1 San Raffaele Scientific Institute, Milan, Italy
2 Stem Cell Research Institute, San Raffaele Scientific Institute, Milan,
Italy
3 Max Planck Institute of Psychiatry, Munich, and GSF-Research Center of
Environment and Health, Institute of Developmental Genetics, Neuherberg,
Germany
4 CEND, Department of Endocrinology, University of Milan, Italy
Author for correspondence (e-mail:
g.consalez{at}hsr.it)
Accepted 18 October 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Olf/Ebf genes, Neurogenesis, Neural development, Neuronal migration, Neuroendocrine, GnRH neurons, Peripheral nerve, Peripheral neuropathy, Dysmyelination, Homologous recombination, Knockout, Targeted inactivation, Gene targeting, COE2, O/E3
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three mammalian Ebf genes have been isolated so far: Ebf1, Ebf2
and Ebf3. An additional member of the family, O/E4, was
isolated more recently (Wang et al.,
2002) and its expression in the embryonic neural tube has not been
mapped to date. In the embryonic mouse nervous system, Ebf1, Ebf2 and
Ebf3 are transcribed in partially overlapping territories
(Garel et al., 1997
), but
display significant differences in their distributions
(Garel et al., 1997
;
Malgaretti et al., 1997
),
suggesting that each may play roles in the morphogenesis of different head
territories. As one example, Ebf1 is the only family member expressed
in the striatal anlage (Garel et al.,
1997
). Second, noticeable differences are observed in the
distribution of Ebf transcripts across the wall of the neural tube (NT):
Ebf1 and Ebf3, which map to mouse chromosomes 11 and 7,
respectively (Garel et al.,
1997
), are co-expressed at most sites in the mantle layer of the
developing NT, whereas Ebf2, which maps to mouse chromosome 14, is
expressed by younger postmitotic neural cells located in the subventricular
layer of the prospective spinal cord (Garel
et al., 1997
; Malgaretti et
al., 1997
). In the developing nervous system, the functions of
Ebf1 and Ebf3 appear to be somewhat redundant, in that
Ebf1-/- mutants feature defects in neuronal
differentiation (Garel et al.,
1999
) and short-range neuronal migration
(Garel et al., 2000
) only in
territories in which Ebf3 is not expressed. However, no genetic
evidence is available regarding the specific role of Ebf2 in the
context of vertebrate neural development. Again, overexpression studies
conducted in Xenopus, where an Ebf1 ortholog has not been
isolated to date, indicate that Xebf2/Xcoe2
(Dubois et al., 1998
;
Pozzoli et al., 2001
) is
expressed earlier than Xebf3
(Pozzoli et al., 2001
), and
suggest that the two genes may play distinct roles in the context of primary
neurogenesis (Pozzoli et al.,
2001
). In particular, gain-of-function experiments suggest that
Xebf2 and Xebf3 differ in their sensitivity to lateral
inhibition (Pozzoli et al.,
2001
). In fact, Notch activation can suppress the ability
of NeuroD (Neurod1 Mouse Genome Informatics) to
promote Xebf2 but not Xebf3 transcription
(Pozzoli et al., 2001
).
Finally, lateral inhibition interferes with the ability of overexpressed
Xebf2, but not Xebf3, to activate transcription of early
(N-tubulin) and late (NF-M) neuronal differentiation markers
(Pozzoli et al., 2001
). These
results indicate that Notch activation interferes in a
cell-autonomous fashion with Ebf2 function, not just with its
transcription.
The features and interactions common to the entire Ebf gene family in
phylogeny, as well as the specific features of the Ebf2 homolog in
Xenopus neurogenesis prompted us to study the contribution of
Ebf2 (Malgaretti et al.,
1997) to neural development and function, using a genetic approach
in the mouse. Ebf2-/- mice were generated by gene
targeting, and revealed a key role for Ebf2 in the embryonic
migration of gonadotropin-releasing hormone (GnRH)-synthesizing neurons from
the vomeronasal organ to the hypothalamus. In addition, our results implicated
this gene in peripheral nerve morphogenesis.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of Ebf2 gene targeting vector
Eleven Ebf2 genomic clones were isolated by screening of a 129/SvJ
mouse genomic library (106 pfu) with an Ebf2-specific
intronic probe. the probe was derived by restriction of a PCR product obtained
with a forward primer from exon 1 and a reverse primer from exon 3 (F:
5'-CTG GGT GCC GAG ATG GAT T; R: 5'-TGT TGG TCT TCT CAT TGC CTT).
We used a modified pPNT vector containing a Neo cassette flanked by
LoxP sites. The construct contains a PGK-tk cassette
downstream of the 3' arm of homology. After homologous recombination,
5.5 kb of genomic sequence were deleted from the Ebf2 gene, including
the putative translation initiation site and the first five exons.
