NuSAP is essential for chromatin-induced spindle formation during early embryogenesis.
ABSTRACT Mitotic spindle assembly is mediated by two processes: a centrosomal and a chromosomal pathway. RanGTP regulates the latter process by releasing microtubule-associated proteins from inhibitory complexes. NuSAP, a microtubule- and DNA-binding protein, is a target of RanGTP and promotes the formation of microtubules near chromosomes. However, the contribution of NuSAP to cell proliferation in vivo is unknown. Here, we demonstrate that the expression of NuSAP highly correlates with cell proliferation during embryogenesis and adult life, making it a reliable marker of proliferating cells. Additionally, we show that NuSAP deficiency in mice leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed in the mitotic cytoplasm. As a result of sustained spindle checkpoint activity, the cells are unable to progress through mitosis, eventually leading to caspase activation and apoptotic cell death. Together, our findings demonstrate that NuSAP is essential for proliferation of embryonic cells and, simultaneously, they underscore the importance of chromatin-induced spindle assembly.
During mitosis, replicated molecules of DNA compacted into
chromosomes must be partitioned equally between daughter cells.
The segregation of chromosomes needs to be precise, because a
single error can lead to aneuploidy or genetic instability, promoting
cell death or disease. This is especially true for pluripotent
embryonic stem (ES) cells, which have the capacity to propagate
and differentiate into a range of specialized cell types, and
eventually produce an entire organism.
Assembly of the mitotic spindle apparatus, which has a key role
in ensuring proper chromosome congression and accurate sister
chromatid separation, relies on two partially redundant mechanisms
– a centrosome-mediated and a chromatin-induced pathway –
whose relative contribution can vary depending on the cell type
(O’Connell et al., 2008). With the first mechanism, microtubules
are nucleated around the centrosomes and subsequently radiate
into the mitotic cytoplasm until they are captured and stabilized by
kinetochores (Kirschner and Mitchison, 1986). The second process
relies on the establishment of a RanGTP gradient around the
chromosomes (Kalab et al., 2006), which regulates the activity of
several target proteins and thereby stimulates the nucleation and
stabilization of microtubules near chromosomes (for a review, see
Kalab and Heald, 2008).
Most RanGTP targets have been extensively characterized in
biochemical experiments, meiotic egg extracts and/or cancer cell
lines. These rather specific approaches have often suggested unique
functions for the proteins involved, which nonetheless proved to
be redundant in more complex experimental settings (Groen et al.,
2009). Moreover, their contribution to spindle assembly in vivo
frequently turned out to be more limited than anticipated from
these in vitro experiments. For example, in vitro experiments have
shown that HURP is crucial for kinetochore fiber stabilization in
human cells (Koffa et al., 2006; Silljé et al., 2006; Wong and Fang,
2006) and essential for chromatin-induced microtubule assembly,
microtubule stabilization and their organization into a bipolar array
in Xenopus egg extracts (Casanova et al., 2008). Nevertheless,
mice deficient in Hurp develop normally (Tsai et al., 2008),
indicating that this protein is dispensable for cell proliferation
during mouse development and in most adult tissues. Instead, Hurp
is required for proliferation of the endometrial stroma in the uterus,
resulting in infertility of Hurp-deficient females owing to
Another example is the chromokinesin Kid. This RanGTP target,
originally characterized as a plus-end-directed microtubule-based
motor (Tokai et al., 1996), contributes to the alignment of
chromosome arms at the metaphase plate in Xenopus egg extracts
(Antonio et al., 2000; Funabiki and Murray, 2000), as well as
human cells (Levesque and Compton, 2001; Tokai-Nishizumi et
al., 2005) by creating a ‘polar ejection force’. Interestingly,
inactivation of Kid in mice resulted in embryonic lethality before
day 9.5 (E9.5) in 50% of the mice, whereas the other 50%
developed into healthy and fertile mice (Ohsugi et al., 2008). This
in vivo study pointed out that Kid contributes to compaction of
anaphase chromosomes, thereby preventing the formation of
multinucleated cells. This function of Kid seems to be crucial only
during oocyte meiosis II and the first embryonic cleavages, whereas
it is essential for neither somatic cell mitosis after the morula
stage, nor for male meiosis (Ohsugi et al., 2008).
Together, these studies nicely illustrate the significance of
investigating the contribution of RanGTP targets to cell proliferation
in vivo, as they unveiled new functions of the proteins concerned
and indicated that their action is essential only at specific
NuSAP is essential for chromatin-induced spindle
formation during early embryogenesis
An Vanden Bosch1, Tim Raemaekers2, Sarah Denayer3, Sophie Torrekens1, Nico Smets1, Karen Moermans1,
Mieke Dewerchin4,5, Peter Carmeliet4,5and Geert Carmeliet1,*
1Laboratory of Experimental Medicine and Endocrinology, 2Laboratory of Membrane Trafficking, 3Laboratory of Molecular Endocrinology and
4Vesalius Research Center (VRC), Katholieke Universiteit Leuven, 3000 Leuven, Belgium
5Vesalius Research Center (VRC), VIB, B-3000 Leuven, Belgium
*Author for correspondence (email@example.com)
Accepted 2 June 2010
Journal of Cell Science 123, 3244-3255
© 2010. Published by The Company of Biologists Ltd
Mitotic spindle assembly is mediated by two processes: a centrosomal and a chromosomal pathway. RanGTP regulates the latter
process by releasing microtubule-associated proteins from inhibitory complexes. NuSAP, a microtubule- and DNA-binding protein, is
a target of RanGTP and promotes the formation of microtubules near chromosomes. However, the contribution of NuSAP to cell
proliferation in vivo is unknown. Here, we demonstrate that the expression of NuSAP highly correlates with cell proliferation during
embryogenesis and adult life, making it a reliable marker of proliferating cells. Additionally, we show that NuSAP deficiency in mice
leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed
in the mitotic cytoplasm. As a result of sustained spindle checkpoint activity, the cells are unable to progress through mitosis,
eventually leading to caspase activation and apoptotic cell death. Together, our findings demonstrate that NuSAP is essential for
proliferation of embryonic cells and, simultaneously, they underscore the importance of chromatin-induced spindle assembly.
Key words: Mitosis, Spindle assembly, Microtubules, Embryogenesis
Journal of Cell Science
developmental stages or at defined sites. Thus, although in vitro
experiments can provide interesting clues about their function,
RanGTP targets are not necessarily essential in vivo, possibly
because of compensation by other proteins.
