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First published online 14 June 2006
doi: 10.1242/dev.02437
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1 San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
2 Department of Cell Biology and Anatomy, Genes and Development Research Group,
and Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary,
Calgary, Alberta T2N 4N1, Canada.
3 Rita Levi Montalcini Center for Brain Repair, Department of Neuroscience,
Section of Physiology, University of Turin, Italy.
4 Department of Pharmacology and Human Physiology, University of Bari School of
Medicine, Italy.
Author for correspondence (e-mail:
g.consalez{at}hsr.it)
Accepted 9 May 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cerebellum, Purkinje neurons, Neuronal migration, Transcription factor, Parasagittal stripes, Zebrin II, Cerebellar map, Olf-1, Early B-cell factor
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
shortly after the birth of deep cerebellar nuclei (DCN) neuron progenitors.
After exiting the cell cycle, PC progenitors migrate along radial glial fibers
away from the VZ and into an intermediate domain known as the cortical
transitory zone (CTZ), while DCN neurons occupy a more dorsal domain termed
the nuclear transitory zone (NTZ). From the CTZ, PC precursors migrate around
NTZ neurons, forming a reproducible array of clusters in the subpial zone.
From as early as E13.5, these clusters can be defined through differential
expression patterns (reviewed by Armstrong
and Hawkes, 2000; Herrup and
Kuemerle, 1997). From E18 onwards, these clusters disperse
rostrocaudally under the influence of reelin/dab1 signaling
(Gallagher et al., 1998;
Heckroth et al., 1989;
Jensen et al., 2004;
Jensen et al., 2002) to form
the PC monolayer of the adult.
The cerebellar cortex is highly compartmentalized: it is first divided into
four transverse zones, and then each zone is further subdivided rostrocaudally
into parasagittal stripes (Ozol et al.,
1999; Sillitoe and Hawkes,
2002). Accordingly, the organization of afferent and efferent
terminal fields is known to match closely the striped distribution of several
molecular markers (Ji and Hawkes,
1994; Ji and Hawkes,
1995; Voogd et al.,
2003; Voogd and Ruigrok,
2004). The most extensively studied marker of parasagittal PC
stripes is Zebrin II (ZII) (Brochu et al.,
1990; Hawkes,
1992), the respiratory enzyme aldolase c
(Ahn et al., 1994;
Hawkes and Herrup, 1995;
Walther et al., 1998).
Alternating stripes of ZII-positive and -negative PCs extend through the
vermis and hemispheres, dividing the cerebellar cortex into a pattern of
parasagittally oriented stripes, which is consistent between individuals and
across species (Sillitoe et al.,
2004).
Our findings implicate a transcription factor, Ebf2COE2,O/E-3,
in cerebellar cortical development. Collier/Olf/Ebf genes (reviewed by
Dubois and Vincent, 2001;
Garel et al., 1997;
Hagman et al., 1993;
Malgaretti et al., 1997;
Wang and Reed, 1993;
Wang et al., 1997), referred
to here as Ebf genes, encode phylogenetically conserved HLH transcription
factors originally characterized for their roles in the immune system
(Hagman et al., 1993), and
subsequently implicated in various aspects of neural development, including
neuronal differentiation (Dubois et al.,
1998; Pozzoli et al.,
2001), migration
(Garcia-Dominguez et al.,
2003; Garel et al.,
2000), and axon fasciculation and guidance
(Garel et al., 1999;
Garel et al., 2002;
Prasad et al., 1998). One
member of this family, Ebf2, is not essential for completion of
embryogenesis, and its mutation leads to a combination of neuroendocrine,
olfactory, and peripheral nerve abnormalities
(Corradi et al., 2003;
Wang et al., 2004). Outside
the nervous system, Ebf2 regulates bone development and homeostasis
non-cell-autonomously, by antagonizing the terminal differentiation of
osteoclasts (Kieslinger et al.,
2005). In the present study, we address the role of Ebf2
in cerebellar cortex formation. Ebf2 regulates (1) the migration and
survival of a specific PC precursor subpopulation during embryonic and
postnatal development; (2) the establishment of a parasagittally striped
molecular pattern in the cerebellar vermis; and (3) the determination of the
ZII-negative phenotype in surviving PCs.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mouse genetics
The targeting construct, described in Corradi et al.
(2003), contains a
lacZ cDNA. lacZ staining distribution in CNS development is
in full agreement with the results of in situ studies
(Garel et al., 1997;
Malgaretti et al., 1997)
(present paper). All experiments were carried out on F1 hybrids
obtained by crossing Ebf2+/- pure-bred FVB/N
(N9) females with Ebf2+/- pure-bred C57BL/6J
males. All studies were conducted using coisogenic control littermates. This
hybrid strain was chosen to obviate the low fertility and poor maternal
behavior of C57BL/6J heterozygous mothers.