Generation of recombinant embryonic stem (ES) cells
ES cells (TBV2 line) were grown in Dulbecco's Modified Eagles Medium (DMEM)
(Gibco), 15% fetal calf serum (Gibco), 10-4 M
ß-mercaptoethanol (Gibco), 2 mM L-Glutamine and 1000 U/ml LIF (Heiko), on
an embryonic fibroblast feeder layer previously inactivated with Mitomycin C.
Electroporation, positive and negative selection were performed as described
(Joyner, 1993). Resistant
colonies were picked after 8-10 days of selection. Genomic DNA was extracted
from expanded clones, digested with HindIII and analyzed by Southern
blotting at the 3' end of the recombinant locus. Homologous recombinant
clones were analyzed at the 5' end by EcoRI digest and Southern
analysis. Out of 1200 ES clones screened, three scored positive for homologous
recombination and were propagated.
Generation of chimeric mice and germline transmission of the Ebf2
targeted allele
The targeted ES clones were injected into blastocysts derived from C57BL/6J
females. The chimeric embryos were then transferred into the uteri of 2.5 day
pseudopregnant foster mothers. Chimeric males with 40-100% agouti color were
test-bred by crossing with wild-type C57BL/6J females and germline
transmission was identified by the presence of agouti offspring. Heterozygous
mice were initially identified by Southern blotting with the 3' probe.
Subsequent genotyping was carried out by PCR amplification with
lacZ-specific and Ebf2-specific primers.
RT-PCR
RNA was extracted by the caesium chloride method from E13 embryos and from
postnatal and adult sciatic nerves. Reverse transcription was conducted with
oligo-dT and random hexamers, as described previously
(Ausubel et al., 1995). A
primer pair (F: 5'-TGG AGA ATG ACA AAG AGC AAG; R: 5'-GGG TTT CCC
GCT GTT TTC AAA) specific for the cDNA region encoding the Ebf2 DNA-binding
domain was used for PCR amplification, according to standard protocols.
Gapdh primers used for normalization were 5'-CGC ATC TTC TTG
TGC AGT G (forward) and 5'-GTT CAG CTC TGG GAT GAC (reverse).
Mouse genetics
The mutation was transferred onto different genetic backgrounds by three
distinct procedures: first, we incrossed the transmitting chimeras with
129/SvJ females to transfer the mutation onto a 129/SvJ background and obtain
pure coisogenic mutants. Second, we intercrossed
(129/SvJxC57BL/6J)F1 mice to obtain F2 homozygotes
of heterogeneous genetic backgrounds. Third, we backcrossed
(129/SvJxC57BL/6J)F1 hybrids with C57BL6/J mice.
Removal of the Neo minigene
The KO construct contains a neomycin minigene
(Joyner, 1993). This cassette
is flanked by loxP sites. In order to exclude a possible effect of
the Neo minigene on the mutant phenotype, we crossed
Ebf2+/- mice with transgenic mice expressing the
Cre recombinase under control of the ß-actin promoter. By
intercrossing Cre+ Ebf2+/- transheterozygotes, we
obtained Neo-negative, Ebf2-/- mice, that were
Cre-negative, formally excluding mosaicism for the Neo
cassette. Phenotypic and pathological abnormalities observed in those mice
were indistinguishable from those scored in Ebf2-/-,
Neo-positive mice (data not shown).
lacZ staining procedures
For whole-mount lacZ staining, whole tissues were removed from
mice anesthetized and transcardially perfused with 0.9% NaCl followed by 4%
paraformaldehyde (PFA). Dissected organs were washed in wash buffer (0.01%
sodium deoxycolate, 0.02% Nonidet P40, 2 mM magnesium chloride in 1x
phoshate buffer) and stained for 2-6 hours at 37°C in staining solution
(50 mg X-gal, 0.106 g potassium ferrocyanide and 0.082 g potassium
ferricyanide were dissolved in 50 ml of wash buffer).
Histology, immunohistochemistry and immunofluorescence
Embryos and mice were respectively fixed by immersion, or anesthetized and
transcardially perfused with 0.9% NaCl followed by 4% PFA. Brain and testes
were postfixed overnight in 4% PFA at 4°C, dehydrated in ethanol, embedded
in paraffin wax and cut into 5-7 µm section using a microtome. Sections
were counterstained with Cresyl Violet (Sigma) or Hematoxylin And Eosin
(Sigma). For immunohistochemistry, tissues were cryoprotected in 30% sucrose,
1xPBS (overnight), included in OCT (Bioptica) and stored at -80°C,
before sectioning in a cryotome (5-15 µm). Immunohistochemical analysis was
conducted on cryosections with the following antibodies: polyclonal anti GnRH
(LR1,1:2000, courtesy of R. Benoit, Montreal), polyclonal anti-peripherin
(1:1000, Chemicon) and anti Tag-1 adhesion molecule (courtesy of A. J. W.
Furley, Sheffield). Sections were immunostained as suggested (Vector
Laboratories), counterstained with Methyl Green (Sigma), dehydrated and
mounted with DPX (BDH-Merck).