Until now, NuSAP [nucle(ol)ar spindle-associated protein] has
only been characterized in biochemical reconstitution experiments
and through loss-of-function studies in vitro. In Xenopus egg
extracts, NuSAP promotes the stabilization and crosslinking of
microtubules near chromatin, activities that are regulated by
RanGTP (Ribbeck et al., 2006; Ribbeck et al., 2007). Together
with the localization of NuSAP to spindle microtubules and its
enrichment around chromosomes (Raemaekers et al., 2003;
Ribbeck et al., 2006), these data suggest a role for NuSAP in
chromatin-induced spindle formation. Consistent with this,
knockdown of NuSAP in HeLa cells causes severe mitotic defects,
i.e. progression through mitosis is delayed as a result of incomplete
chromosome congression to the metaphase plate. Yet, some
cells progress to anaphase, often resulting in chromosome
missegregation or failed cytokinesis and, at later stages, the
appearance of binucleated cells and multipolar spindles
(Raemaekers et al., 2003). Nevertheless, in vitro systems suffer
from a few drawbacks. For instance, HeLa cells and other cancer
cell lines are inherently abnormal and have weakened checkpoint
activity. Hence, our aim was to explore whether NuSAP expression
is crucial for in vivo cell proliferation.
We therefore investigated whether the NuSAP expression pattern
correlates with cell proliferation in developing and adult mice, and
found an excellent association at all stages. Based on these findings,
we hypothesized that NuSAP deficiency in mice could be
detrimental to embryonic development, a process that relies strongly
on cell proliferation. This hypothesis was verified by generating
mice with a systemic NuSAP inactivation, which resulted in early
embryonic lethality, underscoring the importance of NuSAP during
in vivo proliferation.
Tissue-specific expression of NuSAP during adult life
In cultured cells, the expression of NuSAP is restricted to the
proliferation stage (Raemaekers et al., 2003). Based on these
findings, it was assumed that NuSAP expression in vivo would
also be related to cell proliferation. Therefore, NuSAP mRNA and
protein levels were examined in several murine and human tissues
that vary in their proliferation activity. Northern blot and
quantitative RT-PCR (qRT-PCR) analysis of adult mouse tissues
showed abundant expression of Nusap1 in the spleen, testis, thymus,
bone and spine (containing bone marrow), with lower mRNA
levels in the lymph node, muscle, ovary, uterus, small intestine and
stomach (Fig. 1A,B). In general, Nusap1 mRNA levels correlated
well with expression of the histone H4 gene, which is a DNA
replication marker. These data indicated a preferential expression
of NuSAP in organs with persistent cell proliferation in adulthood,
including the immune and reproductive systems. A similar
expression pattern was observed when NUSAP1 mRNA levels
were analyzed in human tissues. In particular, NUSAP1 was highly
expressed in the thymus and testis with lower levels in the ovary,
spleen, small intestine and colon (Fig. 1C). Western blot analysis
of several adult mouse tissues confirmed this expression pattern
and showed a close correlation between the expression of NuSAP
and proliferating cell nuclear antigen (PCNA). Notably, NuSAP
was abundantly expressed in the thymus, long bones (containing
bone marrow) and colon, with lower expression levels in the skin
NuSAP is essential for early embryogenesis
(Fig. 1D). Taken together, these data indicate that NuSAP is highly
expressed in adult tissues that contain a considerable population of
NuSAP expression is confined to cells engaged in mitosis
To substantiate that the expression of NuSAP is limited to
proliferating cells, we examined its expression pattern in murine
tissues typified by localized regions of cell proliferation, including
the small intestine, skin, bone and testis. Immunohistochemistry of
the small intestine showed strong nuclear NuSAP staining, which
was restricted to the epithelial cells localized in the crypts of
Lieberkühn (Fig. 1E). This region contains the mitotically active
cells, as confirmed by the positive staining for phospho-histone H3
(Ser10) (H3-P), a marker of mitotic chromatin. By contrast, the
absorptive epithelial cells of the villi and cells of the lamina
propria, the supporting layer underlying the intestinal epithelium,
showed little or no NuSAP or H3-P expression. High-magnification
confocal imaging of sections immunofluorescently labeled for both
NuSAP and H3-P showed that cells engaged in mitosis stained
positive for both proteins (Fig. 1F). Notably, in early mitosis
(prophase, prometaphase), NuSAP was found around the chromatin,
whereas during anaphase it relocalized to the spindle microtubules,
which is consistent with previous in vitro observations (Raemaekers
et al., 2003). In addition, NuSAP staining was also detected in a
small subset of interphase cells with immunoreactivity localizing
predominantly to the nucleoli. This finding is in agreement with
earlier data showing that NuSAP is expressed during the S-, G2-
and M-phase of the cell cycle, but not during G1-phase
(Raemaekers et al., 2003). Some interphase cells also displayed
foci of H3-P, which might be associated with transcriptional
activation, as previously described (Cheung et al., 2000).
Next, we investigated whether NuSAP showed a confined
expression pattern in murine skin sections. As expected, NuSAP
staining was restricted to the keratinocytes in the basal layer of the
epidermis and to the outer root sheath of the hair follicles, which
harbor mitotically active cells (Fig. 1G). Accordingly, the staining
pattern of NuSAP was largely similar to that of Ki-67, a marker of
proliferating cells, albeit that Ki-67 was more abundantly expressed
than NuSAP. Differentiating keratinocytes and most dermal cells
expressed neither Ki-67 nor NuSAP.
Subsequently, bone sections were analyzed for NuSAP
expression, because proliferation of the hematopoietic cells residing
in the bone marrow continues to be important during adult life.
NuSAP was abundantly expressed in the bone marrow cells, but
not in the differentiated osteocytes embedded in the bone cortex,
an expression pattern that was similar to that of Ki-67, although
Ki-67 staining was more abundant (Fig. 1H).
Finally, we analyzed the expression pattern of NuSAP in the
testis. Surprisingly, little or no NuSAP staining was detected in the
mitotically active spermatogonia of the testis. By contrast, high
expression was confined to the primary spermatocytes undergoing
meiosis (Fig. 1I). The interstitial cells of the seminiferous tubules,
the Sertoli cells and the seminiferous epithelium were negative for
NuSAP expression. Additionally, in sections from the ovary, NuSAP
expression was present in meiotic cells, as indicated by co-staining
with the oocyte marker Stat3 (Murphy et al., 2005), as well as in
proliferating follicle cells (supplementary material Fig. S1A).
To conclude, our analysis of NuSAP expression in adult somatic
as well as gonadal tissues indicated that its expression is confined
to actively proliferating cells in somatic tissues, as observed in the
Journal of Cell Science
small intestine, skin and bone marrow. In the gonads, NuSAP
expression was also found in meiotic cells.
NuSAP is widely expressed during murine embryonic
Given the close correlation of NuSAP expression with cell
proliferation in adult tissues, we hypothesized that NuSAP would
be abundantly expressed from early embryonic development on
and throughout embryonic growth, processes that strongly rely on
cell proliferation. Indeed, confocal imaging of mouse blastocyst
stage embryos clearly showed NuSAP staining in the nuclei of
numerous interphase cells, with enrichment in the nucleoli (Fig.
