Download Msx1 disruption leads to diencephalon defects and hydrocephalus

DEVELOPMENTAL DYNAMICS 230:446 – 460, 2004
RESEARCH ARTICLE
Msx1 Disruption Leads to Diencephalon Defects
and Hydrocephalus
Casto Ramos,1* Pedro Fernández-Llebrez,2 Antoine Bach,3 Benoı̂t Robert,3 and Eduardo Soriano1,4
We have analyzed the expression of the Msx1 gene in the developing mouse brain and examined the brain phenotype
in homozygotes. Msx1 is expressed in every cerebral vesicle throughout development, particularly in neuroepithelia,
such as those of the fimbria and the medulla. Timing analysis suggests that Msx1nLacZ cells delaminate and migrate
radially from these epithelia, mainly at embryonic days 14 –16, while immunohistochemistry studies reveal that some
of the ␤-galactosidase migrating cells are oligodendrocytes or astrocytes. Our results suggest that the Msx1
neuroepithelia of fimbria and medulla may be a source of glial precursors. The Msx1 mutants display severe
hydrocephalus at birth, while the subcommissural organ, the habenula, and the posterior commissure fail to develop
correctly. No label was detected in the mutant subcommissural organ using a specific antibody against Reissner’s
fiber. Besides, the fasciculus retroflexus deviates close to the subcommissural organ, while the paraventricular
thalamic nucleus shows histological disorganization. Our results implicate the Msx1 gene in the differentiation of the
subcommissural organ cells and posterior commissure and that Msx1 protein may play a role in the pathfinding and
bundling of the fasciculus retroflexus and in the structural arrangement of the paraventricular thalamic nucleus.
Developmental Dynamics 230:446 – 460, 2004. © 2004 Wiley-Liss, Inc.
Key words: Msx genes; brain development; glial precursors; astrocytes and oligodendrocytes; diencephalon; hydrocephalus;
axonal guidance
Received 28 July 2003; Revised 12 December 2003; Accepted 30 December 2003
INTRODUCTION
The msh/Msx gene family is one of
the most highly conserved families of
homeobox-containing genes identified in a variety of animal species
from diploblastic organisms to humans (Davidson, 1995). Vertebrate
Msx genes were originally cloned by
homology to the Drosophila muscle
segment homeobox (msh) gene,
and in mammals, there are three
members, Msx1, Msx2, and Msx3,
that share 98% homology in the homeodomain (Holland, 1991; Davidson, 1995).
In Drosophila, msh expression is first
detected in the mesoderm of the
developing somatic musculature of
the embryo and it is later restricted
to the central nervous system (CNS)
and muscle (Lord et al., 1995;
D’Alessio and Frasch, 1996; Isshiki et
al., 1997). Whereas Msx3 expression
is limited to the neural tube (Shimeld
et al., 1996; Wang et al., 1996), Msx1
and Msx2 genes are widely expressed during vertebrate development at many sites of inductive epithelial–mesenchymal interactions,
such as limb buds, branchial arches
1
and craniofacial processes, and
tooth germs (Hill et al., 1989; Robert
et al., 1989; Monaghan et al., 1991;
MacKenzie et al., 1991; Lyons et al.,
1992). Furthermore, experimental
embryology showed that the induction process is required for expression
at these sites (Davidson et al., 1991;
Robert et al., 1991; Brown et al.,
1993; Jowett et al., 1993). Thus, it has
been proposed that Msx1 gene is
involved in epithelial–mesenchymal
signalling and the control of proliferation and differentiation (Davidson,
1995; Hu et al., 2001). Murine Msx1
Department of Cell Biology, University of Barcelona, Barcelona, Spain
Department of Cell Biology, Genetics and Physiology, University of Málaga, Málaga, Spain
Department of Developmental Biology, Institut Pasteur, Paris, France
4
Barcelona Science Park, Laboratory A1-S1, Barcelona, Spain
Grant sponsor: MCYT; Grant number: SAF-2001-3290; Grant sponsor: Caixa Foundation.
*Correspondence to: Casto Ramos, Department of Cell Biology, University of Barcelona, E-08028 Barcelona, Spain.
E-mail: [email protected]
2
3
DOI 10.1002/dvdy.20070
Published online 5 May 2004 in Wiley InterScience (www.interscience.wiley.com).
© 2004 Wiley-Liss, Inc.
Msx1 DISRUPTION AND HYDROCEPHALUS 447
and Msx2 genes are also expressed
in the developing dorsal neural tube
and neural crest derivatives as they
migrate from the neural tube (Hill et
al., 1989; Lyons et al., 1992; Houzelstein et al., 1997). In addition, Msx2
gene is expressed in the rhombencephalon, where it seems to be
linked with apoptosis (Graham et al.,
1994; Maden et al., 1997).
In Drosophila, msh loss-of-function
mutations lead to various alterations
of neuroblasts and glial cells formed
in the dorsal neuroectoderm, such
as abnormal morphology and defects in migration, cell division, and
axon-tract formation, including a
possible dorsal-to-ventral fate switch
(Isshiki et al., 1997). In addition, ectopic expression of msh in the mesoderm leads to a loss and patterning
defects of certain muscles (Lord et
al., 1995; Nose et al., 1998), and inhibits proper differentiation of the
midline and ventral neuroblasts,
causing several alterations, such as
disruption of the axons tracts and an
almost complete absence of commissures (Isshiki et al., 1997). These
results provide evidence in vivo for
the role of the msh/Msx genes in
neural development and suggest
that they may perform phylogenetically conserved functions in the patterning of the neuroectoderm
(D’Alessio and Frasch, 1996; Isshiki et
al., 1997; Maden et al., 1997; Weiss et
al., 1998; Cornell and von Ohlen,
2000). In the mouse, insertional mutation of the Msx1 gene leads to an
arrest of tooth development and
several defects in craniofacial structures, such as cleft palate; reduced
mandible length; abnormalities in
nasal, frontal, and parietal bones;
and defects in the middle ear, suggesting a key role of Msx1 in the formation of osseous tissue of the head
(Satokata and Maas, 1994; Houzelstein et al., 1997; Blin-Wakkach et al.,
2001). Furthermore, Msx1 and Msx2
homeobox genes are known to be
involved in human pathologic conditions (Jabs et al., 1993; Vastardis et
al., 1996; van den Boogaard et al.,
2000; Wilkie et al., 2000).