For dual immunofluorescence, E15 embryo cryosections were treated for 10 minutes with 0.1 M glycine, preincubated in 15% goat serum, 0.2% triton X-100, 1xPBS and incubated overnight at 4°C with the two primary antibodies (polyclonal anti GnRH, 1:1000, monoclonal anti-ß-galactosidase, 1:500 Promega). The sections were washed 6 times for 10 minutes in 0.45 M NaCl, 0.3% triton X-100, 20 mM phosphate buffer, rinsed in 1xPBS and incubated for 1 hour at room temperature with the two secondary antibodies (TRITC anti-mouse, 1:150, Jackson ImmunoResearch Laboratories and FITC anti rabbit, 1:150, Sigma). Controls consisted of replacing either the first primary Ab or the second primary Ab with goat serum. Control sections indicated no cross-reactivity between the first and the second Ab.
Electron microscopy
Sciatic nerve specimens were fixed with 2% glutaraldehyde (2-3 hours),
postfixed in 1% osmium tetroxide, following ethanol dehydration, and finally
embedded in Epon/araldite. Electron microscopy analysis was performed on
ultrathin transverse sections made on a Reichert ultramicrotome, stained with
uranyl acetate and lead citrate. Slides were examined under a Zeiss electron
microscope.
Bromodeoxyuridine labeling and detection
Adult male mice were injected intraperitoneally with 100 mg/kg of
bromodeoxyuridine (BrdU, Sigma) and sacrificed 2 hours later. Anti BrdU
immunohistochemistry was performed as described
(Garel et al., 1997).
Electrophysiological methods
Mice were anesthetized with trichloroethanol, 0.02 ml/g body weight, and
placed under a heating lamp in order to avoid hypothermia.
Electrophysiological tests were carried out as described
(Zielasek et al., 1996). Motor
responses were acquired with a Medelec Sapphire 4 Me electromyograph (Medelec,
Woking, UK). Statistical analysis was performed by using the Student's
t-test for unpaired data.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
RT-PCR experiments conducted on total RNA extracted from wild-type and homozygous mutant E13 embryos provided evidence that homozygous mutants produce no Ebf2 transcript 3' to the lacZ insertion (Fig. 1D). To maximize sensitivity, a similar experiment was conducted on cDNA extracted from the peripheral nerve, with Southern transfer of RT-PCR products followed by hybridization with an Ebf2 probe, yielding concordant results (not shown).
The Ebf2 gene is expressed at numerous sites in the nervous
system, both before and after birth (Garel
et al., 1997; Malgaretti et
al., 1997
). Taking advantage of the promoterless lacZ
gene (encoding a cytoplasmic ß-galactosidase) integrated into the
Ebf2 locus, we analyzed Ebf2 expression by histochemical
lacZ staining. The staining distribution in early development is in
full agreement with the results of previous expression studies
(Garel et al., 1997
;
Malgaretti et al., 1997
). The
present paper will focus on expression sites corresponding to the major
morphological changes observed in the mutants.
Phenotype
Fig. 1E shows the phenotypic
features of two 20-day-old Ebf2-/- males next to a
wild-type male littermate. At birth, Ebf2-/- mice were
hardly distinguishable from Ebf2+/- and
Ebf2+/+ ones after gross examination. The first obvious
abnormalities became detectable starting at 5 days after birth (P5).
Ebf2-/- mice were small compared with their
Ebf2+/+ and Ebf2+/- littermates:
homozygotes weighed less than half the bodyweight of their littermates by P30
(Ebf2+/+, 16.46±1.84 g, n=9;
Ebf2+/-, 15.34±2.45 g, n=12;
Ebf2-/-, 6.87±1.78 g, n=7;
Ebf2+/+ versus Ebf2-/-,
P<0.0001; Ebf2+/- versus
Ebf2-/-, P<0.0001; Ebf2+/+
versus Ebf2+/-, not significant). Studies are in progress
to define the physiological basis of this growth retardation.
Phenotypic and pathological changes described herein have been scored in mice derived through at least five generations of backcrossing on the C57BL6/J background, in order to minimize the effects of genetic background heterogeneity. Once they reached adulthood, Ebf2-/- males and females failed to reproduce when mated to wild-type breeders of the opposite gender. In addition, Ebf2-/- mice were mildly uncoordinated and walked with an unsteady, waddling gait. Most noticeably, they exhibited a `hunchback' posture both at rest and, more prominently, while walking.
GnRH-neurons from Ebf2-null mice fail to migrate into the
hypothalamus
The reproductive failure observed in our mutants suggested several possible
explanations, including an alteration in the hypothalamus-pituitary
gonadotropic axis, which governs sexual maturation
(Wilson et al., 1998). In this
respect, we investigated the development of gonadotropin releasing hormone
GnRH-neurons; these neurons are initially located in the vomeronasal organ
anlage of the nasal placode and subsequently migrate medially and dorsally
through the nasal mesenchyme and cribriform plate of the ethmoid bone along
the vomeronasal and terminal nerve fibers, then penetrate into the rostral
forebrain and move caudally into the septohypothalamic region, where they
extend axons towards the median eminence (ME) and infundibulum of the
pituitary gland (reviewed by Wray,
2001
). In agreement with previous reports
(Wang et al., 1997
),
lacZ staining in heterozygous midgestation embryos revealed
Ebf2 expression in the olfactory epithelium, and particularly in the
vomeronasal organ (Fig. 2A,B).