Tissue preparation
Postnatal mice were anesthetized with Avertin (Sigma) and perfused with
0.9% NaCl followed by 4% paraformaldehyde (PFA). Embryos were fixed overnight
by immersion with either 4% PFA or Carnoy's solution (6:3:1 solution of 100%
ethanol, chloroform and acetic acid). Tissues fixed with 4% PFA were rinsed
three times in 1x PBS, cryoprotected in 30% sucrose overnight, embedded
in OCT (Bioptica), and stored at -80°C, before sectioning on a cryotome
(20 µm); tissues fixed with Carnoy's solution were placed in 96% ethanol
overnight, dehydrated in an ethanol series, embedded in paraffin. Sections of
5-7 µm were cut using a microtome. lacZ staining was conducted as
described (Corradi et al.,
2003).
Immunohistochemistry and immunofluorescence
Cryosections or paraffin-embedded sections were immunostained with the
following antibodies: rabbit or mouse polyclonal anti-calbindin (1:1000,
Swant); rabbit polyclonal anti-beta-galactosidase (1:700, Abcam); rabbit
antineurogranin (1:1500, Chemicon); rabbit monoclonal anti-active Caspase 3
(1:200, BD Pharmingen); rabbit polyclonal anti-sphingosine kinase 1a (1:500),
a gift of N. Terada (Terada et al.,
2004); mouse monoclonal anti-ZII
(Brochu et al., 1990), used in
spent hybridoma supernatant diluted 1:5000; mouse monoclonal
anti-bromodeoxyuridine (1:100, Becton Dickinson); rabbit polyclonal anti-HSP25
(1:500, Stressgen Biotechnologies); rabbit polyclonal
anti-phosphorylated-histone 3 (1:500, Upstate Biotechnology); rabbit
polyclonal anti-Ki67 (1:800, Novo Castra Laboratories) goat polyclonal
anti-EphA4 (1:300, R&D Systems). For immunohistochemistry, sections were
immunostained as recommended (ABC Elite kit, Vector Laboratories), dehydrated
and mounted in DPX (BDH-Merck). For anti-Ki67 and anti-phosphorylated-histone3
immunohistochemistry, a high-temperature antigen retrieval procedure was
required. For dual immunofluorescence, cryosections were rinsed three times in
1x PBS, preincubated in 15% goat serum, 0.2% triton X-100, 1x PBS
and incubated overnight at 4°C with the two primary antibodies. Sections
were then washed 6x10 min in PBS and incubated for 2 h at room
temperature with the two secondary antibodies (Alexa 546 anti-mouse Ig,
1:1000, and Alexa 488 anti-rabbit Ig, 1:1000). In controls either primary
antibody was replaced with normal serum. Wholemount immunocytochemistry was
performed as described (Sillitoe and
Hawkes, 2002).
In situ hybridization
Digoxygenin-labeled riboprobes were transcribed from plasmids containing
Ebf1, Ebf2, Ebf3, Reelin (A. Bulfone), ROR
(B. A.
Hamilton), p57 (L. Muzio and A. Mallamaci), Semaphorin3A
(J.M. Solowska) and Protocadherin10 (S. Hirano) c-DNAs. In situ
hybridizations were performed as described by Pringle and Richardson
(www.ucl.ac.uk/
ucbzwdr/doubleinsituprotocol.htm).
Purkinje cell counts
Paraffin-embedded P15 and P60 wt (n=3) and null (n=3)
cerebella were sagittally sectioned. Complete series of 7 µm sections
spanning the entire cerebellum were immunostained with an anti-CaBP antibody
to visualize PC bodies. Morphometric evaluation of PC numbers was carried out
using Neurolucida software (MicroBrightField) connected to a Nikon E-800
microscope via a color CCD camera. The number of PCs and the length of the PC
layer were estimated from sagittal sections of the cerebellar vermis from
wild-type (wt) and Ebf2 null mice (for details, see
Buffo et al., 1997). For each
animal, three sections close to the midline were analysed. Statistical
analysis was conducted by using a two-tailed t-test with equal
variance.
Apoptotic bodies count
The count of apoptotic bodies (active Caspase 3-positive) at P0 was
performed in triplicate. Using a Zeiss Axioplan upright microscope (5x
objective) and a digital camera, pictures were taken of each complete series
of frontal sections (wt versus mutant) stained with anti-active Caspase 3;
pictures spanning each section were merged with Photoshop 8 to produce
high-definition screen images. Positive cell bodies were counted on screen,
marking each with the paintbrush tool of Adobe Photoshop 8. Statistical
analysis of mean apoptotic cell numbers/section was conducted by using a
two-tailed t-test with equal variance.
Bromodeoxyuridine (BrdU) labeling and detection
Pregnant females were given a single intraperitoneal injection with 100
µg/kg BrdU (Sigma) at gestational ages E11.5 and E12.5, and killed 2 h
later. Embryos were dissected in cold 1x PBS, fixed overnight with
Carnoy's solution and paraffin embedded. Anti-BrdU immunohistochemistry was
performed as described (Garel et al.,
1997). Cell counts were performed in triplicate as described
(Takahashi et al., 1993).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
behavioral abnormalities (Rotarod test, not shown). These results prompted us
to conduct further studies of cerebellar development and function in these
mutants.