3246Journal of Cell Science 123 (19)
2A, left panel). In mitotic cells, NuSAP was detected around the
chromosomes and at the chromosome-proximal microtubules of
the spindle, as expected (Fig. 2A, right panel and magnifications).
Northern blot analysis of whole mouse embryos at later stages
of development showed high Nusap1 mRNA levels from E10.5
until E16.5 followed by a decline in Nusap1 expression, a pattern
that was comparable with that of histone H4 gene expression (Fig.
2B). This developmentally regulated expression pattern was
confirmed by qRT-PCR (Fig. 2C).
The tissue distribution of NuSAP expression was evaluated in
E13.5 embryos, a developmental stage that is characterized by
ongoing organogenesis and is accompanied by extensive cell
Fig. 1. Expression of NuSAP
correlates with cell proliferation in
adult murine and human tissues.
(A–C) Analysis of Nusap1 mRNA
levels in adult mice. Northern blot
analysis showing high similarity
between Nusap1 and histone H4
(H4) gene expression in murine
tissues (A) and a comparable
expression pattern of NUSAP1 in
human tissues (C). Actb (-actin)
and Gapdh levels were used as
loading controls (p. bl. lymph.,
peripheral blood lymphocytes).
(B)Quantification of Nusap1 and
Hprt mRNA levels in murine tissues
via qRT-PCR. Levels are expressed
relative to the Nusap1-to-Hprt ratio
in the brain, which was set to 1.
(D)Western blot analysis of NuSAP,
PCNA and -actin in murine tissues.
analysis showing that NuSAP
expression is confined to
proliferating cells in somatic murine
tissues. (E)In the small intestine,
NuSAP and phospho-histone H3
(Ser10) (H3-P) immunostaining are
restricted to the crypts. (F)Confocal
images show that intestinal crypt
cells engaged in mitosis are double-
stained for NuSAP and H3-P. A
magnification of cells in interphase
(arrowhead), prophase (arrow),
prometaphase (*) and anaphase (**)
is shown in the right panels.
(G,H)NuSAP and Ki-67 display a
comparable expression pattern in the
skin (G) and tibia (H). Scale bars:
50m (E,G,H), 10m (F).
(I)NuSAP immunostaining of
murine testis showing positivity in
meiotic but not mitotic cells
(magnification in right panel). Scale
Journal of Cell Science
proliferation. In situ hybridization and immunohistochemistry
demonstrated that NuSAP was ubiquitously expressed at this
developmental stage (Fig. 2D,E). Positive signals were observed
in several tissues including brain, heart, liver, lung, kidneys and
spine. NuSAP expression seemed therefore not to be confined to
tissues originating from a specific germ layer. To explore the link
with cell proliferation, the expression pattern of NuSAP was
compared with sites of BrdU incorporation and Ki-67 expression,
which are two established markers of cell proliferation. In the
gastrointestinal tract, NuSAP, BrdU and Ki-67 were detected in the
endodermal epithelium, which is still devoid of cytodifferentiation
NuSAP is essential for early embryogenesis
at this embryonic stage. Positive staining for these markers was
also observed in the mesodermal cells that give rise to the lamina
propria, the muscularis mucosae, the submucosa, the circular and
longitudinal muscle layers and the outer mesothelial layer
(oesophagus, Fig. 2F; stomach, Fig. 2G). In the brain, NuSAP, as
well as BrdU and Ki-67 staining, was found adjacent to the
ventricles, where proliferating cells are located that originate from
the ectoderm (Fig. 2H). In the growth plate of E18.5 pups, NuSAP
expression was restricted to the region of the periarticular and
columnar chondrocytes, and was absent from the hypertrophic
zone, which contains terminally differentiated cells (Fig. 2I). BrdU
Fig. 2. NuSAP is widely expressed during murine development. (A)Confocal images of NuSAP immunostaining of blastocysts isolated at embryonic day 3.5
(E3.5) or cultured in vitro for 1 day (E3.5+1). Note that NuSAP redistributes from the nucleoli in interphase cells (late S- and G2-phase) to the spindle microtubules
in the vicinity of the chromosomes in mitotic cells (arrows and magnifications). Scale bar: 20m. (B,C)Analysis of Nusap1 mRNA levels in developing mouse
embryos. (B)Northern blot analysis showing that Nusap1 expression correlates with that of histone H4 (H4) at different stages of development. 18S was used as a
loading control. (C)Quantification of Nusap1 and Hprt mRNA levels via qRT-PCR. Levels are expressed relative to the Nusap1-to-Hprt ratio at E9.5, which was
set to 1. (D)In situ hybridization of Nusap1 on a sagittal section showing extensive Nusap1 expression at E13.5. (E)Immunostaining of NuSAP on a thoracic (top)
and abdominal (bottom) transversal section showing extensive NuSAP expression at E13.5. Scale bar: 500m. (F–J) NuSAP localizes to regions of Ki-67
expression and BrdU incorporation. Positive staining in E13.5 embryos is found in the endodermal epithelium and mesodermal cells of the trachea, oesophagus (F)
and stomach (G) and adjacent to the ventricles in the brain (H), among other sites. Oe, Oesophagus; Tr, trachea; Ve, ventricles. (I,J) Immunostaining of NuSAP,
Ki-67 and BrdU in the growth plate of E18.5 embryos. (I)Immunoreactivity is found in the periarticular and columnar region and is absent from hypertrophic
chondrocytes. (J)Localization of NuSAP- and BrdU-positive cells in subsequent sections (some of the double-positive cells are indicated with arrows).
Pe, periarticular; Co, columnar; Hy, hypertrophic region. Scale bars: 100m (E), 200m (F–H).
Journal of Cell Science
and Ki-67 staining showed an identical distribution. Detailed
analysis of subsequent sections stained for NuSAP and BrdU
showed that nearly all cells displaying NuSAP positivity were also
BrdU-positive cells, supporting previous observations that NuSAP
expression in interphase is restricted to S- and G2-phase (Fig. 2J).
Overall, the pattern of NuSAP expression correlated better with the
extent of BrdU incorporation than with Ki-67 expression.
Taken together, NuSAP expression during murine development
was not confined to a specific germ layer, as tissues originating
from the ectoderm (brain, Fig. 2H), endoderm (gastrointestinal
tract, Fig. 2F,G; lungs and pancreas, supplementary material Fig.
S1B,C) and mesoderm (cartilage, Fig. 2I; heart and kidney,
supplementary material Fig. S1D,E) all contained NuSAP-positive
cells. Rather, its expression correlated with regions of cell
3248Journal of Cell Science 123 (19)
proliferation, as shown by the close parallel of its expression with
the pattern of BrdU incorporation and Ki-67 expression.