We have proposed recently, focusing our study in early embryos,
that Msx genes may regulate Wnt1
expression at the dorsal midline of
the diencephalon and rostral mes-
encephalon (Bach et al., 2003). Taking advantage of the high resolution
and sensitivity of the nLacZ reporter
in a mutant Msx1 allele that we produced previously (Houzelstein et al.,
1997), we show in the present study
that Msx1 gene is expressed in many
cerebral structures, especially neuroepithelia and derivatives. The results were confirmed by in situ hybridization, and as in a previous study
(Houzelstein et al., 1997), we demonstrate that the pattern of expression
of ␤-galactosidase from the targeted Msx1nlacZ closely mimics that
of Msx1 mRNA. Moreover, the study
of the phenotype reveals that brain
anomalies are strictly linked to Msx1
ependymal-derived cells from the
dorsal midline of prosomere 1 and
show that the subcommissural organ
is not functional in homozygous newborns. The possible implication of this
structure in other cerebral disorders,
such as hydrocephalus, maldeveloped posterior commissure, anomalous fasciculus retroflexus, and disorganized paraventricular thalamic
nucleus is discussed.
RESULTS
Whole-Mount Embryos
Expression analyses in whole-mount
embryos revealed that the Msx1
gene, detected by beta-galactosidase activity, was highly expressed
all along the dorsal part of the neural tube at embryonic day (E) 10 and
E12, especially in the area of the diencephalon (Fig. 1A–C). In addition,
analysis of sections showed that, at
E12, Msx1 was also expressed in the
dorsal midline of the neural tube
(Fig. 6D) from the optic stalk to the
tip of the tail (not shown).
Expression in the
Telencephalon
The forebrain contained numerous
structures where Msx1 is expressed
during embryogenesis (Table 1).
Moderate Msx1 expression was
found in the accessory olfactory
bulb at postnatal day (P) 0 (data not
shown). No expression was detected in the neocortex in any layer
at any stage.
High level of Msx1 expression was
found in the dentate gyrus neuroepithelium, particularly at E14 and E16
(Fig. 2A), whereas no label was detected at E12. Only weak Msx1 signals were detected at E18 and P0 in
the hippocampal commissure, while
a weak but stable label was detected in the fimbria and fornix from
E16 onward (Fig. 2B). Msx1 is strongly
expressed in the choroidal ependymocytes and, to a lesser extent, in
the conjunctive tissue and vascular
cells of the choroid plexus of the lateral ventricles, from the beginning of
their formation, i.e., around E12. The
expression remained stable during
organogenesis in these structures
(Fig. 2B; see also Fig. 6B).
Some Msx1 expression sites were
seen in the basal forebrain throughout the development. Thus Msx1 was
moderately expressed in the strionuclear neuroepithelium at E12–E16
and characterized by a marked reduction in the intensity at later
stages. Other weakly labeled sites
were described in the basal forebrain, such as the amygdala hippocampal area and its corresponding neuroepithelium (data not
shown).
One of the most intense sites of
Msx1 expression in the telencephalon was located at posterior levels of
the hypothalamic area. The mammillary and the posterior hypothalamic neuroepithelia were strongly
labeled from E12 onward, with a
peak of intensity at E14 and a slight
decrease at P0 (Fig. 3A–C). Low expression during embryonic life was
also seen in other hypothalamic
structures, such as the intermediate
hypothalamic neuroepithelium, the
supramammillary nucleus, and the
medial mammillary nucleus (data
not shown). At E14, Msx1 expression
was strong in the arcuate nucleus
but decreased thereafter (Fig. 3C).
Previous studies of Msx1 gene expression in the eye by in situ hybridization have mentioned only a label
in the ciliary bodies (Monaghan et
al., 1991). We have also found low
but constant expression in the cornea, in the lens epithelium, and
along the optic nerve throughout
embryogenesis (data not shown).
448 RAMOS ET AL.
Fig. 1. Detection of ␤-galactosidase activity
in heterozygous embryos. A–C: Note the
strong labeling all along the dorsal midline of
the neural tube in embryonic day (E) 10 embryos (A,B) and, particularly in the diencephalon (epiphysis) in E12 embryos (arrow in C).
See also Figure 6D. Scale bars ⫽ 500 ␮m.
Fig. 6. Msx1 expression in the rhombencephalon. A,B,C,E,F: X-Gal histochemistry.
A,B: Embryonic day (E) 16, sagittal sections.
A: The anterior (ap) and posterior (pp) precerebellar neuroepithelia, and the medullary dorsal fissure (df) displayed a strong
expression (see also C,F). c, cerebellum; m,
medulla; circle, see C for explanation. B:
Only the midline of the cerebellar neuroepithelium (ce) is labeled (see Fig. 2A). Some
labeled cells were seen in the cerebellum
(arrows) and very close to the cerebellar
neuroepithelium. Observe that the conjunctive tissue (asterisk) of the choroid plexus
(cp) showed little expression. C: Postnatal
day 0, coronal section. Msx1 expression is
present in the area postrema (ap), the subarea postrema (sap), the medullary dorsal
fissure (df), the hypoglossal nuclei (arrows),
the medullary raphe (mr) and in two clusters of cells in the lateroventral parts all
along the medulla (circle). These cells were
also observed in other sections and stages
(see Fig. 6A,D–F). D: E12, coronal section, in
situ hybridization. Two clusters of Msx1-positive cells were observed in the lateroventral
parts of the medulla (arrowheads), near
the epithelium. The arrow indicates the roofplate was strongly labeled. E: E12, coronal
section. The labeled cells (arrows) of the
ventral medulla seemed to derive from the
close epithelium (me), where expression
was also detected. F: E16, coronal section
across the spinal cord. In this embryo, the
Msx1-expressing cells formed two rows in
each of the ventral horns (vh). Scale bars ⫽
500 ␮m in A,C,100 ␮m in B,E, 250 ␮m in D,F.
Fig. 7. Msx1 is expressed in glial cells. A,B:
Postnatal day (P) 0, coronal sections, X-gal
histochemistry. Some beta-galactosidase
labeled cells (arrows) of the fimbria (f)
showed a glial fibrillary acidic protein
(GFAP) immunoreactivity. C: However, the
P0, sagittal section, was marked with an
anti-O4 antibody (arrows and magnification in the upper left corner) in the ventral
medulla. See circles in Figure 6A,C. Scale
bars ⫽ 100 ␮m in A, 25 ␮m in B, 50 ␮m in C.
Figure 6.
Figure 7.
Msx1 DISRUPTION AND HYDROCEPHALUS 449
Fig. 2. Msx1 expression in the hippocampal formation. X-Gal histochemistry. A: Embryonic day (E) 14, coronal section. In this stage, Msx1 is heavily expressed in the dentate
gyrus neuroepithelium (dt) and in the choroid plexus (cp), as during the whole embryonic life. he, hippocampal neuroepithelium; m, meninges. B: E18, coronal section. The
fimbria (f) is weakly labeled. Observe the strong choroid plexus labeling of the first
ventricle (I). Scale bars ⫽ 250 ␮m in A,B.
strong Msx1 expression (Fig. 4A,B).
Nevertheless, the intensity of staining
in these structures decreased at late
embryonic stages (see Table 1).
Other
circumventricular
organs
were found to express the Msx1
gene. So, the lamina terminalis was
strongly labeled during development, whereas the vascular organ
of the lamina terminalis and the median preoptic nucleus were slightly
labeled, but only at E18 –P0 (Fig.
