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Vol. 13, No. 21, pp. 2801-2810, November 1, 1999
1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G-1X5; 2 Centre for Research in Neurodegenerative Diseases and Department of Medicine, Division of Neurology, University of Toronto. The Toronto Hospital, Toronto, Ontario, Canada M5S-3H2; 3 Merck Research Laboratories, Rahway, New Jersey 07065 USA; 4 Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada M5S-1A8
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Abstract |
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 Top
 Abstract
 Introduction
Results
Discussion
Materials and methods
References
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Genetic studies in worms, flies, and humans have implicated the presenilins in the regulation of the Notch signaling pathway and in the pathogenesis of Alzheimer's Disease. There are two highly homologous presenilin genes in mammals, presenilin 1 (PS1) and presenilin 2 (PS2). In mice, inactivation of PS1 leads to developmental defects that culminate in a perinatal lethality. To test the possibility that the late lethality of PS1-null mice reflects genetic redundancy of the presenilins, we have generated PS2-null mice by gene targeting, and subsequently, PS1/PS2 double-null mice. Mice homozygous for a targeted null mutation in PS2 exhibit no obvious defects; however, loss of PS2 on a PS1-null background leads to embryonic lethality at embryonic day 9.5. Embryos lacking both presenilins, and surprisingly, those carrying only a single copy of PS2 on a PS1-null background, exhibit multiple early patterning defects, including lack of somite segmentation, disorganization of the trunk ventral neural tube, midbrain mesenchyme cell loss, anterior neuropore closure delays, and abnormal heart and second branchial arch development. In addition, Delta like-1 (Dll1) and Hes-5, two genes that lie downstream in the Notch pathway, were misexpressed in presenilin double-null embryos: Hes-5 expression was undetectable in these mice, whereas Dll1 was expressed ectopically in the neural tube and brain of double-null embryos. We conclude that the presenilins play a widespread role in embryogenesis, that there is a functional redundancy between PS1 and PS2, and that both vertebrate presenilins, like their invertebrate homologs, are essential for Notch signaling.
[Key Words: Presenilin; notch; somite segmentation; Alzheimer's Disease]
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Introduction |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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The presenilins belong to a family of highly conserved
proteins that localize largely to the endoplasmic reticulum and Golgi apparatus (Cook et al. 1996
; Kovacs et al. 1996; Walter et al. 1996; De
Strooper et al. 1997). The presenilins have also been found in the
plasma membrane, in the nuclear envelope, and associated with
kinetochores and centrosomes (Dewji and Singer 1997; Li et al. 1997).
They are predicted to have between 6 and 8 transmembrane domains and a
large hydrophilic loop that, along with the amino and carboxyl termini,
is oriented toward the cytoplasm (Doan et al. 1996; Li and Greenwald
1996, 1998). The presenilins exist in a cleaved form as an
amino-terminal fragment (NTF) and a carboxy-terminal fragment (CTF)
(Thinakaran et al. 1996). They are widely expressed in mammalian
embryonic development and in adult tissues, with high expression in the
cell bodies and dendrites of neurons in the mammalian central nervous
system (CNS) (Cook et al. 1996; Lee et al. 1996). One presenilin gene
(Dps) has been identified in Drosophila melanogaster,
two genes (Sel-12 and Hop-1) have been isolated in
Caenorhabditis elegans, and two homologs, called presenilin-1
(PS1) and presenilin 2 (PS2), have been cloned in mammals.
Insights into the role of the presenilins in normal and pathological
processes have emerged from both genetic and biochemical analyses.
Biochemical interactions between the presenilins and a variety of
proteins have been reported, although the significance of these remains
to be determined. For example, several groups have reported
independently that the presenilins bind and affect the stabilization of
members of the 
-catenin family (Zhou et al. 1997; Murayama et al.
1998; Yu et al. 1998; Zhang et al. 1998b; Levesque et al. 1999;
Nishimura et al. 1999a). In addition, cytoskeleton- and
microtubule-associated proteins, calcium-binding proteins, and
G-proteins and GTPases have all been reported to interact with the
presenilins (Buxbaum et al. 1998; Smine et al. 1998; Takashima et al.
1998; Zhang et al. 1998a; Dumanchin et al. 1999; Stabler et al. 1999).
There have also been studies suggesting that the presenilins directly
associate with the amyloid precursor protein (APP) and the Notch
receptor (Weidemann et al. 1997; Xia et al. 1997; Ray et al. 1999).
