (hypoxanthine,
Figure 4 Absence of the EGL in E14.5 Math1 null mice due to lack of proliferation
of granule precursors. a–d, Generation of the EGL by cells migrating from the
rhombic lip (haematoxylin and eosin stain). In coronal sections from wild-type
brain (a,c), the EGL is identified as cells migrating out from the rhombic lip (RL)
over the cerebellar primordium (CbP). In Math1 null mice (b,d), the rhombic lip is
thinner and lacks EGL precursors migrating rostrally. e,f, The absence of the EGL
precursors in Math1 null mice is supported by the lack of proliferating cells in the
rhombic lip of these mice, as demonstrated by BrdU staining. Proliferating cells
are seen at the rhombic lip and EGL of wild-type mice (e), but not in Math1 null
mice (f). Original magnifications: a,b,×40; c,d,×200; e,f,×100.
Figure 5 Inferior olive nuclei are normal in Math1 null mice. Coronal sections from
brains of Math1 heterozygous (a,c) and null mice (b,d) at E18.5 were stained with
haematoxylin and eosin. The inferior olive (IO) nuclei in the medulla oblongata
(MO), which arise from the lower rhombic lip (LRL) appear normal in both animals
(a– d). Arrows indicate the nucleus tractus solitarius (NS) and nucleus ambiguus
(NA), which appear normal in both animals (a,b). This is in contrast to the lack of
the external germinal layer (EGL), and the smaller upper rhombic lip (URL) seen in
Math1 null mice (b). Original magnifications: a,b,×40; c,d,×100.
Nature © Macmillan Publishers Ltd 1997
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aminopterin and thymidine) and FIAU (1-(29-deoxy-29-fluoro-b-D-arabino-
furanosyl)-5-iodouracil). The targeted alleles can be identified by the presence
of a 14.6-kb fragment instead of 10 kb, when DNA is digested with ApaI and
probed with a 59diagnostic probe, and by a fragment of 6.4 kb instead of 8.5 kb,
when digested with EcoRI and HindIII and probed with an external 39probe.
Homologous recombination was detected in 74% (130/175) of the ES clones
screened. Two independent ES cell lines heterozygous for the targeted Math1
allele (AT37-G6 and AT37-G12) were injected into blastocysts and gave rise to
chimaeric mice, which transmitted the deletion through the germline.
Histology, immunohistochemistry and in situ hybridization. Pregnant
females were killed and embryos removed quickly into cold PBS. Extra-
embryonic membranes were discarded, and the yolk sac was collected for
DNA extraction. Embryos or newborn mice were fixed overnight in either
Bouin’s fixativeor 4% paraformaldehyde in PBS at 4 8C, dehydrated and stored
at −208C. Embryoswere embedded inparaffin wax, andsections of7 mm were cut.
Sections were stained in Mayer’s haematoxylin and eosin or cresyl violet. Isotopic
in situ hybridization withRNA probes labelled with [
35
S]UTPhas been described28.
Non-radioactive in situ hybridization using the digoxigenin system
(Boehringer Mannheim) was done according to ref. 29, but with paraffin
embedding. Sections were dewaxed (Histoclear, National Diagnostics), rehy-
drated through a graded series of ethanol concentrations, and processed
according to the protocol. Templates for cRNA synthesis were as follows:
Math1 (PCR-amplified coding region) and RU49 (0.847-kb EcoRI/PstI frag-
ment, a gift from N. Heintz). For immunohistochemical staining, paraffin
blocks were cut at 5 mm, dewaxed in xylene, antigen-retrieved by microwaving
in citrate buffer, blocked and incubatedwith the appropriate antibodies in PBS
containing 10% normal goat serum for several days at 4 8C. Primary antibodies
were detected using the avidin–biotin complex system (ABC, Vector Lab).
Antibodies used were as follows: anti-calbindin-D (1:1,000; Sigma), anti-GFAP
(1:1,000; Dako) anti-nestin (1:150, a gift from R. McKay).
For evaluation of brainstem cranial nerve nuclei, coronal sections (6mm)
were cut from E18.5 fetuses and alternative sections were stained with
haematoxylin and eosin, cresyl violet (Nissl staining) and one-step trichrome
(Gomori’s staining). Cranial nerve nuclei were identified and scored
independently by two individuals. Criteria for the examination were:
localization of each cranial nerve nucleus, symmetry, shape and size, and
neuronal density.
BrdU staining of proliferating cells. Pregnant females were injected with
BrdU (5-bromodeoxyuridine) (50 mg kg
−1
body weight from a stock solution
of 10 mg ml
−1
in 0.1M Tris-HCl, pH 7.5) and killed two hours later. Embryos
were dissected in cold PBS, fixed in 70% ethanol, dehydrated, embedded in
paraffin wax and cut at 7-mm sections. These were treated with 2M HCl for
30 min at 37 8C to denature the DNA partially, then neutralized in 0.1M sodium
borate, pH 8.5, for 10 min, as recommended by the supplier of the anti-BrdU
antibodies (Novocastra Lab). BrdU-labelled cells were detected using the ABC
system.
Received 19 May; accepted 7 August 1997.
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Acknowledgements. We thank B. A. Antalffy for technical assistance; P. R. Cox andJ. Dong for their help;
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tmc.edu).
Impaired mast cell-dependent
natural immunity in
complementC3-deficientmice
Andrey P. Prodeus*, Xiaoning Zhou*, Marcus Maurer†,
Stephen J. Galli*†& Michael C. Carroll*
*Departments of Pathology, Harvard Medical School and †Beth Israel Deaconess
Medical Center, Boston, Massachusetts, 02115, USA
.........................................................................................................................
The complement system is widely regarded as essential for normal
inflammation, not least because of its ability to activate mast
cells1–5. However, recent studies have called into question the
importance of complement in several examples of mast cell-
dependent inflammatory responses6– 9. To investigate the role of
complement in mast cell-dependent natural immunity, we exam-
ined the responses of complement-deficient mice10,11 to caecal
ligation and puncture12, a model of acute septic peritonitis12,13 that
is dependent on mast cells and tumour necrosis factor-a(TNF-a).
We found that C4- or C3-deficient mice10,11 were much more
sensitive to caecal ligation and puncture than wild-type (WT)
controls (100% versus 20% in 24-h mortality, respectively). C3-
deficient mice also exhibited reductions in peritoneal mast cell
degranulation, production of TNF-a, neutrophil infiltration and
clearance of bacteria. Treating the C3-deficient mice with purified
C3 protein enhanced activation of peritoneal mast cells, TNF-a
production, neutrophil recruitment, opsonophagocytosis of bac-
teria and resistance to caecal ligation and puncture, confirming
that the defects were complement-dependent. These results pro-
vide formal evidence that complement activation is essential for