4C,D). Also, very low, steady expression levels were detected in the subfornical organ from E14 onward (Fig.
4D).
Moderate to low expression was
seen in all fiber tracts of the diencephalon (see Table 1). Thus, very
weak signals appeared first in the PC
(Fig. 4A), then from E14 onward in
the habenular commissure. In the
latter structure, the intensity remained stable through the first
stages of embryogenesis, then progressively decreased in later phases.
Similar intensity of label was seen in
the stria medullaris from E16 onward,
becoming somewhat higher at P0.
Msx1 expression was detected in the
stria terminalis only at neonatal
stages (data not shown).
Expression in the
Mesencephalon
Fig. 3. Detection of ␤-galactosidase activity in the hypothalamic area, colliculi, and
cerebellum. A: Embryonic day (E) 14, horizontal section. B,C: E16, sagittal sections. Observe in A that the cerebellar neuroepithelium (ce) and the colliculi (co) neuroepithelia
(arrowheads) are only labeled in the midline. In B,C, Msx1 is heavily expressed in the
mammillary neuroepithelium (arrows) and in the posterior hypothalamic neuroepithelium (ph). c, cerebellum; he, hippocampal neuroepithelium; pg, pituitary gland; t, tectum; te, tectal neuroepithelium; tge, tegmental neuroepithelium; asterisk, arcuate nucleus. See Figure 5B for the explanation of the selected area from B. Scale bars ⫽ 500 ␮m
in A, B, and C (applies to B,C).
Expression in the Diencephalon
Another prominent area of Msx1 expression in the CNS along development was in the dorsal part of the
diencephalon (Table 1). Msx1 transcripts were very abundant in the
subcommissural organ (SCO) from
E14 onward. The SCO is a small secretory gland composed of special-
ized ependymal-derived cells of
neuroepithelial origin, which occupies the diencephalic roof, just beneath the posterior commissure
(PC). Other related structures, such
as the habenula, the pineal gland,
the pretectal neuroepithelium, and
to a lesser extent, the epithalamic
neuroepithelium, were also sites of
In the midbrain, Msx1 expression was
mainly limited to the tectum and the
tegmentum (Table 1). All the tectal
(superior and inferior colliculi) and
the tegmental neuroepithelia displayed strong, nearly constant label
before birth, with a slight decrease in
the latter in older embryos (Figs. 3B,
5B). Only the midline of these neuroepithelia expressed the Msx1 gene
(Figs. 3A, 5A). During development,
Msx1-positive cells appeared to delaminate from the tegmental neuroepithelium, more intensively in the
areas where the expression was the
heaviest, and migrate inside the tegmentum. The ␤-gal⫹ migrating cells
moved radially on the sagittal plane,
sometimes forming rows (Fig. 5B). At
E12, the tegmental neuroepithelium
also strongly expressed Msx1 gene;
however, delaminated ␤-gal⫹ cells
are seen adjacent to the former. The
delamination gradually increased
throughout embryogenesis and be-
450 RAMOS ET AL.
TABLE 1. Msx1 Expression in the Developing Mouse Braina
Telencephalon
Olfactory bulb
Accessory olfactory bulb
Hippocampal formation
Choroid plexus of the lateral ventricles
Dentate gyrus neuroepithelium
Fimbria/fornix
Hippocampal commissure
Hippocampal neuroepithelium
Stratum lacunosum moleculare
Subicular neuroepithelium
Basal forebrain
Amygdala hippocampal area
Amygdaloid neuroepithelium
Preoptic neuroepithelium
Strionuclear neuroepithelium
Hypothalamus
Arcuate nucleus
Intermediate hypothalamic neuroepithelium
Mammillary neuroepithelium
Medial mamillary nucleus
Pituitary gland
Pars nervosa
Pars intermedia
Pars distalis
Posterior hypothalamic neuroepithelium
Supramammillary nucleus
Optic vesicle
Ciliary bodies
Cornea
Lens epithelium
Optic nerve
Diencephalon
Choroid plexus of the first ventricle
Epithalamic neuroepithelium
Habenula
Habenular commissure
Lamina terminalis (LT)
Median preoptic nucleus
Pineal gland
Posterior commissure
Pretectal neuroepithelium
Septal neuroepithelium
Stria medullaris
Stria terminalis
Subcommissural organ
Subfornical organ
Vascular organ of LT
Mesencephalon
Dorsal raphe
Median raphe
Tectal neuroepithelium
Tegmental neuroepithelium
Rhombencephalon
Anterior precerebellar neuroepithelium
Area postrema
Cerebellar cells
Cerebellar germinal trigone
Cerebellar neuroepithelium
Choroid plexus of IV ventricle
Cochlear external germinal layer
Cochlear nuclei (dorsal and ventral)
Cochlear nuclei neuroepithelium
E12
E14
E16
E18
P0
ND
ND
ND
ND
⫹⫹
⫹⫹⫹
⫺
⫺
⫺
⫹⫹
⫺
⫺
⫹⫹⫹
⫹⫹⫹
⫹
⫺
⫹
⫺
⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫺
⫹
⫺
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹
⫹/⫺
⫹/⫺
⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹
⫹/⫺
⫹/⫺
⫹
⫺
⫺
⫹
⫹⫹
⫺
⫹/⫺
⫹
⫹⫹
⫹/⫺
⫹
⫹
⫹⫹
⫹/⫺
⫹
⫺
⫹/⫺
⫹/⫺
⫹
⫺
⫹/⫺
⫹⫹
⫺
⫹⫹⫹
⫹
⫹⫹⫹
⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹
⫹
⫹⫹⫹
⫹
⫹⫹
⫹
⫹⫹⫹
⫹
⫹⫹
⫹
⫹⫹
⫹/⫺
⫹/⫺
⫹⫹
⫹⫹⫹⫹
⫹⫹⫹
⫹
⫹/⫺
⫹⫹
⫹⫹⫹⫹
⫹⫹⫹
⫹
⫹/⫺
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹/⫺
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
ND
ND
ND
⫹⫹
⫹/⫺
⫹⫹⫹
⫹
⫺
⫹
⫹⫹⫹
⫹
⫹/⫺
⫹
⫹⫹⫹
⫹
⫹
⫹
⫹⫹⫹
⫹
⫹
⫹
⫹⫹⫹
ND
⫹
⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫺
⫹⫹⫹
⫺
⫹⫹⫹
⫹/⫺
⫹⫹⫹
⫺
⫺
⫺
⫺
⫺
ND
⫹⫹⫹
⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹⫹
⫺
⫹⫹⫹
⫹/⫺
⫹⫹⫹
⫺
⫺
⫺
⫹⫹⫹⫹
⫹/⫺
ND
⫹⫹⫹
⫹⫹
⫹⫹⫹⫹
⫹
⫹⫹⫹
ND
⫹⫹
⫹/⫺
⫹⫹⫹
⫺
⫹/⫺
⫺
⫹⫹⫹⫹
⫹/⫺
ND
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹/⫺
ND
⫹
⫹
⫹/⫺
⫹⫹
⫹/⫺
⫹/⫺
⫺
⫹⫹⫹⫹
⫹/⫺
⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹/⫺
ND
⫹
⫹
⫹/⫺
⫹⫹
⫹
⫹
⫹
⫹⫹⫹⫹
⫹/⫺
⫹
⫹
⫺
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫺
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹
⫹⫹⫹
⫹⫹
⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹⫹
ND
⫹/⫺
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹
⫺
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹/⫺
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹
⫺
⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹
⫺
⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹/⫺
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹
⫹⫹⫹
Msx1 DISRUPTION AND HYDROCEPHALUS 451
TABLE 1. Msx1 Expression in the Developing Mouse Braina
Hypoglossal nucleus
Medullary dorsal fissure
Medullary neuroepithelium
Medullary raphe
Medullary ventrolateral nuclei
Pontine raphe
Posterior pontine neuroepithelium
Posterior precerebellar neuroepithelium
Subarea postrema
Velum medullare
Vestibular nuclei
Other structures
Meninges
Superior sagittal sinus
a
E12
E14
E16
E18
P0
ND
⫺
⫹⫹⫹
⫺
⫹⫹
⫺
⫺
⫹⫹⫹⫹
ND
⫹⫹⫹
⫺
ND
⫺
⫹⫹⫹
⫺
⫹⫹
⫺
⫹/⫺
⫹⫹⫹⫹
ND
⫹⫹⫹
⫺
⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹
⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹
⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹
⫹⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
ND
ND, not determined; E, embryonic day; P, postnatal day.