Genetic studies of families with familial Alzheimer's disease (FAD),
an autosomal dominant disorder of the CNS have implicated PS1, PS2, and
APP in the pathogenesis of this disease (Chartier-Harlin et al. 1991;
Murrell et al. 1991; Levy-Lahad et al. 1995; Rogaev et al. 1995;
Sherrington et al. 1995). Nearly 50% of FAD patients bear mutations in
one of the presenilin genes, whereas <3% of patients bear APP
mutations (Blacker and Tanzi 1998; Nishimura et al. 1999b). Genetic
studies in C. elegans and D. melanogaster have also
demonstrated a role for the presenilins as positive regulators of the
Notch pathway (Levitan and Greenwald 1995; Li and Greenwald 1997;
Struhl and Greenwald 1999; Ye et al. 1999). Interestingly, FAD patients
have elevated Notch-1 expression (Berezovska et al. 1998).
Thus, Notch signaling might play a role in this disease.
Analyses of presenilin mutations in mammalian cells and flies have
shown that the presenilins function in the processing of membrane-associated proteins such as the Notch-1 receptor and APP (De
Strooper et al. 1998, 1999; Struhl and Greenwald 1999; Wolfe et al.
1999; Ye et al. 1999). APP sustains a series of cleavage events, which
can culminate in the release of a toxic product, called the Abeta1-42
peptide, that is a major component of the extracellular plaques that
are the hallmark of AD. The presenilins appear to control aspects of
APP processing, in that cells lacking PS1 make less of the Abeta1-42
peptide, whereas mice bearing FAD-linked PS1 alleles make more (Duff et
al. 1996; De Strooper et al. 1998).
The Notch signaling pathway controls embryonic cell-fate decisions in a
variety of cell lineages in flies, worms, and mammals (Artavanis-Tsakonas et al. 1999). There is only one Notch gene in
flies, whereas there are two and four Notch genes in worms and mammals,
respectively. The molecular mechanisms of Notch signaling are conserved
in different species and are initiated by the interaction of the Notch
receptor with its ligands [Delta-like-1 (Dll1) or -3, or Jagged-1 or
-2, in mammals]. This interaction induces a series of proteolytic
cleavage events of the Notch receptor. These processing steps culminate
in the release of a Notch intracellular fragment that translocates to
the nucleus and complexes and activates RBP-J
, a transcription
factor regulating a number of downstream genes, including
HES-5 and Dll1 (Tamura et al. 1995; Lu and Lux 1996;
Artavanis-Tsakonas et al. 1999; Ohtsuka et al. 1999). Apparently, the
presenilins positively regulate the release of the Notch intracellular fragment (De Strooper et al. 1999; Struhl and Greenwald 1999; Ye et al. 1999).
Consistent with genetic and biochemical studies in flies and worms that
indicate a role for the presenilins in regulating the Notch pathway, a
targeted inactivation of the PS1 gene in mice results in
somite disorganization characteristic of mice harboring single
mutations in genes that comprise the Notch pathway (Conlon et al. 1995;
Oka et al. 1995; Shen et al. 1997; Wong et al. 1997; Evrard et al.
1998; Zhang and Gridley 1998). In addition to the somite defects,
PS1-null mice exhibit rib-cage abnormalities, and hemorrhaging and
cell-loss in the forebrain, which result in a perinatal lethality.
Interestingly, the developmental abnormalities in mutant PS1-null mice
are mild in comparison with those described in Notch-1 null
mice (Conlon et al. 1995). Furthermore, the expression of
Dll1, normally negatively regulated by the Notch signaling pathway, was reported to be decreased in PS1-null mice (Wong et al.
1997). If Notch signaling is perturbed by PS1-loss, Dll1
expression would be expected to be up-regulated in the PS1-null mice.
Together, these observations raise the likely possibility that PS2 is
partially compensating for the loss of PS1 in maintaining Notch
signaling in these mutant mice. Genetic studies in C. elegans
have indicated that there is a genetic interaction between the two worm
presenilin genes, Sel-12 and Hop-1 (Li and Greenwald
1997; Westlund et al. 1999). Thus, it is likely that the mammalian
presenilins may also have redundant functions, based on their sequence
similarity and overlapping patterns of expression (Lee et al. 1996). To
test this hypothesis, we have generated PS2-null mice and
PS1/PS2 double-null mice. Here, we provide evidence that
murine PS1 and PS2 have overlapping, but nonidentical functions and
that loss of both known mammalian presenilins leads to profound and
widespread developmental defects.