In Ebf2-/- embryos, olfactory and vomeronasal fibers could
be detected at all correct locations in the mutant forebrain at embryonic day
15, and stained positive for peripherin
(Wray et al., 1994
)
(Fig. 2D) and for the Tag-1
adhesion molecule (Wolfer et al.,
1994
) (not shown) in wild-type and mutant mice alike. Furthermore,
by histological analysis, we examined olfactory bulb sections obtained from
P30 wild-type and null mutant mice. No abnormalities were observed in the
cytoarchitecture of this region in Ebf2-/- mice
(Fig. 2F).
|
By dual immunofluorescence, we determined that Ebf2 is expressed
in migrating GnRH-neurons as the onset of their migration, i.e. E11 in the
mouse (Schwanzel-Fukuda and Pfaff,
1989) (Fig. 3).
Fig. 3 shows colocalization
(Fig. 3C) of GnRH
(Fig. 3A) and
ß-galactosidase (B) immunostaining in migrating E15 GnRH-neurons in the
null mutant's nasal mesenchyme. We used ß-galactosidase-like staining as
an unambiguous marker of Ebf2 expression, as polyclonal anti-Ebf2
antibodies available to us crossreact with other Ebf proteins. At embryonic
day 15 (E15), wild-type (wt) GnRH-neurons were mostly located dorsal to the
cribriform plate and were navigating caudally towards the preoptic region;
conversely, in E15 mutant embryos, most GnRH-neurons were still detected in
the nasal mesenchyme (Fig. 4B,
Fig. 3A).
Ebf2-/- neurons were very tightly clustered and in some
cases did not show any obvious leading nor trailing processes typical of
migrating GnRH-neurons. At birth (P0), GnRH-positive fibers were evident in
the ME of the hypothalamus (Fig.
4C), whereas no fibers were detectable in the ME of the mutants
(Fig. 4D). Finally, in P30
mutant brains, hardly any GnRH-positive neurons were detected either in the
septohypothalamic region (Fig.
4F) or in any region spanning the olfactory bulbs through the
anterior commissure. The defect in GnRH-neuron development did not appear to
be secondary to abnormal growth or navigation of the olfactory fibers that
support their neurophilic migration. In fact these fibers are normally present
in null mutants (Fig. 2D).
|
|
Based on the above observations, we investigated the eventual fate of GnRH-neurons in Ebf2-/- mice. As mentioned earlier, at E15 most neurons were abnormally retained in the nasal cavity of Ebf2-/- embryos, unlike in their wild-type counterparts. Hoechst counterstaining of these neurons revealed that in most cases their nuclei had indented borders and fuzzy to undiscernible nucleoli, suggesting that neurons that failed to migrate eventually degenerated (not shown). Neuronal degeneration appeared to be secondary to defective migration, as numerous neurons were still found in the nasal mesenchyme 5.5 days after their birth in the vomeronasal organ. Although migration of most GnRH-neurons was arrested in the nasal mesenchyme or just dorsal to the cribriform plate, a subset of GnRH-immunoreactive neurons were found in the forebrain at birth, at the caudal boundary of the olfactory bulb (Fig. 4G).
Consistent with the observed defect in GnRH-neuron development, inbred
Ebf2-/- mice were hypogonadic and failed to reproduce or
to exhibit any mating behavior when caged with wild-type C57BL6/J breeders.
This statement refers to a cumulative period of over 14 months of random
matings (n=3 homozygous mutant males; n=3 homozygous mutant
females). At birth, homozygous mutants displayed completely formed testes,
that were similar in size to their littermates. Conversely, inspection of
post-pubertal (P45, P60) male gonads showed a dramatic disparity in their
volume in Ebf2-/- and Ebf2+/+ males
(Ebf2+/+: 3.29±0.14 mm, n=4;
Ebf2-/-: 2.22±0.05 mm, n=4;
P=0.0004). Histological examination of Ebf2-/-
male gonads indicated testis hypoplasia without Leydig cell hyperplasia. This
was consistent with hypogonadotropic hypogonadism and argued strongly against
an intrinsic defect of the seminipherous tubule. Testes displayed a reduction
of the interstitial component, similar to what has been observed in
hypogonadal (hpg) mice (Cattanach et al.,
1977), which carry an intragenic deletion in the
Gnrh-gene (Mason et al.,
1986
). While Sertoli cells from normal and
Ebf2-/- mice were morphologically indistinguishable,
mutant seminipherous tubules contained fewer dividing spermatogonia
(Fig. 5D) compared with
wild-type tubules, as assayed by BrdU incorporation. Likewise, mutant
epidydimes had a reduced diameter and contained very few sperm cells
(Fig. 5F), when compared with
control wild-type epidydimes.
|
Morphological and functional defects in the mutant peripheral nervous
system
The defects observed in the development of GnRH-synthesizing neurons were
not the only morphogenetic abnormalities observed in the CNS of
Ebf2-/- mice. In fact, null mutants exhibited substantial
defects in cerebellar morphogenesis that are beyond the scope of the present
study, and are being actively investigated by our group (L. C. and S. Z.,
unpublished). In addition, major defects were also observed in the
Ebf2-/- peripheral nervous system.