NuSAP inactivation results in early embryonic lethality
We next explored whether NuSAP is required during cell
proliferation in vivo by disrupting the gene in murine ES cells.
Homologous recombination and Cre–LoxP technology enabled us
to generate mutant ES cells lacking exon 3 of the Nusap1 locus in
one allele (Fig. 3A). Correct targeting of the mutant construct and
germline transmission was shown by Southern blot analysis of ES
cell and mouse tail DNA, respectively (Fig. 3B). Mice heterozygous
for Nusap1 were morphologically indistinguishable from wild-
type (WT) animals (Fig. 3C). They appeared healthy, gained weight
similarly to WT mice (supplementary material Fig. S2) and were
Fig. 3. Targeted disruption of Nusap1 induces early embryonic lethality. (A)Schematic representation of Nusap1 exon 3 inactivation strategy. Exons are
depicted as small boxes, loxP sites as triangles, the 3? external b-probe as a red bar and primers for genotyping as numbered arrows. At the bottom, DNA fragments
generated by NcoI digestion of either the wild-type (9 kb) or mutant allele (>12 kb) are indicated. (B)Southern blot analysis of offspring from heterozygous
matings by digestion of genomic tail DNA with NcoI and detection with the 3? external b-probe. (C)WT and heterozygous Nusap1 littermates are phenotypically
indistinguishable at 13 weeks. (D)PCR genotyping of WT and mutant alleles. (E)Phase-contrast microscopy shows that Nusap1–/–embryos cultured in vitro grow
slower than WT embryos and show internal disorganization. White arrowheads indicate cells of various sizes that are not connected to the inner cell mass (ICM,
dotted lines) or trophectoderm layer (TE). Black arrowheads indicate very small cells or fragments inside the ICM (magnifications in the insets). After 3 days of in
vitro culture (E3.5+3), WT and heterozygous embryos develop outgrowths composed of an ICM on top of a layer of trophoblastic giant cells (TGC). Nusap1–/–
embryos that reach this stage consist either of a very small ICM, remnant TGC or a combination of both. Scale bars: 50m.
Journal of Cell Science
fertile. However, intercrossing Nusap1+/–mice failed to yield any
Nusap1-null mice among the 478 live births, whereas other
genotypes were present at the expected frequency (Table 1). These
data indicate that one functional Nusap1 allele is sufficient for
normal development, whereas inactivation of both alleles leads to
embryonic lethality, suggesting that Nusap1 is essential for
To determine the stage when NuSAP is crucial for embryonic
development, embryos from Nusap1+/–intercrosses were collected
at different times of gestation and genotyped by PCR (Fig. 3D).
Analysis at E10.5 did not reveal any Nusap1-null embryos; yet, a
larger number of empty decidua was found in the uterus (38%)
compared with the amount observed after mating WT mice (19%)
(Table 1). The presence of these empty decidua suggests that
Nusap1-null embryos could implant into the uterine wall, but failed
to develop into gastrulated embryos and were therefore completely
resorbed at E10.5. Accordingly, all genotypes were present at the
expected frequency in embryos isolated before implantation at
E3.5 (Table 1). In addition, no difference in the morphology of the
blastocysts was observed between genotypes at this stage (Fig. 3E,
left panels). Together, these data indicate that Nusap1-null embryos
died between E3.5 and E10.5.
Nusap1-null mice die at the peri-implantation stage
To define more accurately the stage of embryonic lethality, embryos
were collected at the blastocyst stage (E3.5), cultured in vitro and
closely monitored by phase-contrast microscopy (Fig. 3E). It should
be noted that embryos develop more slowly in vitro than in utero.
WT and heterozygous Nusap1 embryos progressively increased in
size and hatched from their zona pellucida after two to three days
in culture. They subsequently attached to the chamber slide by
spreading of the trophectoderm cells, a behavior that is analogous
to in vivo implantation. However, Nusap1-null embryos were
clearly distinguishable from WT and heterozygous embryos from
E3.5+1 onwards. They were smaller, and the size of their inner cell
mass (ICM) was reduced, suggesting a decreased number of cells.
These differences became more pronounced at E3.5+2 (Fig. 3E,
dotted lines). In addition, the internal structure of the Nusap1-null
embryos was disorganized, because we could detect cells or cellular
fragments that were part of neither the ICM, nor the surrounding
trophectoderm layer (Fig. 3E, white arrowheads). These cells or
fragments were of various sizes, with an excess of very small
structures. Small fragments were also found in the ICM (Fig. 3E,
black arrowheads). Furthermore, after 3 days of culture, the number
of Nusap1-null outgrowths decreased (Table 2), indicating that a
large fraction of them died or were in such an advanced stage of
disintegration that we were unable to retrieve sufficient genetic
material for genotyping.
The surviving Nusap1-null embryos were still able to hatch
from their zona pellucida and attach to the chamber slide with a
normal frequency. Indeed, 33% of the surviving Nusap1-null
embryos attached to the slide at E3.5+2, compared with 29% for
WT embryos (n18 and 28, respectively), and this number increased
to 77% and 80%, respectively, at E3.5+3 (n26 and n45,
respectively). Nevertheless, the Nusap1-null outgrowths remained
smaller than the WT or heterozygous outgrowths and mainly
consisted of residual ICM cells and/or remnant trophectoderm
cells, called trophoblastic giant cells at this stage, because they
become polyploid, non-dividing cells after implantation (Fig. 3E,
right panels). The aberrant appearance of Nusap1-null outgrowths
at E3.5+3 was associated with a strongly reduced mitotic activity,
NuSAP is essential for early embryogenesis
assessed by immunofluorescence staining for H3-P, indicating that
they were devoid of cell proliferation, whereas WT embryos
displayed numerous H3-P-positive cells (supplementary material
Fig. S3). Collectively, these results demonstrate that NuSAP
inactivation induces embryonic death at the peri-implantation stage.
Absence of NuSAP leads to increased apoptosis
The reduced growth of Nusap1-null embryos, their rapid
disintegration and the presence of small cellular fragments
suggested that lack of NuSAP resulted in apoptosis. To explore this
possibility, we closely inspected DNA integrity in embryos that
had been cultured for 3 days (Fig. 4A), a stage at which the
Nusap1-null outgrowths were severely affected. Confocal
microscopy of TO-PRO-3 iodide-labeled WT outgrowths showed
that cells of the ICM had regularly shaped nuclei, were of uniform
size (assessed by phalloidin staining) and grew on top of
trophoblastic giant cells. By contrast, the smaller Nusap1-null
outgrowths lacked a discernible ICM and the remaining cells
contained nuclei of various sizes. Large, irregular nuclei (probably
the remnants of trophoblastic giant cells) as well as very small,
dense DNA fragments were detected in the Nusap1-null outgrowths.
The latter morphological changes are suggestive of nuclear
fragmentation during apoptosis.