Ebf2 expression in the embryonic and postnatal cerebellum
During mouse embryonic development, PCs are born in the VZ between E11 and
E13, when the bulk of PC proliferation is completed
(Hashimoto and Mikoshiba,
2004; Sotelo,
2004), then move into a postmitotic domain known as the cortical
transitory zone (CTZ). Because Ebf genes encode structurally similar proteins
that could play redundant roles in development, we compared the distribution
of Ebf1-Ebf3 mRNAs in the cerebellar primordium at E11.5
(Garel et al., 1997) and
E12.5, when the peak of PC progenitor proliferation occurs, and then analyzed
subsequent stages of embryogenesis and postnatal development.
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At E17.5, the expression domains of Ebf1-Ebf3 in the cerebellar
cortex are nested: Ebf1 is expressed from the midline through the
lateral edge of the developing PC layer; Ebf3 is excluded from the
most lateral domains and Ebf2 labels an even more medially confined
PC subset (Fig. 2D-F). For
comparison, we analyzed the expression of ROR, a universal PC
marker, in adjacent sections (Fig.
2G) and found a similar distribution as compared to Ebf1,
allowing the latter to be considered as a new bona-fide PC-specific
marker.
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No changes in the expression of cell cycle progression and control marker
Having scored a decrease in the total number of PCs in the mutant
cerebellum, we analyzed markers of DNA synthesis, mitosis, and cell cycle exit
in the VZ of the cerebellar primordium. Initially we measured BrdU
incorporation 2 h after a single pulse
(Takahashi et al., 1993)
(Fig. 3A,B) at E11.5 and E12.5
(Fig. 3I). Moreover we counted
progenitors positive for phosphohistone 3, a marker that selectively labels
metaphase chromosomes (Paulson and Taylor,
1982) (Fig. 3C,D).
We also tested the distribution of Ki67, which interacts with members of the
heterochromatin protein 1 family, present in all active phases of the cell
cycle (Scholzen et al., 2002;
Scholzen and Gerdes, 2000)
(Fig. 3E,F). Finally, we
analyzed by in situ hybridization the expression of the Kip2 gene,
encoding p57, a cyclin kinase inhibitor of the Cip/Kip family that regulates
cell cycle progression during development
(Hong et al., 1998)
(Fig. 3G,H). Our results do not
reveal any significant changes in the null VZ, indicating that Ebf2
does not control PC progenitor proliferation or cell-cycle exit. This is in
keeping with observations independently made by others in chick embryos
(Garcia-Dominguez et al.,
2003).
Defective migration of PC precursors in the Ebf2 null cerebellum
We then tackled the possible role of Ebf2 in the control of PC
migration. During normal development, PCs migrate along radial glial fibers,
skirting the DCN neuron clusters in the nuclear transitory zone (NTZ) to reach
their final destination beneath the granule cell progenitors located in the
external granular layer (reviewed in
Sotelo, 2004). Importantly, in
the mutant cerebellum, radial glia are normally developed, as revealed by
immunostaining for several specific markers: BLBP, RC2, GLAST and GFAP (not
shown).
In order for PC migration to be analyzed in the mutant, PCs were
immunolabeled at E15.5 for Calbindin (CaBP), a calcium-binding protein
selectively expressed in PCs at the onset of their migration into the cortex.
Our results indicate that in the mutant a large number of CaBP-positive PCs
accumulate in the anterior CTZ (Fig.
4B,D), while CaBP transcript levels are unchanged, as
assayed by real-time PCR (not shown). Studies of Ebf1-Ebf3
and ROR transcript distribution at the same stage confirmed
these findings, as detailed in Fig. S1 in the supplementary material.
Importantly the expression of Reelin, the best-characterized guidance
cue in PC migration (Jensen et al.,
2002), is unchanged in DCN neurons and in the EGL of Ebf2
mutants (see Fig. S1I,J in the supplementary material).
Our findings raised questions as to whether delayed-migrating PCs undergo cell death. At birth, in the null cerebellum the number of apoptotic bodies positive for active caspase 3 is significantly increased (P=0.00024) (Fig. 5A). Many dying cells are located near the origin of their migratory path (Fig. 5B,F), and colocalize with delayed-migrating lacZ-positive cells (Fig. 5G), while no clustering of dead cells is observed at the same location in the wt (Fig. 5E). By double immunofluorescence for active Caspase 3 and CaBP we found that 51% of the apoptotic cells in the vicinity of the VZ (Fig. 5B) were also positive for CaBP (example in Fig. 5C,D). However, some cell debris immunoreactive for active Caspase 3 was CaBP-negative (arrowheads in Fig. 5D). The DCN are already well developed at birth in the wt and null alike, and no increase in the number of Caspase 3-positive cells could be found in the null. Finally, postmigratory, caspase-3/lacZ-double-positive PCs are observed in the Ebf2 null cerebellar cortex, both in the vermis (Fig. 5I) and in hemispheres (Fig. 5L,M).
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).
E18.5 and P4 cerebella of Ebf2 null and wt mice were stained for
neurogranin. While in the wt, neurogranin labels arrays of PCs located in the
cerebellar cortex (Fig. 6A), in
the null mutant E18.5 cerebellum, neurogranin labels mostly ectopic,
delayed-migrating cells near the VZ (Fig.
6B). By P4, while the wt cerebellum exhibits an ordered array of
neurogranin-positive PC clusters (Fig.