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Results |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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Targeted inactivation of the murine PS2 gene
The PS2 gene was disrupted by homologous recombination in
embryonic stem (ES) cells by replacing four amino acids (RCYK) from exon 5, which includes part of the second transmembrane region, with
the neo resistance gene (Fig. 1A). ES cell
clones that had sustained a homologous recombination event were
identified by Southern blot analysis with 3' external (Fig. 1B) and
5' external (not shown) probes using HindIII (Fig. 1B) or
EcoRI (not shown). Subsequent screening of the resulting mice
by PCR confirmed this conclusion, as DNA from PS2-null mice did not
give rise to any PCR fragments corresponding to the wildtype locus
(Fig. 1C). RT-PCR and sequencing analyses confirmed that
PS2
/ cells only expressed PS2
transcripts lacking exon 5 (not shown). Any translation product from
these transcripts would be predicted to encode only the first 99 amino
acids of the PS2 protein (retaining only one intact transmembrane
domain) plus 5 additional nonsense residues, as the deletion of exon 5 should give rise to out-of-frame transcripts. To confirm the absence of
PS2 protein in the PS2/ mice, brain
extracts from PS2+/+ and
PS2/ mice were examined by Western blot
analysis using three different antibodies directed against the
hydrophilic loop and amino terminus. As expected, neither the PS2 CTF
(Fig. 1D) nor the PS2 NTF (not shown) were detected in PS2/
mice but were observed in PS2+/+ (Fig. 1D) and
PS2+/ mice (not shown). We conclude that
the PS2/ mice are PS2-null.
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Generation of mice lacking both PS1 and PS2
Crosses between the heterozygous PS2+/
mice, maintained on several genetic backgrounds (CD1, 129, and
129/C57Bl6/J F1) gave rise to
expected Mendelian ratios of phenotypically normal PS2-null mice (not
shown). To date, we have not observed any defects in adult mice that
are PS2-null, suggesting that PS1 is compensating for the loss of PS2.
To examine this further, we generated doubly heterozygous
PS1+/; PS2+/
mice (on a C57Bl6/129 F1 hybrid background) by crossing
PS2+/ mice (maintained on a 129 background)
described here with 129/C57B6j F1
PS1+/ mice described previously (Wong et
al. 1997). The doubly heterozygous animals exhibited no apparent
phenotype. Analysis of >290 progeny [at embryonic day 8(E8) to
E9.5] from intercrosses of these doubly heterozygous or doubly
heterozygous crossed to PS1+/;
PS2/ mice demonstrated that loss of
both or even one copy of PS2 on a PS1/
background resulted in an early embryonic lethality prior to E13.5
(Table 1). However, loss of one copy of PS1 on a
PS2-null background had no consequence (Table 1). Thus, vertebrate
presenilins, like their homologs in C. elegans (Li and
Greenwald 1997; Westlund et al. 1999), genetically interact and are
functionally redundant, and PS2 is haploinsufficient in the absence of
PS1. We also conclude that the absence of any discernible phenotype in
the PS2/ mice is due to the presence of PS1.
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Notch signaling defects in embryos lacking all presenilins
At E8.5-E9, double-null embryos failed to show somite segmentation
and exhibited a kinked neural tube (n = 16) (Fig.
2I,L), whereas PS1/;
PS2+/ embryos showed a more variable
phenotype, ranging from no somite segmentation (n = 15) to
the formation of highly disorganized somites (n = 5) (Fig.
2H,K). Mild somite disorganization defects as have been described
previously (Shen et al. 1997; Wong et al. 1997) were observed in
PS1-null, and none were observed in PS2-null embryos at E8.5
(n = 6, n = 15, respectively; data not shown) and
at E13.5 (n = 12, n = 14, respectively; data not
shown). To determine whether the somite abnormalities in the
double-null embryos were attributable to a paraxial mesoderm defect, we
tested the expression of Dll1 (Bettenhausen et al. 1995) in
presomitic embryos. The level and spatial pattern of Dll1
expression in the paraxial mesoderm of E8 double-null embryos was
similar to that in PS1+/;
PS2+/ embryos (n = 3) (Fig.