To confirm that the apoptotic pathway in Nusap1-null embryos
was turned on, we assessed the activation of caspase-3 by
immunostaining. Quantitative analysis was performed on confocal
images through the entire embryo at E3.5+2, the time point when
the phenotype became noticeable (Fig. 4B). Very few cells in the
WT embryos stained positive for active caspase-3 (0 or 1 per
embryo; n6). However, the number of caspase-3-positive cells in
Nusap1-null embryos was strongly increased (8–20 per embryo;
n5; P<0.001) (Fig. 4C). These data indicate that the apoptotic
pathway is triggered at an early stage in Nusap1-null embryos and
that massive apoptosis ultimately results in the disintegration of
the entire embryo.
NuSAP deficiency impairs mitotic progression
A potential explanation for the increased apoptosis is that NuSAP
deficiency leads to mitotic defects. To investigate this possibility,
Table 1. Genotypes of progeny from Nusap1+/–matings
Nusap1+/+ Nusap1+/– Nusap1–/– decidua number
Neonatal Expected 25% 50% 25%
Detected 40% 60% 0% 478
E10.5 Expected 20% 41% 20% 19%a 138
Detected 20% 42% 0% 38% 173
E3.5 Expected 25% 50% 25%
Detected 24% 56% 21% 102
aResult obtained after mating Nusap1+/+mice.
Table 2. Genotypes of progeny from Nusap1+/–intercrosses
harvested at E3.5 and cultured in vitro
Nusap1+/+ Nusap1+/– Nusap1–/– Total number
Expected 25% 50% 25%
E3.5 + 1 day 22% 53% 25% 292
E3.5 + 2 day 29% 50% 21% 84
E3.5 + 3 day 26% 60% 14% 125
E3.5 + 4 day 33% 55% 12% 33
Journal of Cell Science
we analyzed the number of mitotic cells and mitotic progression in
E3.5+1 embryos immunostained for H3-P by confocal imaging
(Fig. 5A). The mitotic cells were identified based on both H3-P
positivity and their DNA staining pattern. Only mitotic cells with
clearly condensed chromosomes (prometaphase until telophase)
were counted, because the speckled H3-P staining pattern in
prophase cells was not clearly discernible from the H3-P staining
that indicates transcriptional activation in interphase cells (Cheung
et al., 2000). Likewise, apoptotic cells, as judged by the presence
of strongly condensed DNA fragments, or cells with extremely
altered chromosome arrangements, were not included.
Mitotic cells were occasionally detected in WT and heterozygous
blastocysts (0–9 per embryo; n10) and the majority of them
(63%) were found in the ICM. By contrast, Nusap1-null embryos
showed a pronounced number of mitotic cells (4–22 per embryo;
n10) (Fig. 5B). This finding could either result from increased
cell proliferation or a mitotic delay. Several observations support
the latter possibility. First, quantification of the total cell number
revealed that Nusap1-null embryos have a decreased total cell
number at this stage compared with WT and heterozygous
littermates, making increased cell proliferation less likely (Fig.
5C). Second, the distribution of the mitotic phases in these embryos
is different between genotypes. In WT and heterozygous embryos,
3250 Journal of Cell Science 123 (19)
19% of the mitotic cells was in prophase, 54% in prometaphase,
18% in metaphase and 9% in anaphase or telophase, a distribution
that is comparable with the one described by Le Cam and colleagues
(Le Cam et al., 2004). In Nusap1-null embryos, more cells were
found in prometaphase (72%), whereas the fraction of cells in
metaphase and in anaphase or telophase was strongly decreased
(10% and 2%, respectively) (Fig. 5D). These data suggest that
Nusap1-null cells can enter mitosis normally but get retarded in
NuSAP is required for proper chromosome alignment
To investigate whether this impaired mitotic progression resulted
from spindle abnormalities, we immunofluorescently stained
embryos for -tubulin and DNA to visualize the mitotic spindles.
As expected, the mitotic spindles appeared properly arranged
during the different stages of mitosis in WT and heterozygous
embryos (Fig. 5E, left panel). More specifically, chromosomes
were precisely aligned at the equatorial plane during metaphase
and were correctly segregated during anaphase.
In Nusap1-null embryos, however, the majority of mitotic cells
resided in a stage that was reminiscent of prometaphase, i.e. a
mitotic spindle was assembled but chromosomes remained
unaligned (Fig. 5E, right panel). Spindle structures were often
distorted and chromosomes were severely dispersed throughout
the cell, suggesting that not all chromosomes could be captured
properly by spindle microtubules. These observations, together
with the localization of NuSAP at chromosome-proximal
microtubules, are consistent with the suggested role of NuSAP in
linking microtubules to mitotic chromosomes, a model that was
proposed by Ribbeck and co-workers (Ribbeck et al., 2007). Note
that we did not observe any multinuclear cell or multipolar spindle
in the ICM of WT, heterozygous or Nusap1-null embryos. Taken
together, these in vivo findings indicate that NuSAP is required for
mitotic progression by ensuring proper chromosome alignment.
NuSAP deficiency causes sustained spindle assembly
We next tried to determine why the cells in Nusap1-null embryos
accumulated in prometaphase. We hypothesized that the spindle
assembly checkpoint (SAC), which retains cells in mitosis by
preventing anaphase onset in the presence of unattached
kinetochores (Musacchio and Salmon, 2007), could not be satisfied
in NuSAP-deficient embryos because of impaired chromosome
congression. We tested this hypothesis in several ways. First, we
reasoned that in embryos with a properly functioning SAC, cells
would accumulate in mitosis in response to artificial induction of
sustained SAC activation. In other words, when the SAC is
operative in Nusap1-null embryos, the mitotic index will (further)
increase when SAC activity is stimulated. This was verified by
culturing WT and Nusap1-null embryos in the presence of either
nocodazole or taxol. Both drugs prevent normal spindle assembly
and thus cause prolonged SAC activity as soon as the cells enter
mitosis. Interestingly, after 18 hours of culture in the presence of
either of these compounds, the mitotic index of the embryos
increased markedly, irrespective of their genotype (Fig. 6A,B).
More specifically, following nocodazole treatment, the mitotic
index in WT and heterozygous embryos increased 20-fold compared
with 13-fold for Nusap1-null embryos. Taxol treatment caused a
sixfold increase of the mitotic index in WT and heterozygous
embryos and a threefold increase in Nusap1-null embryos. These
findings demonstrated that the SAC was indeed capable of arresting
Fig. 4. Nusap1-null blastocysts display increased apoptosis. (A)Confocal
images of DNA integrity in blastocysts harvested at E3.5 and cultured in vitro
for 3 days (E3.5+3). The majority of nuclei in Nusap1–/–outgrowths display
fragmentation (magnification in inset). Note that these embryos consist mainly
of remnant trophoblastic giant cells (TGC) (Nusap1–/–, left panel) and/or a
small residual inner cell mass (ICM) (Nusap1–/–, right panel). Scale bar:
10m. (B)Maximum intensity Z-projection of confocal images of E3.5+2
blastocysts stained for cleaved caspase-3. Scale bar: 10m. (C)The
percentage of cells positive for caspase-3 cleavage is increased in Nusap1–/–
embryos. Values are means ± s.e.m., n5–6; **P<0.01.