came maximum at E14 –E16, where
the migration was also the most intense. At E18 –P0, fewer cells delaminated adjacent to the tegmental
neuroepithelium, in accordance
with the decrease of Msx1 expression in this structure. This scenario of
delamination and migration of Msx1expressing cells from Msx1-labeled
neuroepithelia was observed at several places during brain development, i.e., in the fimbria (Fig. 2B), in
the cerebellum (Fig. 6B), and in the
medulla (Fig. 6E,F). All these structures showed a similar expression
profile with a peak of delamination/
migration activity at E14 –E16.
Expression in the
Rhombencephalon
Msx1 expression in the hindbrain was
similar to that observed in the foreand midbrain, i.e., all the Msx1-labeled structures retained a constant
label throughout embryogenesis.
Maximal intensity was observed in
the anterior and posterior precerebellar neuroepithelia, the medullary neuroepithelium, the cerebellar
germinal trigone, and the cerebellar
neuroepithelial midline (Table 1). This
last structure showed lower intensity
around birth. Some labeled cells
were located very close to this neuroepithelium, whereas others were
scattered through the ventral cerebellum, on the midline plane (Fig.
6A,B).
In the pons, Msx1 expression was
strong and stable during development in the cochlear nuclei neuroepithelium. Msx1 transcripts were
also abundant in the dorsal and
ventral cochlear nuclei at E12 but
decreased substantially from E14 onward. At E12–E16, no label was detected in the cochlear external germinal layer. However, Msx1 expression in
this structure was moderate at E18 –
P0. Msx1 was weakly expressed in the
posterior pontine neuroepithelium
and in the pontine raphe from E14
and E16 onward, respectively, with a
peak of expression at birth (data not
shown).
The periventricular organs of the IV
ventricle, the velum medullare and
the choroid plexus, like those of the
first and lateral ventricles, displayed
strong steady Msx1 expression in the
fetus (Fig. 6B). Msx1 was also moderately and strongly expressed in the
area postrema and in the subarea
postrema, respectively, from E16 onward (Fig. 6C).
At more posterior levels, Msx1 was
heavily expressed in the medullary
dorsal fissure at E16 –P0 stages. Low
expression was detected in the
medullary raphe, in the vestibular
nuclei and in the hypoglossal nucleus, from E16 onward (Fig. 6C). At
E12, two clusters of Msx1-expressing
cells were located at each side of
the midline of the ventral neural
tube close to the neuroepithelium,
from the medulla backward (Fig.
6D). Some Msx1-positive cells were
also detected within the neuroepi-
thelium facing the clusters (Fig. 6E).
As development proceeded, X-gal–
positive cells were scattered through
the ventral part of the medulla and
along the longitudinal axis in the
ventral horns of the spinal cord (Fig.
6C,F). These groups of labeled cells
were named ventrolateral nuclei, as
they were not determined.
Msx1nLacZ Delaminating Cells
From Fimbria and Medulla
Epithelia Are Glial Cells
To determine the nature of Msx1-expressing cells apparently derived
from some neuroepithelia (i.e., those
from the fimbria, the tegmentum,
the cerebellum, and the medulla;
see above), an immunohistochemistry study was performed by using different neuronal (calbindin, calretinin, Tuj-1, or NeuN) and glial markers
(glial fibrillary acidic protein [GFAP]
or O4) on X-gal free-floating sections. None of the neuronal markers
labeled Msx1-positive cells originating from those epithelia. Nevertheless, some Msx1-expressing cells of
the hippocampus showed colocalization by using an anti-NeuN antibody (data not shown). On the
other hand, when an anti-GFAP antibody was used some ␤-gal⫹ cells of
the fimbria displayed double immunostaining (Fig. 7A,B). Msx1 expression in the mature fimbria closely resembles that observed at P0.
Ultrastructural study in adult heterozygotes revealed that ␤-gal⫹
452 RAMOS ET AL.
cells of the fimbria were exclusively
glial cells, i.e., astrocytes and oligodendrocytes (data not shown). Colocalization was also detected in
some Msx1-positive cells in the ventral medulla by using anti-GFAP and
anti-O4 antibodies (Fig. 7C). These
double-labeled cells corresponded
to the two clusters of cells located in
the ventrolateral parts of the medulla and ventral horns of the spinal
cord (see Fig. 6A,C–F).
Homozygous Mutant
Phenotype
Morphological study showed that
the forebrain of the homozygous
newborns was, from a dorsal view,
triangular, in contrast to the characteristic roundness of the wild-type
brain (Fig. 8). No difference in size
between the two genotypes was
detected.
Some mutants (six of eight) exhibited severe hydrocephaly (Table 2)
of the lateral and third ventricles, accompanied by a drastic decrease in
size of some structures, such as the
septum, the caudate putamen, and
the cerebral cortex (Fig. 9A,B). Hydrocephalus showed no direct relation with aqueduct stenosis (see Table 2). Immunohistochemical study
using anti-calretinin, calbindin, L1,
and TAG1 antibodies on sections
from homozygous P0 revealed no
histological abnormalities in those
structures. Preliminary analysis indicated that cortex thickness decrease is mainly caused by a significant decrease of cell number in
each of its layers, rather than by
compression due to cerebrospinal
fluid overpressure. Furthermore, cortical cell death increased appreciably in mutants, especially in areas
where hypoplasia was more marked,
which was proportional to ventricular
enlargement severity (data not
shown). In coronal sections, the mesencephalic region in mutant embryos
was somewhat triangular (Fig. 9C,D).