3A,B). However, at E8.5, whereas Dll1
expression remained high in the paraxial mesoderm of double-null
embryos, Dll1 expression was undetectable in the segmental
plate (n = 4) (Fig. 3D). Instead, ectopic expression of
Dll1 was observed in the neural tube, hindbrain, and forebrain
of double-mutant embryos (n = 4) (Fig. 3D). Ectopic neural
tube Dll1 expression was also observed in some
PS1/; PS2+/
(n = 4) and PS1-null embryos (n = 3) (Fig. 2F,H),
whereas others of these genotypes showed the expected somitic or
segmental plate expression at E9 at levels comparable to those in
PS1+/; PS2+/
embryos (Fig. 2K,L). These observations should be contrasted with a
previous report of decreased Dll1 expression in PS1-null mice
(Wong et al. 1997).
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To confirm the absence of somites in the double-null embryos, we
analyzed expression of UncX4.1, a marker for posterior somitic compartments (Mansouri et al. 1997). E9 embryos were purposely overstained to ensure detection of any UncX4.1 expression in
double-null embryos (Fig. 3M,N). Expression of UncX4.1 was
undetectable in the segmental plate of double-null embryos
(n = 3) (Fig. 3N), concurrent with a lack of somitic
identity in the mutants. Loss of somite identity was confirmed by the
lack of Dll1 expression in a metameric somitic pattern in
presenilin double-null embryos (Fig. 2D). In addition, the expression
of murine Twist, a mesodermal marker, was also absent from
presomitic mesoderm adjacent to the neural tube in double-null embryos
(data not shown).
Because other Notch pathway mutant mice show ectopic Dll1
expression in the neural tube (de la Pompa et al. 1997), it is likely that Notch signaling was ablated by the loss of both presenilin genes.
To test this hypothesis further, we analyzed expression of
HES-5, a murine homolog of Enhancer of split, a gene
that lies in the Notch pathway in D. melanogaster, and is
down-regulated in Notch pathway mutant mice (de la Pompa et al. 1997).
HES-5 expression was reduced in PS1/ and
PS1/; PS2+/
embryos (data not shown), whereas it was completely abolished in the
presenilin double-null embryos (n = 3) (Fig. 3, cf. O and P), consistent with lack of Notch signaling caused by presenilin loss.
Pleiotropic defects of presenilin double-null embryos
The somite segmentation defects of presenilin double-null embryos
were anticipated in light of the fact that mice carrying mutations in
genes that comprise the Notch pathway also exhibit similar
abnormalities (Conlon et al. 1995; Oka et al. 1995; Evrard et al. 1998;
Zhang and Gridley 1998). However, presenilin double-null embryos also
exhibited a wide range of additional phenotypes. For example, there was
a delay in the closure of the anterior neuropore at E8.5
(n = 16) (Fig. 4A) and E9
(n = 3) (Fig. 5C), as well as cell loss in
the mesenchyme of the presumptive midbrain at E8.5 (n = 4)
(Fig. 4, cf. B and C). Furthermore, presenilin double-null embryos had
underdeveloped second branchial arches (Fig. 5C) and unlooped hearts at
E8.5 (n = 16) and E9 (n =4) (Fig. 5C,F). Some
PS1/; PS2+/
exhibited heart looping delays at E8.5 and E9 (n = 12 and 5, respectively) (Fig. 5B,E), whereas others exhibited partial heart looping at E9 (n = 2) (not shown), and no cardiac defects
were observed in PS1-null (Shen et al. 1997; Wong et al. 1997) or
PS2-null embryos (Fig. 5A). Cardiac looping defects generally lead to
cardiovascular failure and probably contribute to the lethality in the
double-null embryos at E9-E9.5. In addition, we observed
chorioallantoic fusion defects in double-null (n = 10), but
not PS1/;
PS2+/ embryos (n = 14) (not
shown), which would also lead to the demise of the double-null embryos.
Our data do not exclude the possibility that the heart looping,
branchial arch and neuropore closure defects are due to a developmental
delay arising from a primary defect of mesoderm patterning.
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Finally, E8.5 double-null embryos exhibit a severe disorganization of
the trunk ventral neural tube (n = 4) (Fig.