Journal of Cell Science
cells in mitosis in Nusap1-null embryos and thus that NuSAP
inactivation did not affect the integrity of the SAC.
Second, we examined the presence of a crucial SAC component,
BubR1, on the kinetochores of unattached chromosomes. Despite
technical limitations, BubR1 staining was found on the kinetochores
in pro(meta)phase cells of control as well as Nusap1-null embryos
(Fig. 6C). These data suggested that NuSAP is not essential for
recruitment of BubR1 to unattached chromosomes. Additionally,
the SAC was shown to be activated in at least a subset of the
pro(meta)phase cells found in Nusap1-null embryos at E3.5+1.
Finally, we treated embryos with SP600125, a compound that
disrupts the SAC by inhibiting Mps1 (Schmidt et al., 2005). We
found that the mitotic index in WT and heterozygous embryos was
not altered, but it strongly decreased (–47%) in Nusap1-null
embryos, indicating that sustained SAC activity was indeed
responsible for retaining the cells in Nusap1-null embryos in
mitosis (Fig. 6D,E).
To conclude, our data support the following sequence of events
in Nusap1-null embryos: incomplete chromosome congression to
the metaphase plate delays cells in prometaphase and keeps the
SAC activated, causing a steep rise in the mitotic index; this
prolonged stay in mitosis eventually triggers the apoptotic pathway
NuSAP is essential for early embryogenesis
and leads to cell death. Together, our findings show that NuSAP is
essential for embryogenesis.
In the present study, we demonstrate that NuSAP, a protein shown
to contribute to chromatin-induced spindle formation in vitro by
generating microtubules near chromatin, is essential for cell
proliferation during murine embryonic development. NuSAP
deficiency impairs mitotic progression in early embryos because
chromosomes fail to align at the metaphase plate, resulting in
sustained SAC activity and ultimately, cell death (Fig. 7). Loss of
NuSAP function clearly cannot be compensated by any other
protein, underscoring the unique function of NuSAP in chromatin-
induced spindle assembly.
Apart from validating that NuSAP is essential for mitotic spindle
assembly in vivo, our study confirms the requirement of chromatin-
induced spindle assembly for cell proliferation in vivo. NuSAP is
one of the few proteins, in addition to Rae1 and Cdk11 (Yokoyama
et al., 2008; Blower et al., 2005), shown thus far to be crucial to
RanGTP-mediated spindle assembly in vivo, as demonstrated by
the early lethality of mice lacking either of these proteins (Babu et
al., 2003; Li et al., 2004). Indeed, other RanGTP targets seem to
Fig. 5. Impaired mitotic progression in NuSAP deficient embryos. (A)Confocal images of phospho-histone H3 (Ser10) (H3-P) staining in E3.5+1 blastocysts,
showing a high number of mitotic cells in Nusap1–/–blastocysts. Scale bar: 10m. (B)The mitotic index is increased in Nusap1–/–embryos. Values are means ±
s.e.m.; n10; **P<0.01. (C)The total cell number of E3.5+1 embryos is comparable between genotypes. Values are means ± s.e.m.; n10. (D)Cells in
prometaphase are enriched in E3.5+1 Nusap1–/–embryos. Values are means ± s.e.m.; n14–30; *P<0.05. (E)Confocal images showing mitotic spindles in E3.5+1
embryos (Nusap1+/+and Nusap1+/–left to right: prometaphase, metaphase, anaphase, telophase, cytokinesis). In Nusap1–/–embryos, the majority of the cells
display abnormal spindle organization and chromosome dispersion (arrows). Scale bar: 10m.
Journal of Cell Science
be dispensable for spindle assembly in vivo, as exemplified by the
survival of mice lacking either Hurp or Kid (Ohsugi et al., 2008;
Tsai et al., 2008). The fact that NuSAP is crucial for chromatin-
induced spindle assembly might be explained by its ability to
interact with microtubules and DNA (Ribbeck et al., 2006; Ribbeck
et al., 2007), whereas other proteins, such as Hurp, only act on
microtubules (Koffa et al., 2006; Silljé et al., 2006; Wong and
The chromosome dispersion and inefficient chromosome
congression observed in Nusap1-null embryos is consistent with
its proposed role in the generation of microtubules near
chromosomes, thereby increasing the likelihood that a chromosome
gets incorporated into the spindle (Ribbeck et al., 2007). The exact
molecular mechanism underlying the interaction between NuSAP,
chromatin and microtubules remains unresolved.
When chromosomes fail to align at the metaphase plate in
Nusap1-null cells, the SAC remains active for a prolonged period,
after which caspase-mediated apoptosis is induced. Although our
3252Journal of Cell Science 123 (19)
data are not conclusive about the mechanism that triggers cell
death in early embryos, we speculate that cells lacking NuSAP
either die in mitosis, or in G1-phase after exiting mitosis, without
satisfying the spindle checkpoint, referred to as ‘mitotic slippage’
(Weaver and Cleveland, 2005; Rieder and Maiato, 2004). In the
latter case, the cells would bypass the SAC as a result of gradual,
proteasome-mediated degradation of cyclin B1 (Brito and Rieder,
An intriguing, yet not fully explained observation is that Nusap1-
null embryos are able to reach the blastocyst stage without
displaying morphological abnormalities or obvious cell proliferation
defects. Analogous findings of peri-implantation lethality are also
noticed when cell-cycle-related genes are inactivated (Artus and
Cohen-Tannoudji, 2008). A possible explanation is the plasticity of
the mammalian pre-implantation embryo, which has the ability to
adapt its development in response to various perturbations.
Alternatively, this phenotype might indicate that NuSAP is still
present in early Nusap1-null embryos, either in the form of protein
Fig. 6. Analysis of spindle checkpoint activity in NuSAP-deficient embryos. (A)Confocal images of E3.5 blastocysts cultured in the presence of nocodazole or
taxol and stained for phospho-histone H3 (Ser10) (H3-P). Scale bar: 10m. (B)The mitotic index of the embryos is increased by nocodazole or taxol treatment
irrespective of the genotype of the embryo. Values are mean ± s.e.m.; n10 (untreated), n>9 (nocodazole), n3 (taxol); ***P<0.001 vs untreated of same genotype.