6C,E), no neurogranin immunoreactivity is detectable in the null
mutant cerebellum (Fig. 6D,F).
This suggests that neurogranin-positive PC precursors fail to migrate, and
subsequently die between E18.5 and P4. Importantly, in the heterozygous
cerebellum, neurogranin is coexpressed with beta-galactosidase (not shown),
suggesting a cell-autonomous effect of Ebf2 on the
development/survival of neurogranin-positive PCs. Likewise, cerebella were
stained for HSP25, another PC marker expressed in parasagittal stripes in the
vermis (Armstrong et al.,
2001b). Hardly any immunostaining for HSP25 can be observed at P3
in the anterior vermis of Ebf2 null mice
(Fig. 6H). The loss of two
independent striped markers, neurogranin and HSP25, in the vermis is
suggestive of PC loss rather than transcriptional downregulation.
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), that
is widely coexpressed with Ebf2, and the receptor tyrosine kinase
EphA4 (Rogers et al., 1999),
showing that EphA4-positive stripes are considerably reduced in size
(asterisks in Fig. 7I, control
in H), and that a large number of Sema3A-expressing PCs are lost in
the mutant (arrows in Fig. 7F
and O, controls in E and N).
In parallel, we performed in situ hybridizations of P6 frontal sections to
evaluate the distribution of two markers of cerebellar hemispheres,
protocadherin 10 (Pcdh10)
(Hirano et al., 1999) and
Sema3A (L.C. and G.G.C., unpublished observations). Cerebellar
hemispheres exhibit complex folding defects in the Ebf2 null mutant.
Our results indicate that the number of Pcdh10-expressing PCs is
hardly or not reduced, whereas the number of Sema3A-expressing PCs is
sharply decreased in the hemispheres (supplemental Fig. S2). Overall, our
result speak for a massive reduction in the number of a molecularly defined PC
subset in the Ebf2 null cerebellum.
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): the anterior zone (AZ: approximately lobules I-V), the
central zone (CZ: approximately lobules VI and VII), the posterior zone (PZ:
approximately lobules VIII and dorsal IX), and the nodular zone (NZ:
approximately dorsal lobule IX and lobule X). Each zone includes several
lobules, and interdigitates at their boundaries, but zonal boundaries do not
coincide with lobule boundaries (Hawkes
and Eisenman, 1997; Ozol et
al., 1999; Eisenman,
2000). We analyzed the distribution of the Ebf2-lacZ transgene in adult Ebf2+/- cerebella, which develop normally (Fig. 8). In the NZ, flocculus and paraflocculus, there is no transgene expression. In the PZ (e.g. lobule VIII), the transgene is expressed in the vermis by two pairs of PC stripes, and an additional array of broad expression stripes lies in the hemispheres (l. paramedianus). More rostrally, in the CZ (e.g. lobules VIa,b) and in its hemispheric extension, expression is either absent or weak. The ansiform lobule is negative. Finally, in the AZ (lobules I-V) most PCs in the vermis express the transgene (e.g. lobule III), but narrow transgene-negative stripes can be seen (e.g. arrows in lobule III).
We systematically compared the expression of Ebf2-lacZ in the
heterozygote to the expression of ZII
(Brochu et al., 1990)
(Fig. 8B-H). In general, the
transgene is expressed at high levels in the ZII-immunonegative PC population,
as shown for the posterior hemispheres (the hemispheric PZ:
Fig. 8B,D,E), posterior vermis
(both PZ, alternating stripes, and NZ, all ZII-immunopositive/lacZ-negative:
Fig. 8C,F), anterior hemisphere
(hemispheric AZ: Fig. 8G) and
anterior vermis (the AZ: Fig.
8H). Rare exceptions to this rule were also found
(Fig. 8F).
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): this pattern is unaltered in the Ebf2 null mutant
cerebellum (not shown). In the AZ, an alternating pattern of ZII expression
can still be defined (lobule IV (Fig.
9E,E'), III (Fig.
9F,F'), I (Fig.
9G,G'), but the spacing between the ZII+ stripes is
significantly reduced (e.g. in lobule III from 1050 µm in the +/- to
700 µm in the null; see also wholemounts in
Fig. 9M-P).
The effect of the Ebf2 deletion is not restricted to ZII
expression. The striped expression of sphingosine kinase 1a (SPHK1a), which is
co-expressed in the same stripe arrays as ZII
(Terada et al., 2004), is
similarly affected (Fig.
9H': control in 9H), strongly suggesting that the effect is
not mediated via the direct control of ZII gene expression by EBF2, but rather
reflects a wider alteration in the specification of PC subtypes. The effect of
the null mutation on cerebellar patterning in the anterior cerebellum can be
explained by the loss of a large fraction of the ZII-immunonegative PC
population. However, the effect in the posterior cerebellum clearly suggests
that Ebf2 is required for a subset of ZII-positive PCs to switch and
acquire the ZII-negative phenotype. A large subset of Ebf2+ (i.e.
beta-galactosidase-positive) PCs, normally fated to become ZII-negative during
the third week of life, fail to switch, and remain ZII positive instead. For
example, `transdifferentiated' PCs are plentiful in the paramedian lobe of the
homozygous mutant (e.g. Fig.