6C,D). In some instances, the notochord was
completely surrounded by disordered ventral neural tube cells
(n = 3) (Fig. 6E). To address whether notochord defects
might be the primary cause of the ventral neural tube disorganization
in the presenilin double-null embryos, we analyzed expression of Sonic
Hedgehog (Shh). At E9, Shh is expressed in the notochord and the
ventral midline of the neural tube (i.e., floor plate) in both the head
and trunk (Echelard et al. 1993). Expression of SHH in the notochord of
E9 double-null embryos was comparable to that of
PS1+/; PS2+/
embryos (n = 3) (Fig. 6G-J), suggesting that the ventral
neural tube defect observed in those mutants is not secondary to
notochord abnormalities. However, ventral neural tube expression of Shh in the double-null embryos was restricted to the head region (Fig. 2H)
and was completely absent in the trunk (Fig. 6J) (n = 3). The ventral neural tube in the trunk of presenilin double-null embryos
also failed to express Nkx2.2 (not shown), a gene whose expression pattern in wild-type embryos defines a population of presumptive neurons in the midline of the ventral neural tube. These
observations support the hypothesis that distinct mechanisms govern the
patterning of the neural tube along the rostrocaudal axis (Dale et al.
1997; Ensini et al. 1998) and suggests that the consequences of presenilin
loss is restricted to ventral neural tube patterning in the trunk region.
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Discussion |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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The mammalian presenilins have overlapping but not identical functions
Mice lacking PS1 die at birth with a truncated skeleton, rib-cage
defects, and forebrain hemorrhaging and cell loss, whereas mice lacking
PS2, and even those lacking PS2 and one copy of PS1, are viable and
phenotypically normal. However, loss of even one copy of PS2 in the
absence of PS1 greatly exacerbates the PS1-null phenotype, in that
those embryos die between E9.5 and E13.5 of pleiotropic defects. Loss
of both presenilins results in a severe, complex phenotype, and
lethality before E9.5. These results show that PS1 and PS2 have
different, but partially overlapping, activities. Studies in C. elegans have also demonstrated overlapping, but distinct
requirements for Sel-12 and Hop-1, the two
presenilins in the worm genome (Li and Greenwald 1997; Westlund et al. 1999).
Both PS1 and PS2 positively regulate Notch signaling
Presenilin double-null embryos exhibit a complex phenotype
demonstrating that the presenilins are widely required in
embryogenesis. Certain aspects of the double-null phenotype, such as
the complete lack of somite organization, an undulating neural tube,
anterior neuropore closure defects, and chorioallantoic fusion
abnormalities, could be attributed to perturbations in Notch signaling.
Similar defects are exhibited by mice mutated in RBP-Jk, a
gene that encodes a transcription factor downstream in the Notch
pathway (Oka et al. 1995; de la Pompa et al. 1997). Furthermore, two
genes downstream of Notch signaling, HES-5 and Dll1,
are misexpressed in both presenilin double-null and
RBP-Jk-null embryos, demonstrating that presenilin loss in
mammals directly affects Notch signaling. Because the RBP-Jk-null-like phenotypes were only observed when both PS1
and PS2 were mutated, we conclude that both presenilins play a role in
the Notch pathway.
The complexity of the presenilin-null phenotype
Embryos lacking both PS1 and PS2 exhibit a variety of defects not
described previously in mice with single mutations in Notch pathway
genes, including defects in heart looping, branchial arch development,
midbrain mesenchyme cell loss, and trunk ventral neural tube
disorganization. This pleiotropy may indicate that the presenilins
affect the processing of all four mammalian Notch proteins, and
therefore, that the presenilin-null phenotype reflects a complete
absence of all mammalian Notch signaling activity. To date, only mice
carrying a mutation in the Notch-1 gene have been described
(Conlon et al. 1995), and these mice exhibit a less complex set of
phenotypes than those described here in presenilin-null embryos. It is
possible that mice with a combination of targeted mutations in all four
Notch genes would exhibit phenotypes identical to those of the
presenilin double-null embryos. However, the RBP-Jk transcription
factor, the only known mammalian member of its family, is thought to
act downstream of all four Notch genes. Hence, a complete loss of
RBP-Jk activity might be expected to phenocopy a complete loss of all
Notch genes. However, loss of both presenilins leads to more profound
developmental defects than even those observed in
RBP-Jk/ embryos, raising the
possibility that mammalian presenilins might function within and
outside the Notch pathway. For example, the presenilins can associate
with multiple proteins (see above) and may have a role in apoptosis,
the regulation of calcium homeostasis, or the regulation of G proteins.
In addition, the presenilins can associate with proteins that belong to
the Armadillo family and can affect -catenin stabilization and
trafficking, suggesting that they may play a role in the
Wnt/Wingless pathway (Zhou et al. 1997; Murayama et al.