In a systematic analysis of Ebf2 gene expression, we found
Ebf2 expression in the embryonic and postnatal spinal cord, dorsal
root ganglia and peripheral nerve (Fig.
6). A detailed analysis of lacZ expression in the mutant
mice provided us with new information in addition to that stemming from
previous studies. At E12.5, Ebf2 was expressed in dorsal root ganglia
(DRG), as reported previously (Garel et
al., 1997; Malgaretti et al.,
1997
). However, its expression (blue signal in
Fig. 6A) was clearly confined
to presumptive satellite cells negative for the heavy chain neurofilament
(NF-H). Likewise, dorsal root fibers positive for NF-H were negative for
cytoplasmic lacZ, while presumptive peripheral glial progenitors
intercalated to those fibers were positive (blue signal in
Fig. 6B). In agreement with
this, E16.5 immature glial progenitors positive for the low-affinity nerve
growth factor receptor p75 were also positive for lacZ staining
(Fig. 6C). In P15 sciatic
nerves from null mice, Ebf2 was expressed in both myelin forming
(msc) (arrows in Fig. 6D,E) and
non-myelin forming Schwann cells (nmsc) (arrowhead in
Fig. 6D), while in heterozygous
mice Ebf2 expression was detected only in nmsc (arrows in
Fig. 6F). In the P30 spinal
cord, Ebf2 was expressed in dorsal interneurons (lamina 2), in the
commissural gray matter and in other as yet undefined neural cells localized
in dorsal, and commissural areas of the spinal cord (blue signal in
Fig. 6G). Finally, in
Fig. 6H a high magnification of
ventral region of the spinal cord revealed colocalization of Ebf2 (in
blue) with choline acetyltransferase, a motoneuron marker (brown). Unlike in
postnatal development, Ebf2 was not obviously expressed in islet
1-positive motoneuron precursors at E10
(Garel et al., 1997
) or at
subsequent midgestation embryonic stages (L. C. and G. G. C.,
unpublished).
|
In keeping with the evidence of Ebf2 expression throughout glial
cell differentiation and in postnatal motoneurons, Ebf2-null sciatic
nerves showed various abnormalities in their postnatal development. At gross
examination, sciatic nerve trunks came apart spontaneously during surgical
manipulation. Histological analysis of Toluidine Blue-stained semi-thin
sections revealed that axons, especially large caliber ones, were considerably
hypomyelinated in the Ebf2-/- sciatic nerve
(Fig. 7). Likewise,
ultrastructural analysis of Ebf2-/- nerves revealed
several abnormalities. As previously described
(Webster et al., 1973), by P15
wild-type sciatic nerves display a complete segregation of large axons (
1
µm across) from smaller ones. The former establish a one-to-one
relationship with MSC and become myelinated; the latter are fasciculated
together by each NMSC and wrapped by cytoplasmic lamellae, remaining
unmyelinated (Fig. 8A).
Conversely, examination of mutant nerves revealed many unsorted medium to
large axons (1-4 µm) that failed to be myelinated. Even in adult nerves
(P30), these were fasciculated into anomalous bundles next to smaller axons
(Fig. 8B). Occasionally,
unsorted axons still wrapped by NMSC cytoplasm became abnormally myelinated
(Fig. 8C). Longitudinal
sections clearly demonstrated that in some cases myelinated and unmyelinated
segments belonged to consecutive internodes of the same axon, constituting a
case of segmental dysmyelination (Fig.
8D). In addition, some myelinated fibers featured abnormally thin
myelin sheaths (Fig. 8E). With
nerve maturation, signs of axonopathy became apparent: starting at about P15,
it became possible to observe features of axonal damage, such as accumulations
of vesicular, membrane-bound material in the axon
(Fig. 8F).
