DEVELOPMENTAL DYNAMICS 238:110 122, 2009

RESEARCH ARTICLE

BMP2 Is Required for Cephalic Neural Tube Closure

in the Mouse
*
Trisha Castranio1 and Yuji Mishina1,2

BMPs have been shown to play a role in neural tube development particularly as dorsalizing factors. To explore the possibility
that BMP2 could play a role in the developing neural tube (NT) beyond the lethality of Bmp2 null embryos, we created Bmp2
chimeras from Bmp2 null ES cells and WT blastocysts. Analysis of Bmp2 chimeras reveals NT defects at day 9.5 (E9.5). We found
that exclusion of Bmp2 null ES cells from the dorsal NT did not always prevent defects. For further comparison, we used a Bmp2
mutant line in a mixed background. Phenotypes observed were similar to chimeras including open NT defects, postneurulation
defects, and abnormal neural ectoderm in heterozygous and homozygous null embryos demonstrating a pattern of dose-
dependent signaling. Our data exposes BMP2 as a unique player in the developing NT for dorsal patterning and identity, and
normal cephalic neural tube closure in a dose-dependent manner.

Developmental Dynamics 238:110 122, 2009. Published 2008 Wiley-Liss, Inc.

Key words: BMP2; neural tube defects; neurulation; neural tube closure; NTD; chimera

Accepted 13 November 2008

INTRODUCTION
Bone morphogenetic proteins (BMPs) were
originally identified by their ability to
cause bone differentiation more than 40
years ago (Urist, 1965). Together with
growth and differentia-tion factors (GDFs),
nodal and lefty, BMPs compose a large
subgroup within the transforming growth
fac-tor- (TGF- ) gene superfamily
(Kingsley, 1994; Zhao, 2003). How-ever,
recent studies of several organ-isms
demonstrate that several BMPs have other
roles during embryogene-sis, notably in
dorsoventral (DV) and/or anterior
posterior (AP) axis formation and
patterning (Hogan, 1996; Mishina, 2003;
Whitman, 1998).
Although BMPs and their signaling
molecules are important factors in
patterning in early embryonic devel-
opment (Kishigami and Mishina, 2005),
BMP2 function during early mouse
development is poorly under-stood due to
the early lethality of tar-geted null mutant
embryos (Zhang and Bradley, 1996). Very
recently, it has been reported that BMP2
has a unique functional role in the develop-
ing embryo for the migration but not
induction of neural crest cells (Correia et
al., 2007). Although there are still many
important questions unan-swered as to the
role of BMP2 in early organogenesis, a
floxed mouse line for Bmp2 has recently
become available
and BMP2 function in later stages of
development has begun to emerge (Ma et
al., 2005; Tsuji et al., 2006). A lim-ited
number of attempts to reveal the function
of BMP2 in early stages in-cluding neural
tube development has been reported (Ybot-
Gonzalez et al., 2002, 2007).
During the 24 hr between E8.5 and E9.5,
a mouse embryo goes through rapid and
dramatic development (Kaufman et al.,
1998). Developmen-tal events at this stage
include fore-limbs budding, otic and optic
pits in-vaginating, heart looping, along
with embryonic turning and primitive gut
evolution. Simultaneously, the neural tube
(NT) closes and three distinct re-
 Additional Supporting Information may be found in the online version of this article.
1
Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
2
University of Michigan, School of Dentistry, Ann Arbor, Michigan
Grant sponsor: Intramural Research Program of the NIEHS/NIH; Grant number: ES071003-10.
*Correspondence to: Trisha Castranio, Molecular Developmental Biology Group, Laboratory of Reproductive and Develop-mental Toxicology,
National Institute of Environmental Health Sciences, National Institutes of Health, 111 T. W. Alexander Dr., Research Triangle Park, NC 27709. E-
mail: castran1@niehs.nih.gov
DOI 10.1002/dvdy.21829
Published online 17 December 2008 in Wiley InterScience (www.interscience.wiley.com).

Published 2008 Wiley-Liss, Inc.