1998; Yu et al. 1998; Zhang et al. 1998b; Nishimura et al. 1999a).
Alternatively, the more pleiotropic effects observed in presenilin
double-null mice, in comparison to those observed in
RPB-Jk-null mice, may indicate that Notch receptor signaling
may funnel into the nucleus to other transcription factors besides
RBP-Jk. Consistent with this hypothesis, studies in D. melanogaster have indicated that Notch can also function
independently of Suppressor of Hairless, the RBP-Jk homolog
(Matsuno et al. 1997).
The complex phenotypes observed in the presenilin-null embryos may be
attributable to a global mesoderm-patterning defect. For example,
disorganization of the paraxial mesoderm could affect both somite
segmentation and the patterning of the trunk ventral neural tube.
Furthermore, the disorganization of head mesenchyme, as evidenced by
cell loss in the presumptive midbrain region of presenilin double-null
embryos, could contribute to the anterior neuropore closure defects, as
proposed by genetic analysis of the murine Twist gene (Chen
and Behringer 1995). The branchial arch and heart looping defects may
also be due to the mispatterning of cephalic and lateral plate
mesoderm, respectively. Interestingly, we have not observed any obvious
morphological differences or changes in gene expression in primitive
streak mesoderm in E6.5-E7 presenilin double-null embryos (not shown).
Presenilin double-null embryos and Alzheimer's disease
It is interesting that mutant mice that completely lack PS2 have no
discernible phenotype. In contrast, humans with a point mutation in
only one copy of PS2 develop FAD. There are several possible
explanations for these contrasting effects of PS2 mutations in mice and
humans. First, it is possible that the mutant alleles in FAD, which are
point mutations, are actually gain-of-function alleles that confer a
novel activity onto the PS2 protein. Second, it is also possible that
the nervous system in humans is significantly more sensitive to
presenilin gene dosage than in mice, such that hypomorphic
loss-of-function mutations in even one allele of PS2 is sufficient to
result in severe pathology. If this is the case, a more severe
reduction in murine presenilin gene dosage, such as in the viable
PS1+/; PS2/
mice generated in this study, might result in AD-like pathologies. A
third possibility is that mutant FAD alleles may be acting in a
dominant-negative fashion interfering with the activity of the remaining three wild-type alleles. Complementation studies involving the introduction of FAD alleles of PS1 into mice and worms argues against this latter possibility (Levitan et al. 1996; Baumeister et al.
1997; Davis et al. 1998; Qian et al. 1998).
Regardless of whether the FAD-linked mutations cause a gain or a loss of function, the availability of cells and tissues derived from embryos lacking both presenilins will significantly facilitate functional studies aimed at understanding the role of these proteins in Notch signaling and in normal and disease cellular processes.
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Materials and methods |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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Generation of PS2-targeted mice
P1 clones containing the murine PS2 gene were isolated
from a 129 genomic library (Genome Systems) by PCR with primers from exon 4 of the published murine PS2 sequence (5'-TCCTCCACTGGGCAGTG, 5'-GGAAGAGGTGTGTGATGAGC). A 12-kb HindIII fragment
containing PS2 exons 4-6 was cloned into the pZERO vector (Invitrogen)
and mapped by Southern analysis and sequencing. A
StuI-EcoRI 2.2-kb fragment from intron 5 was
blunt-cloned into the BamHI and KpnI sites of
pPNT-loxP-Neo vector, followed by the insertion of the XbaI-ClaI 2.4-kb fragment containing exon 4 and most
of exon 5 into the corresponding sites in the vector. This vector was
electroporated into R1 ES cells using standard techniques (Pirity et
al. 1998). Following positive/negative selection,
homologous recombination events were detected at a 1:250 frequency,
as determined by Southern analyses with probes generated by PCR using
primers designed to exons 3 (5'-ATGTCAGCCGAGAGCCCCACATC,
5'-GGCTGGACAGATGGCTCAGCAGT) and 6 (5'-CATGGCTGGCTGATCATGTCCT,
5'-GGACAGCATACAGAGTCTACTC). PCR genotyping of mice and embryos was
performed with primers for the Neo gene
(5'-GCCTGAAGAACGAGATCAGCA), PS2 exon 5 (5'-AAGTATCGATGCTACAAGGTGAGG), and intron 5 (5'-CCCACATGATAAAAGGAGAGC). Two independent targeted lines were
used in aggregation with CD1 morulae (Pirity et al. 1998). Progeny from
backcrosses of founder mice (which transmitted the 129 ES-cell derived
allele of PS2 through the germ line) to 129svcp mice were pure 129.