|
|
Based on the morphological abnormalities observed in mutant nerves, we conducted electrophysiological tests on adult, age- and gender-matched homozygous mutants (n=9) and controls (n=8). We stimulated the sciatic nerve at the ankle and at the ischiatic notch; the compound motor action potential (cMAP) was recorded from the paw muscles with a pair of needle electrodes to measure motor nerve conduction velocity (NCV). The mean values of NCV, compound motor axon potential (cMAP) latencies and amplitude, and F-wave latencies are summarized in Table 1. The main finding emerging from our studies was a striking reduction (by 40%) of NCV in the knockout group when compared with the wild-type group. All members of the knockout group scored lower in terms of NCV than controls. The mean amplitude of motor responses was also significantly decreased in comparison with controls, although cMAP amplitude data should be interpreted cautiously when dealing with needle recordings. Likewise, although the mean value of F-wave latency was found to be significantly higher in mutant nerves, this finding may simply reflect the overall decrease in NCV. Actually, the finding of measurable F-wave recordings in the mutant group ruled out a conduction block at the level of proximal nerve segments or spinal roots.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our data are consistent with the notion that proteins belonging to the Ebf
network play some redundant roles. Overall, Ebf2-/- mice
complete embryonic development successfully, albeit displaying subtle
abnormalities that are beyond the scope of the present study. However, our
evidence also indicates that some specific morphogenetic processes clearly
require Ebf2, despite the fact that other Ebf genes are co-expressed
with it. For example, although Ebf1 is coexpressed with Ebf2
in migrating GnRH-neurons (Wray,
2001), their migration is defective in Ebf2 mutants.
Likewise, although both Ebf1 and Ebf3 are co-expressed with
Ebf2 in the postnatal nerve (L. C. and G. G. C., unpublished), nerve
organization and conduction are clearly altered in Ebf2-/-
mice.
The study of Ebf2-/- embryos indicates that
Ebf2 is not required for the birth of GnRH-neurons, or that redundant
mechanisms are in place to offset the lack of Ebf2 in this function.
However, our mutants show a clear impairment in GnRH-neuron migration from the
olfactory epithelium to the diencephalon. In particular, GnRH-neurons exit the
nasal mesenchyme later than in wild-type mice, and show signs of degeneration.
Those mutant neurons that manage to reach dorsal to the cribriform plate, fail
to deflect caudally into the ventral forebrain. Instead, they move dorsally
through the rostral telencephalon, possibly along the dorsal branch of the
vomeronasal nerve. Altered migration of GnRH-neurons has been observed in
other animal models. For example, it has been reported that enzymatic removal
of polysialic acid from NCAM-rich vomeronasal nerves at E12 significantly
inhibits the migration of GnRH-neurons, without affecting the vomeronasal
tract itself (Yoshida et al.,
1999). The same authors found that NCAM180 knockout mutants show a
shift in the migratory route of GnRH cells, resulting in an excess number of
these neurons in the accessory olfactory bulb. Likewise, Dcc (deleted
in colorectal cancer, which encodes a netrin-1 receptor) knockout mice
revealed GnRH neurons that were misrouted into the cerebral cortex
(Schwarting et al., 2001
).
However, these mutants could not be studied at later stages, because of
postnatal lethality. In both genetic mutants cited above, the impairment of
GnRH-neuron migration is accompanied by malformation of the vomeronasal nerve.
Conversely, no alterations have been observed in Ebf2-/-
vomeronasal nerve axons, either by histology or using specific surface markers
(Wolfer et al., 1994
;
Wray et al., 1994
). Likewise,
no changes have been found in the olfactory bulbs of null mutants
(Fig. 2F). In other mutants
(Yoshida et al., 1999
), a
significant number of GnRH-neurons appear to follow a normal migratory route
into the diencephalon and to send projections to the ME, in keeping with the
existence of different GnRH-neuron subpopulations or heterogeneity of the
migratory pathway (Skynner et al.,
1999
; Tobet et al.,
1996
; Wray et al.,
1994
). In Ebf2-/- mice we observed a virtually
complete lack of immunoreactive fibers in the ME, and a complete lack of
immunoreactivity throughout the septohypothalamic region; this evidence, along
with the observed expression of Ebf2 in GnRH neurons, strongly
supports a fundamental role for the Ebf2 protein in GnRH-neuron development.
Previous studies have shown that mutation of another Ebf family member
(Ebf1) (Lin and Grosschedl,
1995
) results in a subpopulation of facial branchiomotor neurons
adopting a rhombomere 6-specific short-range migration pattern in rhombomere
5, in the embryonic hindbrain (Garel et
al., 2000
). Not only are our findings consistent with those
observations, but they clearly implicate Ebf2 as a primary factor in
the long range developmental migration of an entire population of
neuroendocrine neurons. As the Ebf2 gene is expressed both in GnRH
and vomeronasal neurons, our results allow no conclusions to be drawn as to
whether the observed migration defect is cell-autonomous, or secondary to the
lack of axonal cues. Transgenic approaches should make it possible to solve
this riddle, shedding further light on the role of Ebf2 in neuronal
migration.
To the authors' knowledge this is the first report of a single genetic
defect that directly affects GnRH-neuron migration with no gross alterations
of their migration substrate. In this respect, our findings differ
substantially from those reported in a fetus with Kallmann Syndrome (KS),
where the defect in GnRH-neuron migration is accompanied by atrophy of the
olfactory bulb (Schwanzel-Fukuda et al.,
1989). Several genes have been proposed as potential candidates
for other forms of KS or for idiopathic hypogonadotropic hypogonadism (HH).