10A-C,D-F); conversely, in Ebf2 heterozygous littermates,
no double-labeled cells are seen (Fig.
10G). These results confirm that the cerebellar stripe topography
is profoundly disrupted in Ebf2 mutants.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Impaired migration and death of a PC subpopulation
The Ebf2 null mutation does not overtly affect cell proliferation
or cell cycle exit, nor does it appear to significantly disrupt the
development of cerebellar radial glia. Conversely, the mutant cerebellum
displays a clear defect in the migration of a subset of PC precursors. The
migration abnormalities observed in Ebf2 null mutants are probably
not secondary to a defect in early stages of the canonical Reelin pathway, and
reveal the existence of a Reelinindependent network presiding over PC
precursor migration.
The effects of nullisomy for Ebf2 on PC migration become obvious by embryonic day 15. At birth, numerous delayed-migrating PCs accumulate in the proximity of the VZ, due to an arrest in their migration prior to reaching the cortex, and die by apoptosis. Dying cells colocalize with delayed-migrating lacZ-positive neurons, suggesting a cell-autonomous role of Ebf2 in regulating PC migration/survival. In addition, many PCs that do reach the null mutant cortex also die, possibly due to a lack of intrinsic survival mechanisms; alternatively, mislocalized PCs may fail to connect properly to their postsynaptic targets. Importantly, no marker misexpression has been scored in mutant DCN labeled with Sema3A, Ebf3, Cdh8 or Pcdh10 riboprobes (not shown).
The considerable increase in perinatal PC death observed in the mutant is
in keeping with the 38% PC loss scored at P15 and P60 in null cerebella. A
large share of the apoptotic bodies observed at birth are clearly positive for
the PC-specific marker CaBP, while others are not, potentially representing
dying PCs that have ceased expressing CaBP
(Vig et al., 1998), PCs that
do not yet express CaBP, or possibly other unrelated neural cell types.
A high percentage of apoptotic cells are represented by delayedmigrating,
neurogranin-positive PCs, that are lost between E18.5 and P4. Neurogranin is
an abundant thyroid hormone-dependent protein kinase C substrate, that
regulates Ca2+ signaling modulating Ca2+/calmodulin
availability, and lessens the extent to which calcium-calmodulin-dependent
enzymes become or stay activated
(Gerendasy, 1999).
Neurogranin-positive PCs, in which the Ca2+ buffering strength of
calmodulin is attenuated, may fail to respond to specific
Ca2+-dependent developmental signals such as retinoic acid, thyroid
hormone or (potentially) Notch signaling
(McKenzie et al., 2005).
Incidentally, the gross abnormalities observed in Ebf2 null cerebella
are reminiscent of, albeit more severe than, those observed in hypothyroid
mice (Koibuchi and Chin,
2000).
Neurogranin is not the only subtype-specific marker lost in the mutant
cerebellum: during early postnatal development HSP25, normally expressed in
six narrow parasagittal stripes in the vermis of the anterior lobe
(Armstrong et al., 2001b), is
lost in the anterior lobe of the Ebf2 null mutant. PCs lost around
birth are positive for generic PC markers, and for genes with established
roles in axon guidance, such as Sema3A and EphA4, that are
expressed in parasagittal stripes.
|
). The fact that in the Ebf2 null cerebellum
neurogranin-positive PCs are selectively lost by P4 translates into a loss and
underrepresentation of ZII-negative PCs in the mature cerebellum. The same
applies to other parasagittally striped markers coexpressed with ZII, such as
SPHK1a. The development of cerebellar patterning extends from before E10 to
P20. Evidence from multiple routes concurs that most major elements of the
fundamental cerebellar topography are established at the time of PC birth,
between E11 and E13, in the VZ of the 4th ventricle (reviewed by
Armstrong and Hawkes, 2000;
Herrup and Kuemerle, 1997;
Oberdick et al., 1998). From
E13.5 onwards, molecular markers reveal evidence of PC compartmentation [e.g.
the restricted distribution of the OMP transgene
(Nunzi, 1999)]. Incidentally,
the OMP promoter contains two binding sites for OLF1/EBF transcription factors
(Kudrycki et al., 1993). Thus,
Ebf2 is one of the earliest-known functional links to the molecular
compartmentalization of the adult cerebellar cortex. PC migration defects
restricted to the ZII-negative population have been previously described, e.g.
in the cerebellar-deficient folia mutant (cdf)
(Beierbach et al., 2001;
Park et al., 2002). Selective
PC ectopia has also been described within a ZII-positive population in the
weaver mouse (Armstrong and Hawkes,
2001). However, the Ebf2 null mouse reveals the first
evidence of the mechanisms through which the prospective ZII phenotype is
determined in postmitotic PC precursors. Ebf2 expression in the wt
cerebellum is restricted to ZII-negative PCs, and in the Ebf2 null
cerebellum ZII-negative PC stripes are selectively disrupted, with profound
consequences on cerebellar topography. In the vermis, lobules VIII and VII,
which normally display alternating ZII-positive/-negative stripes
(Fig. 9C and D, respectively),
now contain exclusively ZII-positive PCs
(Fig. 9D'), or an
unpatterned admixture of ZII-positive and -negative cells
(Fig. 9C'). Likewise, in
the anterior zone, where neurogranin-positive PCs are lost, the characteristic
stripes of ZII-positive PCs are much more narrowly spaced than normal. Taken
together, these abnormalities show that the Ebf2 null mutation
results in hypotrophy, defective lobulation and an incomplete failure of PC
dispersal.