Angular outline of the tegmentum
was also observed in one mutant embryo in sagittal sections (Fig. 11).
Other striking morphological anomalies in the brain of Msx1 mutants were
present in the diencephalon. They
consisted of a drastic reduction in size
of the SCO and abnormal develop-
Fig. 4. Detection of ␤-galactosidase activity in the diencephalon and other structures.
A: Embryonic day (E) 16, sagittal section. Msx1 is heavily expressed in the subcommissural
organ (sco), habenula (h), and choroid plexus (cp). Moderate to little expressions were
found in the pineal gland (pig), habenular commissure (hc), and posterior commissure
(pc; see magnification in the upper right corner). B: E18, coronal section. The epithalamic
neuroepithelium (en) displayed little expression. C: Postnatal day 0, coronal section. ac,
anterior commissure; dt, dentate gyrus neuroepithelium; hp, hippocampal commissure;
mnpo, median preoptic nucleus. D: E18, coronal section. The vascular organ of the
lamina terminalis (ovlt) and the median preoptic nucleus showed weak label. Very weak
expression was found in the subfornical organ (sfo). III, third ventricle. Scale bars ⫽ 500 ␮m
in A,C, 250 ␮m in B, 100 ␮m in D.
ment of the posterior commissure in all
mutants (PC; Fig. 10A,B). In addition,
the paraventricular thalamic nucleus
(PVT) showed a lax histological organization, i.e., less compact tissue,
while the bundling of the fasciculus
retroflexus (FR; formed at the p1/p2
prosomere boundary) near the SCO
was severely affected (Fig. 10A–D). Diencephalon defects were present independently of ventricular dilatation
rate (see Table 2). In one knockout
(KO) neonate, the SCO, the habenula, and the PC were totally missing,
as revealed by the study of the ␤-galactosidase activity. Instead of SCO, a
monolayer ependyma was found
(Fig. 11). In this case, Msx1 expression
was absent in the pretectum midline,
affecting thus the structures that derive from the prosomere 1 (p1), ac-
cording to the prosomeric model of
forebrain organization (Puelles and
Rubenstein, 1993). The pineal gland
and habenular commissure seemed
to develop normally in homozygous
embryos.
When remaining, the SCO from
Msx1-deficient embryos was not immunoreactive using a specific
marker (AFRU; Fig. 12A,B). Similar
results were obtained using a GFAP
marker (data not shown). By using
an anti-L1 antibody, labeled fibers
of the FR from homozygous embryos showed abnormal pathway
and fasciculation close to the SCO,
while the PC showed much less
staining than that from wild-types
(Fig. 12C,D). Except for pretectum,
Msx1nLacZ expression pattern was
similar in heterozygotes and ho-
Msx1 DISRUPTION AND HYDROCEPHALUS 453
Fig. 5. Msx1 expression in the mesencephalon. X-gal histochemistry. A: Postnatal day 0,
coronal section. Only the midline of the tegmental (tge) and tectal (te) neuroepithelia
expressed Msx1. The expression in the median raphe (arrow) is weak to moderate. The
asterisk indicates weak label was found in the cochlear nuclei. B: Embryonic day 16,
sagittal section (see Fig. 3B for reference). The ␤-galactosidase cells (arrows) seem to
originate from the tegmental neuroepithelium (tge) and migrate inside the tegmentum
(tg) in a radial manner, forming rows (arrowheads). Scale bars ⫽ 500 ␮m in A, 100 ␮m in
B.
mozygotes. Ependymal denudation was also observed in the III
ventricle of P0 mutants. Furthermore, the sections of affected
ependyma coincided with those
where Msx1 was normally expressed. On the other hand, not
all the ␤-galactosidase–labeled
ependyma showed denudation in
homozygotes (data not shown). No
gross defects were detected in
other cerebral areas than the diencephalon itself, and no label difference was detected by using O4
and GFAP in fimbria or medulla between wild-type and homozygotes.
DISCUSSION
Msx1 Is Expressed in the
Circumventricular Organs
This study shows that the Msx1 gene
is expressed in a variety of structures
lining the murine ventricular cavities.
The pineal gland, the SCO and habenula, and the ependyma of the
plexus choroid are all well-labeled
during development. Msx1 is, nevertheless, much less expressed in other
periventricular organs, such as the
median preoptic nucleus or the subfornical organ. We also demonstrated that Msx1 is expressed on the
dorsal surface of the medulla oblon-
gata, in particular in the area and
subarea postrema.
The finding that Msx1 is expressed
early and in postnatal development (our observations) in the circumventricular organs implicates
this gene not only in the initiation
and differentiation of the circumventricular organs but also in neurohemal functions. In accordance
with this, Msx1 transcripts were detected in differentiated thyrotropic
cells, and MSX1 protein binds to a
consensus site on the glycoprotein
hormone alfa-subunit promoter
(Sarapura et al., 1997). Furthermore, mutation of the Msx1-binding sites decreased alpha-subunit
promoter activity in these cells by
approximately 50% when compared with the wild-type promoter
(Sarapura et al., 1997). On the
other hand, high Msx1 expression
was also detected in Rathke’s
pouch in the mouse (MacKenzie et
al., 1991; our observations), closely
preceding expression of the glycoprotein hormone alpha-subunit
gene, which is the first hormone
gene to be detected at E11 in the
rat (Simmons et al., 1990). In addition, Msx1 is also expressed in other
noncerebral structures of high secretory activity, such as the ciliary
bodies of the eyes (Monaghan et
Fig. 8. Morphological comparison between wild-type (left) and Msx1-/- (right)
whole brains. Dorsal view. Note the straightshaped Msx1 mutant cerebral hemisphere
brain (arrows) compared with the roundshaped brain from a normal mouse. Scale
bar ⫽ 1,500 ␮m.
al., 1991; Civan et al., 1996; Mirshahi et al., 1999; Takamatsu et al.,
2000; this study), the uterus (Lyons
et al., 1992; Pavlova et al., 1994),
and the mammary gland (Phippard
et al., 1996). Accordingly, it has
been proposed that Msx1 and also
Msx2 are linked to exocrine secretions by this gland (Friedmann and
Daniel, 1996).
The implication of Msx1 in a secretory function is reinforced by
the finding that it is highly expressed in the SCO, considered as
a cerebral gland (Rodriguez et al.,
1998), during embryogenesis and
throughout the lifetime of the
mouse (our observations). Appealing with this, the homozygous embryos fail to form a correct SCO,
and the remaining organ displays
no label in the presence of a specific antibody against its secretion
products.