Western blot analysis
Brains from PS2+/+, PS2+/, and
PS2(/ adult mice were dissected and
homogenized (Dounce) five times in lysis buffer A (Anafi et al. 1997),
followed by sonication on ice 3 times for 20 sec. The homogenate was
spun for 15 min, and the supernatant was quantitated using the BCA
reagent assay (Bio-Rad), according to manufacturer's protocols. Total
protein (75 µg) was loaded in each lane of a 10% polyacrylamide gel, followed by transfer onto nitrocellulose and blocking in 5%
nonfat milk in TBS overnight. Two different antibodies directed against
the hydrophilic loop of human PS2, antiPS2L1 (Fig. 1D) (Oyama et al.
1998) and PS2 Ab-1 (Oncogene) (not shown), were diluted in milk at
1:500 and 1:100, respectively, and incubated with the blot for
1 hr, followed by 4 washes (10 min) with TBS and 2 washes (10 min) with
TBS-T. This was followed by an incubation for 45 min with
HRP-conjugated goat-anti-rabbit antibody (Bio-Rad) diluted 1:20,000
in TBS-T. After 4 washes (10 min) in TBS-T, detection of
immunocomplexes was revealed using ECL reagents (Amersham). Both
anti-PS2 antibodies gave consistent results in multiple experiments with independent mice and primary cells derived from these mice.
Analysis of mutant mice
Mice were kept on a 12-hr light/dark cycle. Noon on
plug date was designated day 0.5, although the embryos were staged
according to standard morphological landmarks. Embryos were dissected
in HEPES-buffered Dulbecco's modified Eagle medium (DMEM) containing 10% serum, their yolk sacs were removed directly into PCR lysis buffer, whereas the embryos were fixed in 4% paraformaldehyde for
histology or in situ analysis. Embryos were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Unless indicated otherwise, all sections shown were transverse. In situ analysis was
performed as described (Conlon and Rossant 1992), using probes for
Dll1 (Bettenhausen et al. 1995), UncX4.1 (Mansouri et
al. 1997), Hes-5 (Akanawa et al. 1992), and Shh (Echelard et
al. 1993). Scanning electron microscopy (SEM) was performed as
described (Hayat 1974).
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Acknowledgments |
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We thank P. Hunter and S. Vesely for assistance with in situ and Western analysis; S. Tondat for ES cell aggregations; K. Harpal for sectioning of embryos; D. Holmyard for SEM; E. Fan for RNA analysis; P. Cheung for Southern blot analyses; F. Oyama, Y. Ihara, and T. Iwatsubo for PS2 antibodies; J. Rossant for in situ probes, helpful discussions, and comments on the manuscript; R. Conlon for in situ probes and helpful discussions; J. Cross, S. Cordes, and C.C. Hui for help with the analysis of the histological sections; S. Egan and G. Boulianne for critical reading of the manuscript; and S. Osadchuk for assistance with the genotyping. This work was supported by grants from the Medical Research Council of Canada to A.B. and P.H.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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Received July 27, 1999; revised version accepted September 3, 1999.
5 Present address: Huffington Center on Aging, Baylor College of Medicine, Houston, Texas 77030 USA.
6 Corresponding author.
E-MAIL donoviel{at}mshri.on.ca; FAX (416) 586-8857.
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References |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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),
would yield a 12-kb fragment from the untargeted locus and a 5.6-kb
from the targeted locus. Primers (arrows) designed to amplify both the
unmodified and the modified loci were used for genotyping by PCR. (NEO)
PGK-Neomycin resistance gene; loxP sites (
) flank the NEO gene;
(TK) thymidine kinase gene; (E) EcoRI, (H) HindIII,
(X) XbaI, (C) ClaI, (S) StuI, (B)
BamHI, (K) KpnI. (B) Southern analysis of
HindIII-digested genomic DNA from targeted ES cell clones
probed with a PS2 3' external probe, showing the expected sized
fragment for the correct integration of the PS2 targeting vector into
the murine PS2 locus in two independent cell lines designated by
+/
. (C) PCR analysis on tail DNA from
weaning-age mice from PS2 +/