|
;
Leclerc et al., 1988;
Wassef et al., 1990). Previous
attempts to alter cerebellar topography surgically
(Armstrong et al., 2001a;
Leclerc et al., 1988) or
genetically (Armstrong and Hawkes,
2001; Edwards et al.,
1994; Eisenman et al.,
1998; Gallagher et al.,
1998) have proven ineffectual: the gross morphology may be
affected but the underlying topography is normal. These data suggest a model
for PC phenotype determination: while the default phenotype is ZII-positive,
the presence of Ebf2 - which is expressed from at least E11.5 - affects the
development of Ebf2-expressing PCs fated to acquire the ZII-negative
phenotype. When Ebf2 is deleted then some PCs fated to become ZII-negative
fail to migrate and die, and others default to a ZII-positive phenotype.
What could be the functional relevance of the observed alterations in
cerebellar circuits documented in the present paper? The cerebellar striped
markers' distribution has been found to reflect the ordered arrangement of the
afferent and efferent terminal fields (Ji
and Hawkes, 1994; Ji and
Hawkes, 1995; Voogd et al.,
2003; Voogd and Ruigrok,
2004). Preliminary results (F.R. and S.G., unpublished) clearly
indicate that climbing fiber terminals spread laterally in the
Ebf2-/- molecular layer across ZII+/-
boundaries, a finding that faithfully mimics the effect of olivocerebellar
denervation (Rossi et al.,
1991). Further studies are in progress to characterize these
events and their molecular basis in due detail.
To date, our results allow us to propose Ebf2 as a factor regulating short-range cerebellar patterning, organizing orderly molecular domains and intraparenchymal boundaries. The abnormalities scored in the Ebf2 null cerebellum strengthen the hypothesis that EBF transcription factors are key players in a phase of development that links neuronal differentiation, morphogenetic movements, cell survival and the early functional activation of fledging neuronal circuits.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/14/2719/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ahn, A. H., Dziennis, S., Hawkes, R. and Herrup, K. (1994). The cloning of zebrin II reveals its identity with aldolase C. Development 120,2081 -2090.[Abstract]
Armstrong, C. L. and Hawkes, R. (2000). Pattern formation in the cerebellar cortex. Biochem. Cell Biol. 78,551 -562.[CrossRef][Medline]
Armstrong, C. and Hawkes, R. (2001). Selective Purkinje cell ectopia in the cerebellum of the weaver mouse. J. Comp. Neurol. 439,151 -161.[Medline]
Armstrong, C. L., Krueger-Naug, A. M., Currie, R. W. and Hawkes, R. (2000). Constitutive expression of the 25-kDa heat shock protein Hsp25 reveals novel parasagittal bands of purkinje cells in the adult mouse cerebellar cortex. J. Comp. Neurol. 416,383 -397.[CrossRef][Medline]
Armstrong, C. L., Krueger-Naug, A. M., Currie, R. W. and Hawkes, R. (2001a). Constitutive expression of heat shock protein HSP25 in the central nervous system of the developing and adult mouse. J. Comp. Neurol. 434,262 -274.[CrossRef][Medline]
Armstrong, C. L., Krueger-Naug, A. M., Currie, R. W. and Hawkes, R. (2001b). Expression of heat-shock protein HSP25 in mouse Purkinje cells during development reveals novel features of cerebellar compartmentation. J. Comp. Neurol. 429, 7-21.[Medline]
Beierbach, E., Park, C., Ackerman, S. L., Goldowitz, D. and Hawkes, R. (2001). Abnormal dispersion of a purkinje cell subset in the mouse mutant cerebellar deficient folia (cdf). J. Comp. Neurol. 436,42 -51.[CrossRef][Medline]
Brochu, G., Maler, L. and Hawkes, R. (1990). Zebrin II: a polypeptide antigen expressed selectively by Purkinje cells reveals compartments in rat and fish cerebellum. J. Comp. Neurol. 291,538 -552.[CrossRef][Medline]
Buffo, A., Holtmaat, A. J., Savio, T., Verbeek, J. S., Oberdick,
J., Oestreicher, A. B., Gispen, W. H., Verhaagen, J., Rossi, F. and Strata,
P. (1997). Targeted overexpression of the neurite
growth-associated protein B-50/GAP-43 in cerebellar Purkinje cells induces
sprouting after axotomy but not axon regeneration into growth-permissive
transplants. J. Neurosci.
17,8778
-8791.
Corradi, A., Croci, L., Broccoli, V., Zecchini, S., Previtali,
S., Wurst, W., Amadio, S., Maggi, R., Quattrini, A. and Consalez, G. G.
(2003). Hypogonadotropic hypogonadism and peripheral neuropathy
in Ebf2 null mice. Development
130,401
-410.