Some Neuroepithelia Produce
Msx1nLacZ-Positive Glial Cells
The analysis of the timing of Msx1
expression in some neuroepithelia
(fimbria, medulla) and underlying
cells throughout embryogenesis
strongly suggests a process of delamination/migration of ␤-galactosidase positive (Msx1-␤-gal⫹) cells
from those epithelia, with a bulk of
activity at E14 –E16 stages. The immunohistochemical study revealed
that some of the Msx1-positive cells
of the fimbria at P0 coexpressed
GFAP. Consistent with this, by using
transmission electronic microscopy,
454 RAMOS ET AL.
TABLE 2. Hydrocephalus and Diencephalon Defects in Homozygotesa
Mutant
Hydrocephaly
SCO
Labeled
SCOb
PC
FR
deviation2
Aqueduct
stenosis
1
2
3
4
5
Severe
Severe
Moderate-severe
Moderate-severe
Severe
Absent
Absent
Reduced
Reduced
Reduced
No
No
No
Absent
Absent
Disorg.
Disorg.
Disorg.
ND
Yes
Yes
Yes
Yes
6
7
8
Moderate
Moderate-severe
Moderate
Reduced
Reduced
Reduced
No
ND
ND
Disorg.
Disorg.
Disorg.
Yes
Yes
Yes
Yes
Yes
No
Narrowed
No (even
dil.)
No
Yes
No
a
FR, fasciculus retroflexus; ND, not determined; PC, posterior commissure; SCO, subcommissural organ; Disorg, disorganized;
even dil., evenly dilated.
b
Using an anti-AFRU antibody.
c
Close to the subcommissural organ.
Fig. 9. Description of brain abnormalities in homozygous newborns. A,B: Coronal sections, cryostat, Nissl stain. A: Brain from a wild-type mouse. B: Some homozygous embryos
displayed severe hydrocephalus with a drastic decrease in size of the cerebral cortex
(cc), the corpus callosum (cca), the septum (s), and the caudate putamen (cp).
C,D: Coronal sections, paraffin, Nissl stain. Observe in D (Msx1-/- subject) the triangularshaped form of the brain and the abnormal enlargement of the third ventricle (arrow) at
the diencephalic level in comparison with those from a wild-type brain (C). Scale bars ⫽
500 ␮m in A–D.
we found that Msx1-␤-gal⫹ cells of
the fimbria of the adult mouse are
astrocytes and oligodendrocytes
(unpublished data). The ultrastructural study also revealed the presence of X-Gal precipitate in oligodendrocyte-like cells. An interesting
feature to emphasize is the presence of double-labeled Msx1/PSANCAM–positive cells in the mature
fimbria (unpublished data). It has
been shown in cell culture conditions that PSA-NCAM is involved in
motility and differentiation of oligodendrocyte progenitors into myelinating cells (Decker et al., 2000).
Likewise, we showed that some of
the Msx1-␤-gal⫹ cells located in the
ventral aspect of the medulla and
spinal cord are oligodendrocytes,
because they were stained by an
anti-O4 antibody. These cells originate in vertebrates from two narrow
columns located at either side of the
ventral neuroepithelium, a region
also known to generate somatic mo-
toneurons (Marti and Bovolenta,
2002). Taken together, our results
suggest that some of the ␤-galactosidase–labeled neuroepithelia may
constitute a source of precursors of
glial progenitors that delaminate
and move to differentiate later in astrocytes and oligodendrocytes, because those showed no colocalization using neuronal markers. That no
defects were detected in fimbria
and medulla epithelia-derived cells
may be explained by functional redundancy with Msx2, as already
proposed for other structures during development (Houzelstein et
al., 1997).
Msx1 has been linked to proliferation/differentiation processes in migratory cells from distinct epithelia
(Thomas et al., 1998; Houzelstein et al.,
1999, 2000; Christ et al., 2000). In addition, it has been proposed that BMP,
Wnt, and Msx genes are interrelated
at different sites of epithelial–mesenchymal interactions (reviewed in Davidson, 1995; Peters and Balling, 1999).
Furthermore, Wnts and BMPs genes
have been implicated in the control
of the development of glial cells of
neuroepithelial origin (Jin et al., 2001;
Ochiai et al, 2001; Mekki-Dauriac et
al., 2002). Finally, msh gene in Drosophila is required for the migration
and the development of neuroblasts
from neuroectoderm (Isshiki et al.,
1997). These findings indicate that
Msx1 gene may contribute to this
mechanism and be linked to glial lineage.
Msx1 DISRUPTION AND HYDROCEPHALUS 455
Fig. 10. Diencephalon defects of homozygotes. A–D: Postnatal day 0, coronal sections,
paraffin, Nissl. B: Observe the drastic decrease of the subcommissural organ (sco), the
reduced posterior commissure (pc), and the severe bundling defects of the fasciculus
retroflexus (FR, arrows). D: Abnormal presence of numerous cells (arrows) in the FR and lax
histological organization of the paraventricular thalamic nucleus (asterisk) were observed in Msx1-deficient mice. Compare with the normal organization in A,C. Scale bars ⫽
100 ␮m in A,B, 50 ␮m in C,D.
Msx1 Deficient Mice Display a
Severe Hydrocephalus
The most evident anomaly detected
in the brain of Msx1 mutants was severe hydrocephalus, accompanied
by a drastic reduction in size of the
cerebral cortex, the caudate putamen, the septum, and the corpus
callosum. The immunohistochemical
study showed no alterations in these
structures. Nevertheless, an increase
of cell death was detected in the
cerebral cortex of mutants. Different
morphostructural anomalies have
been described in hydrocephalic
subjects, independently of the etiology, such as atrophy of the cortex
and anterior commissure (Doublier
et al., 2000), of the corpus callosum
with loss of axons (Dahme et al.,
1997; Del Bigio et al., 1997; Del Bigio
and Zhang, 1998; Doublier et al.,
2000), aberration in the pathway of
the corticospinal tract (Fransen et
al., 1998), periventricular white matter
edema (Makiyama et al., 1997; Del
Bigio and Zhang, 1998), cell death
(Del Bigio and Zhang, 1998), and
damage in ependyma (Kiefer et al.,
1998), in capillaries endothelial cells,
and in the choroid plexus (Del Bigio,
1993; Makiyama et al., 1997). Msx1
mutant mice displayed neither structural nor ultrastructural anomalies in
the choroidal cells, nor in the tela choroidea (results not shown). However,
alterations of the secretory activity or
cerebrospinal fluid (CSF) reabsorption
cannot be ruled out, taking into account (1) the strong Msx1 expression in
these structures and the presence of
specific binding sites for Reissner’s fiber-glycoproteins in ependymal cells
of the choroid plexus (Miranda et al.,
2001), and (2) the ependymal detachment, which seems to be, in certain cases, the primary cause of hydrocephalus (Jiménez et al., 2001).