These include GnRH receptors, the LH and FSH genes, and
DAX1, etc. (Layman,
1999
). The evidence reported in the present paper indicates the
human ortholog of Ebf2 as a solid functional candidate for genetic
studies of HH. Curiously, by computer analysis of the human genome sequence,
we determined that the EBF2 gene maps within 450 kb of the
GNRH in human chromosomal band 8p21.
In agreement with the observed CNS defects, examination of post-pubertal
mutant mice shows clear evidence of testis hypoplasia in the absence of Leydig
cell hyperplasia. The presence of testes, albeit hypoplastic, in these animals
excludes an essential role for Ebf2 in the prenatal stages of gonadal
development, and is coherent with the observation that, during fetal
development, differentiation and proliferation of both Sertoli and Leydig
cells are independent of endogenous gonadotropins
(Baker and O'Shaughnessy, 2001;
Griswold, 1993
;
Lejeune et al., 1998
). Not
surprisingly, testicular abnormalities seen in Ebf2-/-
mice are superimposable with those observed in hypogonadal (hpg) mice, which
carry a null mutation of the Gnrh gene, and, therefore, fail to
produce gonadotropins (Mason et al.,
1986
).
The second most striking abnormality in Ebf2-/- mutants
resides in the peripheral nerve. Ebf2 is expressed in peripheral
glial cell progenitors starting at E12.5, and remains expressed throughout
birth in immature Schwann cells, and through adulthood in nmsc. The defect in
the developmental downregulation of Ebf2 transcription observed in
msc from null mutant mice suggests an important role for this gene in late
embryonic and postnatal development of those cells. In addition to Schwann
cells, Ebf2 is also expressed in postnatal motoneurons. The sciatic
nerve of Ebf2 mutants features glial and axonal defects. These
include incomplete axon sorting, which results in defective axonal
fasciculation. The notion of an evolutionarily conserved role for Ebf family
members in axon fasciculation is supported by the abnormal organization
observed in the ventral nerve cord of unc-3 mutant nematodes, that
carry a mutation within an Ebf homolog
(Prasad et al., 1998).
In parallel to a clear defect in axonal sorting, Ebf2-/- nerves feature signs of segmental dysmyelination and hypomyelination. In fact, the finding of large unsorted axons in transverse nerve sections and that of segmentally unmyelinated axons in longitudinal sections may be linked features of a common defect, whereby large axons fail to establish contacts with msc throughout their length, and as a consequence, are myelinated discontinuously. However, persistent expression of the lacZ reporter gene in Ebf2-/- msc may also suggest a direct role for Ebf2 in msc terminal differentiation (which may, in turn, explain the finding of hypomyelinated axons in the adult nerve).
Consistent with the observed signs of dysmyelination, electrophysiological tests reveal a 40% decrease in NCV in mutant nerves, suggesting that currents may leak across the unmyelinated membrane, hampering an efficient action potential propagation. Again, in keeping with the segmental nature of the defect, our conduction studies do not disclose any electrophysiological hallmarks of widespread demyelination, such as a temporal dispersion and polyphasia of motor responses, or a significant proximal-to-distal amplitude decrement. Although segmental dysmyelination is expected to slow down conduction, altered expression or defective clustering of Na+ or K+ channels in the nodal and juxtaparanodal regions, respectively, may offer an alternative or additional explanation. Further studies are required to address this point.
In conclusion, through a genetic approach, we have implicated the Ebf2 gene in the pathogenesis of two phenotypic defects of potential relevance in medical genetics, namely hypogonadotropic hypogonadism and peripheral neuropathy with segmental dysmyelination. In addition, our data provide evidence for a pivotal role of Ebf2 in the formation of the neuroendocrine axis, which supports pubertal development, and in several morphogenetic events required for peripheral nerve maturation. Molecular and biochemical studies are now required to dissect the genetic circuits involved in those processes.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ausubel, F. M., Brent, R., Kingstone, R. E., Moore, D. D., Smith, J. A. and Struhl, K. (1995). Current Protocols in Molecular Biology. New York: J. Wiley and Sons.
Baker, P. J. and O'Shaughnessy, P. J. (2001).
Role of gonadotrophins in regulating numbers of Leydig and Sertoli cells
during fetal and postnatal development in mice.
Reproduction 122,227
-234.
Cattanach, B. M., Iddon, C. A., Charlton, H. M., Chiappa, S. A. and Fink, G. (1977). Gonadotrophin-releasing hormone deficiency in a mutant mouse with hypogonadism. Nature 269,338 -340.[Medline]
Crozatier, M. and Vincent, A. (1999).
Requirement for the Drosophila COE transcription factor Collier in formation
of an embryonic muscle: transcriptional response to notch signalling.
Development 126,1495
-1504.
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.[Medline]
Dubois, L. and Vincent, A. (2001). The COECollier/Olf1/EBFtranscription factors: structural conservation and diversity of developmental functions. Mech. Dev. 108,3 -12.[CrossRef][Medline]
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.
Garel, S., Garcia-Dominiguez, M. and Charnay, P.