Dubois, L. and Vincent, A. (2001). The COE-Collier/Olf1/EBF-transcription factors: structural conservation and diversity of developmental functions. Mech. Dev. 108, 3-12.[CrossRef][Medline]
Dubois, L., Bally-Cuif, L., Crozatier, M., Moreau, J., Paquereau, L. and Vincent, L. (1998). XCoe2, a transcription factor of the Col/Olf-1/EBF family involved in the specification of primary neurons in Xenopus. Curr. Biol. 8, 199-209.
Edwards, M. A., Leclerc, N., Crandall, J. E. and Yamamoto, M. (1994). Purkinje cell compartments in the reeler mutant mouse as revealed by Zebrin II and 90-acetylated glycolipid antigen expression. Anat. Embryol. 190,417 -428.[Medline]
Eisenman, L. M. (2000). Antero-posterior boundaries and compartments in the cerebellum: evidence from selected neurological mutants. Prog. Brain Res. 124, 23-30.[Medline]
Eisenman, L. M. and Hawkes, R. (1993). Antigenic compartmentation in the mouse cerebellar cortex: zebrin and HNK-1 reveal a complex, overlapping molecular topography. J. Comp. Neurol. 335,586 -605.[CrossRef][Medline]
Eisenman, L. M., Gallagher, E. and Hawkes, R. (1998). Regionalization defects in the weaver mouse cerebellum. J. Comp. Neurol. 394,431 -444.[CrossRef][Medline]
Gallagher, E., Howell, B. W., Soriano, P., Cooper, J. A. and Hawkes, R. (1998). Cerebellar abnormalities in the disabled (mdab1-1) mouse. J. Comp. Neurol. 402,238 -251.[CrossRef][Medline]
Garcia-Dominguez, M., Poquet, C., Garel, S. and Charnay, P.
(2003). Ebf gene function is required for coupling neuronal
differentiation and cell cycle exit. Development
130,6013
-6025.
Garel, S., Marin, F., Mattei, M. G., Vesque, C., Vincent, A. and Charnay, P. (1997). Family of Ebf/Olf-1-related genes potentially involved in neuronal differentiation and regional specification in the central nervous system. Dev. Dyn. 210,191 -205.[CrossRef][Medline]
Garel, S., Marin, F., Grosscheld, R. and Charnay, P. (1999). Ebf1 controls early cell differentiation in the embryonic striatum. Development 126,5285 -5294.[Abstract]
Garel, S., Garcia-Dominiguez, M. and Charnay, P. (2000). Control of the migratory pathway of facial branchiomotor neurones. Development 127,5297 -5307.[Abstract]
Garel, S., Yun, K., Grosschedl, R. and Rubenstein, J. L. (2002). The early topography of thalamocortical projections is shifted in Ebf1 and Dlx1/2 mutant mice. Development 129,5621 -5634.[Medline]
Gerendasy, D. (1999). Homeostatic tuning of Ca2+ signal transduction by members of the calpacitin protein family. J. Neurosci. Res. 58,107 -119.[CrossRef][Medline]
Hagman, J., Belanger, C., Travis, A., Turck, C. and Grosschedl,
R. (1993). Cloning and functional characterization of early
B-cell factor, a regulator of lymphocyte-specific gene expression.
Genes Dev. 7,760
-773.
Hashimoto, M. and Mikoshiba, K. (2004).
Neuronal birthdate-specific gene transfer with adenoviral vectors.
J. Neurosci. 24,286
-296.
Hawkes, R. (1992). Antigenic markers of cerebellar modules in the adult mouse. Biochem. Soc. Trans. 20,391 -395.[Medline]
Hawkes, R. and Herrup, K. (1995). Aldolase C/zebrin II and the regionalization of the cerebellum. J. Mol. Neurosci. 6,147 -158.[Medline]
Hawkes, R. and Eisenman, L. M. (1997). Stripes and zones: the origins of regionalization of the adult cerebellum. Perspect. Dev. Neurobiol. 5, 95-105.[Medline]
Heckroth, J. A., Goldowitz, D. and Eisenman, L. M. (1989). Purkinje cell reduction in the Reeler mutant mouse: a quantitative immunohistochemical study. J. Comp. Neurol. 279,546 -555.[CrossRef][Medline]
Herrup, K. and Kuemerle, B. (1997). The compartmentalization of the cerebellum [Review]. Annu. Rev. Neurosci. 20,61 -90.[CrossRef][Medline]
Hirano, S., Yan, Q. and Suzuki, S. T. (1999).
Expression of a novel protocadherin, OL-protocadherin, in a subset of
functional systems of the developing mouse brain. J.
Neurosci. 19,995
-1005.
Hong, Y., Roy, R. and Ambros, V. (1998). Developmental regulation of a cyclin-dependent kinase inhibitor controls postembryonic cell cycle progression in Caenorhabditis elegans. Development 125,3585 -3597.[Abstract]
Jensen, P., Zoghbi, H. Y. and Goldowitz, D.
(2002). Dissection of the cellular and molecular events that
position cerebellar Purkinje cells: a study of the math1 null-mutant mouse.
J. Neurosci. 22,8110
-8116.
Jensen, P., Smeyne, R. and Goldowitz, D.