Some cases of hydrocephalus
have been attributed to stenosis of
the aqueduct of Sylvius (revised in
Pérez-Fı́gares et al., 2001). In contrast, other authors considered that
aqueductal stenosis is due to a secondary effect of hydrocephalus and
not the cause of the latter (Rolf et
al., 2001). On the other hand, hydrocephalus can also develop in sub-
jects with a normal aqueduct, such
as L1 homozygous mice; therefore, it
does not appear to cause hydrocephalus (reviewed in Kamiguchi et
al., 1998). In accordance with this
view, in the normal developing
brain, axon–axon adhesion generates tension in the white matter that
is sufficient to balance the hydrostatic pressure of the CSF. Thus, in
L1-/- mutants, loss of adhesion and
loss of axons in the corpus callosum
and corticospinal tract would increase brain compliance, resulting
in ventricular dilatation (reviewed in
Kamiguchi et al., 1998). According
to our immunohistochemical study in
Msx1⫺/⫺-deficient mice, only the L1
marked axons close to the SCO displayed anomalies and no other axonal defects were detected by Nissl
dye. However, as discussed above,
the possibility of a relation between
Msx1 transcription factor and loss of
axonal adhesion cannot be ruled
out.
Our study shows that hydrocephalus occurs also in absence of aqueduct stenosis. According to the literature, overproduction or defective
absorption of CSF (Kasubuchi et al.,
1977), abnormal vascular formation
in the brain mantle (Kameyama et
al., 1972), increased permeability of
the blood– brain barrier or blood
brain–CSF barrier (Uno et al., 1997),
and damage to the overlying brain
parenchyma (Inouye and Kajiwara,
1990) could also be causes of hydrocephalus. In Msx1 homozygotes, hypoplasia of brain parenchyma
might lead to compensatory enlargement of cerebral ventricles
and, hence, to hydrocephalus development. This hypothesis is supported by the fact that cerebral
thickness decrease is mainly caused
by a drastic lowering of cortex cell
number. In contrast, when ventricular dilation is moderate, mantle hypoplasia is reduced. Alteration of CSF
composition may have a potential
role in the developmental process of
the cortex (Aolad et al., 2000;
Mashayekhi et al., 2002; Miyan et al.,
2003; Owen-Lynch et al., 2003). Furthermore, it is known that (1) SCO
glycoproteins play a role during cortical development in vitro (Monnerie
et al., 1998), and (2) dysfunctional
SCO may lead to aqueduct stenosis
456 RAMOS ET AL.
and consequent hydrocephalus (revised in Pérez-Fı́gares et al., 2001,
and in Galarza, 2002).
Taken together, these data suggest
that, in Msx1 mutants and according
to our data, hydrocephalus may
have different origins: (1) aqueduct
closure, (2) alteration of CSF composition caused by ependymal denudation and/or SCO and choroid plexus
malfunction. Our results suggest that
multiple origins may interfere.
Subcommissural Organ May
Be Involved in Posterior
Commissure and Fasciculus
Retroflexus Development
The PC is an axonal bundle that
forms some days before the SCO differentiates or secretes anything (Mastick and Easter, 1996; Estivill-Torrús et
al., 2001). Thus, it seems likely that the
roof plate expression of Msx1 before
SCO formation would be involved in
PC and RF development, when one
considers that signalling molecules
are present in this area. Msx2 is also
expressed at E10.5 in the dorsal p1,
but more weakly, and down-regulated at E12.5 (Bach et al., 2003). In
Msx1 mutants, the lack of Msx1 protein may be compensated by Msx2,
at least in the first steps of brain organogenesis, taking into account
the functional redundancy of Msx1
and Msx2 (Houzelstein et al., 1997). In
addition, in Drosophila, a role in
axon guidance for Wnt5 (Yoshikawa
et al., 2003) and a role in RF formation for semaphorin 3F, expressed
early in the rostral p1 of the rat, have
been demonstrated (Funato et al.,
2000).
On the other hand, it is also likely
that, at later stages, abnormal PC
and FR in Msx1 mutants are the consequence of a dysfunctional SCO.
Indeed, this glandular structure secretes glycoproteins, especially
SCO-spondin, which when released
into the CSF form Reissner’s fiber (RF;
revised in Rodriguez et al., 1998). Recently, it has been shown, by in vitro
analysis, that an oligopeptide derived from a thrombospondin type 1
repeat (TSR) of SCO-spondin is implicated in cell differentiation processes, considering the promoting
effects of TSR oligopeptides on neurite outgrowth and cell aggregation
Fig. 11. Detection of ␤-galactosidase activity in homozygous newborns. Sagittal section.
The subcommissural organ, the habenula, and the posterior commissure are missing in
this embryo (see selected area, which corresponds to the anatomical place for these
structures in wild-type mice). Compare with Figure 4A (heterozygous embryo). Note the
angular-shaped form of the mesencephalon (arrows). The habenular commissure (hc) is
normal in homozygotes. Co, colliculus; f, fimbria. Scale bar ⫽ 500 ␮m.
(O’Shea et al., 1991; Neugebauer et
al., 1991; Leung-Hagesteijn et al.,
1992; Osterhout et al., 1992; Adams
et., 1996). On the other hand, we
have shown that the neural cell adhesion molecule L1 is expressed in
the PC and in the FR in both wildtype and mutants. It seems coherent, according to our results, to propose that MSX1 protein is directly
involved in axonal pathfinding and
axonal bundling by means of SCOsecreted glycoproteins. These latter
entities would interact with other
factors, such as cell adhesion molecules like L1, diffusible factors, or extracellular matrix molecules, as suggested elsewhere (Gobron et al.,
1996; Meiniel, 2001; Norlin et al.,
2001). Furthermore, it is well established that L1 is critical to nervoussystem development (revised in
Hortsch, 1996). These results favor the
hypothesis that the SCO is involved,
by means of SCO-spondin or other
glycoproteins release, in the formation of the PC (Gobron et al., 2000;
El-Bitar et al., 2001). Our results thus
support the implication of Msx1
gene in neural differentiation processes, as proposed by other authors in different non-neural tissues
(reviewed in Davidson, 1995; Lincecum et al., 1998; Odelberg et al.,
2000; Blin-Wakkach et al., 2001).
We have also shown that Msx1 mutants displayed histological disorganization of the paraventricular thalamic
nucleus (PVT). Interestingly, the presence of binding sites for RF glycoproteins in the PVT has been demonstrated (Miranda et al., 2001). These
results suggest that the dysfunctional
secretory SCO of homozygotes is related to these neurohistological disorders, especially because the PVT
does not express the Msx1 gene in
normal conditions (or in KO mice).
It is not clear, however, how secreted SCO glycoproteins would be
released from the RF and influence
neurite outgrowth or axonal pathfinding in vivo. It has been postulated that SCO glycoproteins may
have a local effect, being directly
secreted into the extracellular matrix
(Rodriguez et al., 1998). This secretion could be responsible for the formation of the PC, the fasciculation
and directional guidance of the FR,
and the structural organization of
the PVT.