(C)Confocal images showing BubR1 staining in several pro(meta)phase cells in control as well as Nusap1–/–blastocysts cultured in vitro for 1 day (E3.5+1). Scale
bar: 10m. (D)H3-P staining in E3.5 embryos cultured overnight in the presence of SP600125. Scale bar: 10m. (E)The mitotic index of Nusap1–/–embryos is
strongly decreased after treatment with SP600125. Values are mean ± s.e.m.; n3–6.
Journal of Cell Science
originating from the oocyte or as the product of maternally inherited
mRNA. Generally, this maternal mRNA persists only until the mid
two-cell stage, however, the proteins encoded by this mRNA can
be present beyond this time point (Bachvarova and De Leon,
1980). Nevertheless, this possibility is less likely in the case of
NuSAP, because it is degraded at the end of mitosis. In agreement,
we could not demonstrate its presence in Nusap1-null blastocysts
at E3.5 by confocal microscopy, whereas it was clearly detectable
in wild-type embryos (supplementary material Fig. S4), indicating
that the amount of maternally derived protein is negligible at this
stage in Nusap1-null embryos. An alternative explanation is that
chromatin-induced microtubule nucleation is not essential for the
early cleavage divisions or that, because of the slow cycling speed
of ES cells before implantation (Ciemerych and Sicinski, 2005),
chromosomes have sufficient time to become aligned at the
metaphase plate, even though chromosome congression is
The present study also highlights the parallels and contradictions
between in vivo inactivation and in vitro depletion of NuSAP
(Raemaekers et al., 2003). Several similarities are prominent; for
instance, in both cell types, chromosome congression to the
metaphase plate is impaired, leading to accumulation of cells in
mitosis. However, differences in the way HeLa cells and ES cells
react to NuSAP depletion are even more pronounced. Whereas
HeLa cells accumulate in mitosis without becoming fully blocked
in the cell cycle, the progression through the cell cycle of virtually
all embryonic cells is precluded once they have passed a crucial
stage, presumably around E3.5. As a result, no abnormal anaphases,
binucleated interphase cells or multipolar spindles are detected in
NuSAP is essential for early embryogenesis
the ICM of Nusap1-null embryos, whereas their number is greatly
increased among NuSAP-depleted HeLa cells. Rather than
progressing through mitosis and producing abnormal daughter
cells, the ES cells are delayed in prometaphase and eventually die
from apoptosis. A likely explanation is that HeLa cells are only
partially depleted by RNAi, whereas the ES are completely devoid
of NuSAP. Alternatively, these differences might reflect the intrinsic
capability of HeLa cells to bypass the SAC in the presence of a
limited number of unaligned chromosomes (Wong and Fang, 2006).
The crucial contribution of NuSAP to cell proliferation in vivo
is also suggested by its expression pattern in vivo, which we
extensively characterized. NuSAP expression in vivo is confined
to proliferating cells, as evidenced by its restricted localization to
areas where proliferating cells are present, such as the crypts of the
intestine in adult mice and the cells lining the ventricles in the
brains of developing embryos. The strong correlation of the pattern
of NuSAP expression with that of BrdU incorporation and Ki-67
staining confirms the proliferation-restricted expression of NuSAP.
Moreover, its colocalization with the mitotic marker H3-P, together
with its presence in only a subset of interphase cells, strongly
suggest that the cell-cycle-dependent expression of NuSAP is
similar in vivo and in vitro. Consistent with this, NuSAP expression
is generally less abundant than Ki-67 expression. We argue that
NuSAP more accurately predicts the number of proliferating cells,
because it is only expressed from late S-phase, in cells that have
undergone DNA replication and are therefore committed to enter
mitosis (Raemaekers et al., 2003), whereas Ki-67 is also expressed
during G1-phase (Gerdes et al., 1984). These G1-phase cells
represent a large fraction of the total cell population; however,
their fate can range from entering G0-phase and staying quiescent
for a longer period, over proceeding to S-phase, to becoming
apoptotic. Consequently, quantification of Ki-67-positive cells
generally leads to an overestimation of the number of proliferative
Remarkably, in the gonads, NuSAP expression was also found
in meiotic cells. More precisely, in the testis, NuSAP was not
expressed in the proliferating spermatogonia, but instead, it was
present in spermatocytes undergoing meiosis. By contrast, in the
ovary, NuSAP expression was found both in proliferating follicular
epithelial cells and in oocytes. The role NuSAP has in meiosis
remains to be elucidated.
Proliferation is not only essential during development and tissue
homeostasis during adult life, but it is also a hallmark of
malignancy. Consistent with this, NuSAP was detected in four
independent studies aiming to identify genes related to clinical
outcome of breast cancer (Lauss et al., 2008). Thus, apart from
being a reliable proliferation marker, NuSAP holds the potential of
becoming a diagnostic marker for breast cancer and potentially
also other tumor types.
To conclude, the expression of NuSAP is restricted to
proliferating cells in developing as well as adult mice, establishing
its potential as a reliable proliferation marker. Importantly, we
show that NuSAP is an essential protein in vivo and hereby
emphasize the importance of the chromatin-induced mechanism of
spindle assembly for cell proliferation in vivo.
Materials and Methods
Dissected bones (E18.5), soft tissues and whole embryos (E13.5) were fixed in 2%
paraformaldehyde in PBS. Bones were decalcified in 0.5 M EDTA-PBS (pH 7.4) for
3 days, before paraffin embedding. To detect NuSAP and Ki-67, antigens were
retrieved in Tris–EDTA solution (10 mM Tris-HCl, 1 mM EDTA, pH 9) at 98°C.
Fig. 7. Schematic interpretation of the effects of NuSAP inactivation on
mitosis in embryonic stem cells. In wild-type (Nusap1+/+) cells, both
centrosome- and chromatin-induced microtubule formation contribute to
spindle assembly, resulting in efficient spindle assembly and chromosome
congression. The spindle assembly checkpoint (SAC) ensures accurate
chromosome segregation by preventing anaphase onset until all chromosomes
are properly attached to microtubules from both spindle poles. One of the
proteins involved in this checkpoint, BubR1, localizes to unattached
kinetochores. In NuSAP-deficient (Nusap1–/–) cells, the chromatin-induced
pathway of spindle assembly is severely affected. Consequently, chromosome
capture by spindle microtubules is highly inefficient and the chromosomes
remain dispersed in the mitotic cytoplasm. The SAC therefore cannot be
satisfied, as witnessed by the presence of BubR1 on the kinetochores.
Eventually, caspases are activated and cell death is induced.