(2004). Analysis of cerebellar development in math1 null embryos
and chimeras. J. Neurosci.
24,2202
-2211.
Ji, Z. and Hawkes, R. (1994). Topography of Purkinje cell compartments and mossy fiber terminal fields in lobules II and III of the rat cerebellar cortex: spinocerebellar and cuneocerebellar projections. Neuroscience 61,935 -954.[CrossRef][Medline]
Ji, Z. and Hawkes, R. (1995). Developing mossy fiber terminal fields in the rat cerebellar cortex may segregate because of Purkinje cell compartmentation and not competition. J. Comp. Neurol. 359,197 -212.[Medline]
Kieslinger, M., Folberth, S., Dobreva, G., Dorn, T., Croci, L., Erben, R., Consalez, G. G. and Grosschedl, R. (2005). EBF2 regulates osteoblast-dependent differentiation of osteoclasts. Dev. Cell 9,757 -767.[CrossRef][Medline]
Koibuchi, N. and Chin, W. W. (2000). Thyroid hormone action and brain development. Trends Endocrinol. Metab. 11,123 -128.[CrossRef][Medline]
Kudrycki, K., Stein-Izsak, C., Behn, C., Grillo, M., Akeson, R.,
Margolis, F. L., Wang, M. M., Tsai, R. Y., Schrader, K. A. and Reed, R. R.
(1993). Olf-1-binding site: characterization of an olfactory
neuron-specific promoter motif. Mol. Cell. Biol.
13,3002
-3014.
Larouche, M., Che, P. and Hawkes, R. (2006). Neurogranin expression identifies a novel array of Purkinje cell parasagittal stripes during mouse cerebellar development. J. Comp. Neurol. 494,215 -227.[Medline]
Leclerc, N., Gravel, C. and Hawkes, R. (1988). Development of parasagittal zonation in the rat cerebellar cortex: MabQ113 antigenic bands are created postnatally by the suppression of antigen expression in a subset of Purkinje cells. J. Comp. Neurol. 273,399 -420.[CrossRef][Medline]
Malgaretti, N., Pozzoli, O., Bosetti, A., Corradi, A.,
Ciarmatori, S., Panigada, M., Bianchi, M., Martinez, S. and Consalez, G.
G. (1997). Mmot1, a new helix-loop-helix
transcription factor gene displaying a sharp antero-posterior expression
boundary in the embryonic mouse brain. J. Biol. Chem.
272,17632
-17639.
McKenzie, G. J., Stevenson, P., Ward, G., Papadia, S., Bading, H., Chawla, S., Privalsky, M. and Hardingham, G. E. (2005). Nuclear Ca2+ and CaM kinase IV specify hormonal- and Notch-responsiveness. J. Neurochem. 93,171 -185.[CrossRef][Medline]
Nunzi, M. G., Grillo, M., Margolis, F. L. and Mugnaini, E. (1999). Compartmental organization of Purkinje cells in the mature and developing mouse cerebellum as revealed by an olfactory marker protein-lacZ transgene. J. Comp. Neurol. 404,97 -113.[Medline]
Oberdick, J., Baader, S. L. and Schilling, K. (1998). From zebra stripes to postal zones: deciphering patterns of gene expression in the cerebellum. Trends Neurosci. 21,383 -390.[CrossRef][Medline]
Ozol, K., Hayden, J. M., Oberdick, J. and Hawkes, R. (1999). Transverse zones in the vermis of the mouse cerebellum. J. Comp. Neurol. 412,95 -111.[CrossRef][Medline]
Park, C., Falls, W., Finger, J. H., Longo-Guess, C. M. and Ackerman, S. L. (2002). Deletion in Catna2, encoding alpha N-catenin, causes cerebellar and hippocampal lamination defects and impaired startle modulation. Nat. Genet. 31,279 -284.[Medline]
Paulson, J. R. and Taylor, S. S. (1982).
Phosphorylation of histones 1 and 3 and nonhistone high mobility group 14 by
an endogenous kinase in HeLa metaphase chromosomes. J. Biol.
Chem. 257,6064
-6072.
Pozzoli, O., Bosetti, A., Croci, L., Consalez, G. G. and Vetter, M. L. (2001). Xebf3 is a regulator of neuronal differentiation during primary neurogenesis in Xenopus. Dev. Biol. 233,495 -512.[CrossRef][Medline]
Prasad, B. C., Ye, B., Zackhary, R., Schrader, K., Seydoux, G. and Reed, R. R. (1998). unc-3, a gene required for axonal guidance in Caenorhabditis elegans, encodes a member of the O/E family of transcription factors. Development 125,1561 -1568.[Abstract]
Rogers, J. H., Ciossek, T., Menzel, P. and Pasquale, E. B. (1999). Eph receptors and ephrins demarcate cerebellar lobules before and during their formation. Mech. Dev. 87,119 -128.[CrossRef][Medline]
Rossi, F., Wiklund, L., van der Want, J. J. and Strata, P. (1991). Reinnervation of cerebellar Purkinje cells by climbing fibres surviving a subtotal lesion of the inferior olive in the adult rat. I. Development of new collateral branches and terminal plexuses. J. Comp. Neurol. 308,513 -535.[CrossRef]