EXPERIMENTAL PROCEDURES
Mouse Strains
Gene targeting of the Msx1 locus
with the nlacZ (n, nuclear localization signal) reporter gene (Msx1nlacZ)
was described previously (Houzel-
Msx1 DISRUPTION AND HYDROCEPHALUS 457
stored at ⫺20°C. Alternatively,
brains were sectioned on a freezing microtome and immunolabeled with neuronal markers (anticalretinin, 1:3,000; anti-calbindin,
1:5,000; anti–Tuj-1, 1:1,000; and antiNeuN, 1:250) and glial markers (antiGFAP, 1:1,500 and anti-O4, 1:50) after
X-Gal development. Signals were detected by using an immunoperoxidase reaction (see under Immunohistochemistry section).
In Situ Hybridization
Msx1 antisense riboprobe was labeled with digoxigenin-dUTP (Boehringer-Mannheim) by in vitro transcription of a 0.7-kb fragment
encoding mouse Msx1 using T3 polymerase (Ambion). Sense probe (using T7 polymerase) was used as control.
Sections.
Fig. 12. Diencephalon defects and immunohistochemistry. Postnatal day 0, coronal
sections. A.B: Immunostaining using an anti-Reissner's fiber serum (AFRU). B: Although
present, the subcommissural (sco) was not labeled in mutant mice (compare with A,
wild-type). C,D: Immunostaining using an anti-L1 antibody. Observe in D the aberrant
pathway and lack of bundling of the fasciculus retroflexus (arrows) from a Msx1-/embryo close to the SCO in contrast with those from a wild-type mouse (C). Observe also
in D the weak staining in the posterior commissure (pc). Scale bars ⫽ 100 ␮m in A,B, 250
␮m in C,D.
stein et al., 1997). The mutant allele
has been maintained on a C57BL/6J
background. Mutant embryos were
first recognized by the phenotype,
i.e., primarily by detecting a cleft
palate (Satokata and Maas, 1994;
Houzelstein et al., 1997) and then
confirmed genotypically. Homozygous, heterozygous, and wild-type
embryos were distinguished by polymerase chain reaction using tissue
from the embryo itself or from the
extraembryonic membranes.
Beta-Galactosidase Staining
For in toto beta-galactosidase
staining, embryos (E10, E12) were
excised, washed in 0.1 M phosphate buffer saline (PBS), and fixed.
The mating day was considered as
E0. After washing, embryos were
stained in a 0.1 M PBS solution con-
taining 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM magnesium chloride,
0.02% Nonidet P-40 (w/v), and 400
␮g/ml X-Gal (5-bromo-4-chloro-3indolyl-beta-D-galactopyranoside)
as substrate at 32°C overnight. For
descriptive purposes, histological
sections were obtained on a cryostat, stained as above, counterstained with eosin, and mounted
with Eukitt. For this, whole E8 –E12
embryos and heads from E14 –P0
transcardially perfused embryos
were fixed, washed, cryoprotected
in 30% sucrose, and frozen. The day
of birth was considered P0. In all
cases, the fixative was 2% paraformaldehyde for 30 min to 4 hr depending on the size of the embryo.
X-Gal stocks were prepared in dimethyl sulfoxide at 40 mg/ml and
In situ hybridization was performed
on free-floating tissue sections essentially as described (de Lecea et al.,
1994, 1997). Sections were prehybridized at 60°C for 3 hr in a solution
containing 50% formamide, 10%
dextran sulfate, 5⫻ Denhardt’s solution, 0.62 M NaCl, 10 mM ethylenediaminetetraacetic acid, 20 mM
PIPES, 250 ␮g/ml sheared salmon
sperm DNA, and 250 ␮g/ml yeast
tRNA. Labeled riboprobe cRNA was
added to the prehybridization buffer
(500 –1,000 ng/ml), and hybridization
was performed at 60°C overnight.
After stringent washes, sections were
incubated at 4°C overnight with an
anti-digoxigenin antibody conjugated to alkaline phosphatase, and
developed with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3indolyl-phosphate toluidinium salt
(BCIP). Tissue sections were mounted
on gelatinized slides and coverslipped with Mowiol.
Whole-Mount.
Embryos (E10 –E12) were dissected
out,
removing
extra-embryonic
membranes, and fixed with 4% paraformaldehyde in PBS 0.1 M for 24 hr
at 4°C. After washes in PBT (0.1%
Tween-20 in PBS), embryos were successively washed in 25%, 50%, 75%
methanol in PBT for 10 min, then
twice in 100% methanol overnight.
458 RAMOS ET AL.
Embryos can be stored for several
months at ⫺20°C in methanol. After
that, embryos were rehydrated by
passage through the methanol/PBT
series in reverse and washed in PBS,
treated with hydrogen peroxide and
proteinase K, and refixed with 0.2%
glutaraldehyde/4% paraformaldehyde in PBT for 20 min. Thereafter,
embryos were washed in PBT and
immersed in a prehybridization mix
(50% formamide, 5⫻ standard saline
citrate, pH 5.0, 1% sodium dodecyl
sulfate, 100 ␮g/ml yeast RNA, and
100 ␮g/ml heparin) at 70°C for 1 hr
with rocking. After that, 1 ␮g/ml
digoxigenin-labeled cRNA probe
was added to the mix and embryos
were hybridized at 70°C overnight
with rocking. Then, embryos were
washed in stringent conditions and
incubated overnight with an antidigoxigenin antibody conjugated to
alkaline phosphatase. Label was developed as above (tissue sections).
Paraffin Procedure
Neonatal mice were anesthetized
and perfused by means of the cardiac left ventricle with Bouin, and
brains were excised and immersed
in the same fixative solution. After
washes in distilled water, brains were
dehydrated in an ethanol series and
embedded in paraffin wax. Sections
were obtained on a microtome and
contrasted with hematoxylin– eosin
or with Nissl.
Immunohistochemistry
Embryos were anesthetized and
transcardially perfused with 4%
paraformaldehyde in PBS. Brains
were removed, postfixed, and
cryoprotected in 30% sucrose.
Brains were then frozen and sectioned at 50 ␮m on a freezing microtome. Sections were incubated
with different antibodies (anti-L1,
1:2,000; anti-TAG1, 1:2,000; anticalretinin, 1:3,000; anti-calbindin,
1:5,000; anti-GFAP, 1:1,500; antiAFRU, 1:1,000) overnight, washed
and incubated with the secondary
antibodies. Signals were detected
by using the streptavidin–peroxidase complex followed by an enzymatic reaction with diaminobenzidine and H2O2.
ACKNOWLEDGMENTS
We thank Dr. Jeús Pérez-Clausell for
work on the analysis of Msx1 expression, Dr. Yvan Lallemand for the
preparation of some embryos, and
Dr. Soledad Alcántara for technical
assistance. O4 antibody is a gift from
Carlos Paino. We also thank Robin
Rycroft for critical reading of the
manuscript and linguistic advice.
E.S. was funded by MCYT (SAF-20013290) and the Caixa Foundation.
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