Journal of Cell Science
3254Journal of Cell Science 123 (19)
Endogenous peroxidase activity was blocked with 3% H2O2 in methanol and
unspecific binding with TNB blocking buffer (TSA Biotin System kit, Perkin Elmer,
Waltham, MA). Sections were incubated overnight with rabbit anti-NuSAP (1:1000)
(Raemaekers et al., 2003) or rabbit anti-Ki-67 antibody (Novocastra, Newcastle
upon Tyne, UK; 1:2000). Antibody binding was visualized with the Dako Envision
System (Dako, Glostrup, Denmark). For H3-P immunostaining (rabbit anti-H3-P
antibody; Upstate, Charlottesville, VA; 1:100), antigens were retrieved in Target
Retrieval Solution (Dako; pH 6) at 90°C; endogenous peroxidases were blocked
with 0.3% H2O2and unspecific binding was blocked with 20% normal goat serum
(Dako) in TNB blocking buffer. BrdU injection and staining were performed as
described (Maes et al., 2004). Immunofluorescent labeling of NuSAP, H3-P and
Stat3 (rabbit anti-Stat3 antibody; Santa Cruz Biotechnology, Santa Cuz, CA; 1:100)
was performed essentially as described for immunohistochemistry. Antibody binding
was detected with Alexa-Fluor-488- or -546-labeled secondary antibodies (1:500;
Invitrogen, Carlsbad, CA). Stat3 detection required amplification with the Tyramide
Signal Amplification (TSA; Cy3) System (Perkin Elmer). Hoechst 33342 was applied
to stain nuclei. Bright-field images were acquired on an Axioplan 2 microscope
(Zeiss, Jena, Germany) and fluorescence images on a FV1000 confocal microscope
(Olympus, Tokyo, Japan).
Isolation of RNA and real-time quantitative RT-PCR analysis
RNA isolation from adult mouse tissues and whole embryos as well as qRT-PCR
analysis was performed essentially as described (Maes et al., 2004). Primer and
probe sequences are outlined in supplementary material Table S2.
Targeted disruption of the Nusap1 locus
A mouse bacterial artificial chromosome library was screened with Nusap1 cDNA
as a probe. One clone, encompassing exons 3, 4 and 5 of the Nusap1 gene, was
subjected to restriction mapping, subcloning and DNA sequencing. To delete exon
3 (containing the start codon), a targeting construct was generated by inserting
homologous regions of the Nusap1 gene into a pNT-lox-2b vector (Fig. 3A).
Following electroporation into 129/Sv mouse ES cells, G418- and gancyclovir-
resistant clones were screened for homologous recombination. Correctly recombined
clones were transiently transfected with a Cre-expressing plasmid to remove the neo
cassette together with exon 3. Selected clones were introduced into Swiss mouse
embryos via morula aggregation, implanted into pseudopregnant foster mothers
(129/Sv) and bred for germline transmission with Swiss mice, to eventually obtain
mice. All mice were bred in our animal housing facilities
(Proefdierencentrum Leuven). Experiments were approved by the ethical committee
of the Katholieke Universiteit Leuven.
Genomic DNA isolation and PCR genotyping
Genomic DNA from mouse-tail biopsies was prepared as described (Maes et al.,
2006). Genomic DNA from early mouse embryos was obtained by incubating them
in 20 l lysis buffer (10 mM Tris–HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.1 g/l
gelatin, 0.45% NP-40, 0.45% Tween-20, 200 g/ml proteinase K) for 6 hours at
55°C. Semi-nested PCR primers (arrows in Fig. 3A; sequences in supplementary
material Table S1) were designed to generate a product specific for either the wild-
type or the mutant Nusap1 allele.
Isolation of preimplantation embryos and in vitro blastocyst culture
Nusap1+/–mice were mated overnight and successful matings were detected by the
presence of a vaginal plug. Pregnant mice were killed at E3.5, their uterus was
removed and embryos were flushed out of the oviducts with ES cell medium
(Schoonjans et al., 2003). For in vitro culture, embryos were transferred to gelatin-
coated chamber slides containing ES cell medium. Where mentioned, nocodazole,
taxol (Sigma) or SP600125 (Enzo Life Sciences) was added to the medium to a final
concentration of 200 ng/ml, 200 nM and 20 mM respectively. The embryos were
inspected and photographed regularly with a Zeiss Axiovert 25 inverted microscope.
Immunofluorescence analysis of embryos
When needed, embryos were treated briefly with acidic Tyrode’s solution (Sigma)
to remove their zona pellucida. They were fixed in 4% paraformaldehyde in PBS
and permeabilized in 1% Triton X-100 in PBS. Unspecific binding was blocked with
0.5% Tween-20 in PBS containing 5% bovine serum albumin. Subsequently, the
embryos were incubated overnight with either rabbit anti-NuSAP (1:500), rabbit
anti- H3-P (1:500), rabbit anti-caspase-3 (Cell Signaling Technology, Danvers, MA;
1:500) or sheep anti-BubR1 antibody (Abcam, Cambridge, UK; 1:200). Unspecific
binding was blocked with 50% normal goat serum, followed by incubation with an
Alexa-Fluor-488- or -546-labeled secondary antibody (Invitrogen; 1:500). Finally,
the embryos were incubated with Alexa-Fluor-488- or -546-conjugated phalloidin
(Invitrogen; 1/100) and mounted in fluorescence mounting medium (Dako) containing
TO-PRO-3 iodide (Invitrogen; 1:200). For -tubulin staining (mouse anti--tubulin;
Sigma, clone DM1A; 1:200), embryos were incubated in prewarmed (37°C)
extraction buffer (80 mM PIPES pH 6.8, 1 mM MgCl2, 5 mM EGTA, 0.5% Tween-
20, 25% glycerol) before permeabilization in 1% Tween-20 in TBS.
Embryos were imaged at several z-positions by confocal microscopy, on average
every 1.5 m. Images were acquired either on an inverted Diaphot 300 microscope
(Nikon, Tokyo, Japan) (Plan Apo 60?/1.40 oil) connected to an MRC1024 confocal
imaging system (Bio-Rad Laboratories, Hercules, CA) or on an inverted Eclipse
E800 microscope (Nikon) (Plan Apo 60?A/1.40 oil) connected to a Bio-Rad
Radiance 2100 confocal imaging system using LaserSharp software. Quantification
of cell numbers was performed using the Cell Counter plug-in of ImageJ.
Comparison between quantitative data of two groups was done by two-tailed
Student’s t-test following an F-test using statistical software (NCSS, Kaysville, UT).
For multiple comparisons, Fisher’s LSD was performed after ANOVA.
The authors thank T. Van de Putte for advice on the isolation and
culture of preimplantation embryos, L. Le Cam for helpful suggestions
on blastocyst manipulations and immunofluorescent staining, L.
Schoonjans for providing ES cell medium, M. Depypere, I. Stockmans,
R. Van Looveren and M. Van Camp for technical assistance. This work
was supported by grants from the Fund for Scientific Research Flanders
(FWO; G.0508.05 and G.0587.09) and fellowships from the Institute
for the Promotion of Innovation through Science and Technology in
Flanders (IWT) (A.V.) and FWO (T.R.).
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