(2000). Control of the migratory pathway of facial branchiomotor
neurones. Development
127,5297
-5307.
Griswold, M. (1993). Action of FSH on mammalian Sertoli cells. In The Sertoli Cell (ed. L. D. Russel and M. D. Griswold), pp. 493-508. Clearwater: Cache River Press.
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.[Abstract]
Joyner, A. L. (1993). Gene Targeting A Practical Approach. Oxford: IRL Press.
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.[Abstract]
Layman, L. C. (1999). The molecular basis of human hypogonadotropic hypogonadism. Mol. Genet. Metab. 68,191 -199.[CrossRef][Medline]
Lejeune, H., Habert, R. and Saez, J. M. (1998).
Origin, proliferation and differentiation of Leydig cells. J. Mol.
Endocrinol. 20,1
-25.
Lin, H. and Grosschedl, R. (1995). Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature 376,263 -267.[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.
Mason, A. J., Hayflick, J. S., Zoeller, R. T., Young, W. S., 3rd, Phillips, H. S., Nikolics, K. and Seeburg, P. H. (1986). A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse. Science 234,1366 -1371.[Medline]
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.
Schwanzel-Fukuda, M. and Pfaff, D. W. (1989). Origin of luteinizing hormone-releasing hormone neurons. Nature 338,161 -164.[CrossRef][Medline]
Schwanzel-Fukuda, M., Bick, D. and Pfaff, D. W. (1989). Luteinizing hormone-releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome. Mol. Brain Res. 6,311 -326.[Medline]
Schwarting, G. A., Kostek, C., Bless, E. P., Ahmad, N. and
Tobet, S. A. (2001). Deleted in colorectal cancer (DCC)
regulates the migration of luteinizing hormone-releasing hormone neurons to
the basal forebrain. J. Neurosci.
21,911
-919.
Skynner, M. J., Slater, R., Sim, J. A., Allen, N. D. and
Herbison, A. E. (1999). Promoter transgenics reveal multiple
gonadotropin-releasing hormone-I- expressing cell populations of different
embryological origin in mouse brain. J. Neurosci.
19,5955
-5966.
Tobet, S. A., Chickering, T. W., King, J. C., Stopa, E. G., Kim, K., Kuo-Leblank, V. and Schwarting, G. A. (1996). Expression of gamma-aminobutyric acid and gonadotropin-releasing hormone during neuronal migration through the olfactory system. Endocrinology 137,5415 -5420.[Abstract]
Vervoort, M., Crozatier, M., Valle, D. and Vincent, A. (1999). The COE transcription factor Collier is a mediator of short-range Hedgehog-induced patterning of the Drosophila wing. Curr. Biol. 9,632 -639.[CrossRef][Medline]
Wang, M. M. and Reed, R. R. (1993). Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast. Nature 364,121 -126.[CrossRef][Medline]
Wang, S. S., Tsai, R. Y. L. and Reed, R. R.
(1997). The characterization of the Olf-1/EBF-like HLH
transcription factor family: implications in olfactory gene regulation and
neuronal development. J. Neurosci.
17,4149
-4158.
Wang, S. S., Betz, A. G. and Reed, R. R. (2002). Cloning of a novel Olf-1/EBF-like gene, O/E-4, by degenerate oligo-based direct selection. Mol. Cell. Neurosci. 20,404 -414.[CrossRef][Medline]
Webster, H. d., Martin, J. R. and O'Connell, M. F. (1973). The relationships between interphase Schwann cells and axons before myelination: a quantitative electron microscopic study. Dev. Biol. 32,401 -416.[Medline]
Wilson, J., Foster, D., Kronenberg, H. and Larsen, P. (1998). Williams' Textbook of Endocrinology. London: WB Saunders Company.
Wolfer, D. P., Henehan-Beatty, A., Stoeckli, E. T., Sonderegger, P. and Lipp, H. P. (1994). Distribution of TAG-1/axonin-1 in fibre tracts and migratory streams of the developing mouse nervous system. J. Comp. Neurol. 345,1 -32.[Medline]
Wray, S. (2001). Development of luteinizing hormone releasing hormone neurones. J. Neuroendocrinol. 13,3 -11.[CrossRef][Medline]
Wray, S., Key, S., Qualls, R. and Fueshko, S. M. (1994). A subset of peripherin positive olfactory axons delineates the luteinizing hormone releasing hormone neuronal migratory pathway in developing mouse. Dev. Biol. 166,349 -354.[CrossRef][Medline]
Yoshida, K., Rutishauser, U., Crandall, J. E. and Schwarting, G.
A. (1999). Polysialic acid facilitates migration of
luteinizing hormone-releasing hormone neurons on vomeronasal axons.
J. Neurosci. 19,794
-801.
Zielasek, J., Martini, R. and Toyka, K. V. (1996). Functional abnormalities in P0-deficient mice resemble human hereditary neuropathies linked to P0 gene mutations. Muscle Nerve 19,946 -952.[CrossRef][Medline]