This article is a US Government work and, as such, is in the
public domain in the United States of America.
 Bmp2 AND NEURAL TUBE DEFECTS 111

TABLE 1. Phenotypes of Heterozygous and Null Embryos Separated Into Four Categoriesa

Categories Nulls Heterozygotes (%) Homozygous (%)

1. Normal 14 (21) 0 (0)
2. Postneurulation, partial open NT 40 (62) 0 (0)
3. Hypomorphism, heart defects, open NT, failure to turn 9 (14) 9 (43)
4. No development beyond the 5-somite stage 2 (3) 12 (57)

a
Images in Figure 1 show heterozygous mice with phenotypes similar in kind to the chimeras with low to moderate range contribution while the nulls
matched the high to very high contribution chimeras.
 2002; Zohn well as RESULT slight the somite
et al., earlier stage S abnormalitie heterogene count.
2007), for events s and -ity Homozygou
gions of the its unique involving ectoderm Embryos postneurulat For some, increased s null em-
brain take closure neural crest important Show ion defects there were survival bryos (Fig.
shape. issues and cells only for roof Cephalic in the no somites beyond 1KO)
Closure in differentiat (Fukuda et plate Neural forebrain formed and E8.5. showed
the mouse ion al., 2007; formation Tube region (Fig. no Homozygo open NT de-
begins at 3 requiremen Kanzler et and Defects 1B); recognizable us mutant fects,
distinct ts. al., 2000; developmen middle-high neural embryos hypomorphi
points, the t after the BMP2 tissue. We
Sailer et al., contribution for Bmp2 sm, failure
second of closure of chimeras used 4
2005; chimeras displayed to turn,
which varies the neural generated independent
In mid- Sakuta et show a more phenotypes failure to
among tube? In this from Bmp2 Bmp2 / ES
gestation, al., 2006; severe that mimic produce
strains, and re-port, we null ES cell lines
BMPs Timmer et phenotype the limb buds,
then the will cells and and all
function in al., 2002; including phenotypes heart-
neural tube describe wild type showed
the Yanagisawa open NT of the looping
zips shut how the (WT) blas- similar
progressio et al., 2001). defects and chimeras defects, and
(Copp et al., level of tocysts pheno-
n, Numbers of some (Fig. 1, reduced
2003). BMP2 were types. No
differentiat BMPs are posterior Table 1). somite
While the expression harvested abnormal
ion, and expressed defects (Fig. Some count. Table
process of is inversely at E9.5. ES embryos
developme with 1C); high heterozygo 1 displays
neurulation propor- cell were re-
nt of distinctive contribution us and all these pheno-
or neural tional to the derived covered
certain patterns in chimeras homozygo types
tube closure severity of cells in when wild
aspects of the cephalic show open us null grouped into
can be defects in chimeras type ES
the brain. re-gion prior NT defects, embryos 4 categories
described, the carry-ing cells were
For to and failure to showed based on
its developing one copy used at any
example, during turn, overt abnormality.
mechanisms mouse of level of
BMP4 neural tube posterior abnormal From 17
and the embryo ROSA26
along with closure defects, contribution phenotypes litters with
players in- implicating (Soriano,
FGFs are (Furuta et failure to (data not similar in normal
volved are a dose- 1999) were
involved in al., 1997). form limb shown). kind to Mendelian
still unclear. dependent visible
neural Notably, buds, and chimeras ratios, we
Dorsal-ven- requirement after X-gal We also
induction Bmp2 is heart- with mid- found 79%
tral (DV) for de- stain-ing. transferred
(Jessell and expressed looping dle to very of
patterning, velopment Embryos at the targeted
Sanes, exclusively defects (Fig. high heterozygot
cell shaping, during early every level Bmp2
2000; in the 1D). Very contributio es and 100%
cell organogene- of contri- mouse line
Linker and surface high n of null of nulls
proliferation sis. bution of (Zhang and
Stern, ectoderm contribution ES cells. In showed
, and cell Furthermore null ES Bradley,
2004). adjacent to chimeras Figure 1F some type
movement , we propose cells 1996) from
BMP2 has the dor-sal showed J, of
are all that BMP2 verified 129SvEv to
established neuroectode complete heterozygo abnormal-
actively signaling that ES a mixed
expression rm (Furuta failure to us mice ity. Among
involved in from and cells 129/B6
patterns in et al., 1997) develop be- showed these
normal NT within the incorporate background
the dorsal suggesting a yond the postneurul embryos,
de- surface into all since mixed
mid-line of unique role E8.0 E8.5 ation (Fig. 3% of het-
velopment ectoderm tissue types ge-netic
the brain, for BMP2 in stage (Fig. 1F) erozygotes
and closure propels NT at this backgrounds
specifically the cephalic 1E). (category and 57% of
(Brook et closure stage. In are
the roof region. But 2) and nulls
al., 1991; regardless Figure 1A, generally
plate of the is the open NT showed the
Copp et al., of the very low less
telencephal expression defects most severe
1988; establishme (less than vulnerable
on (Furuta of BMP2 in (Fig. 1GJ) phenotype:
Estibeiro et nt of the 30%) to genetic
et al., the surface (category no devel-
al., 1993; dorsal contributio defects and
1997). 3). A small opment
Padmanabha identity and n chimeras show less
BMP2 is number beyond the
n, 2006), but supports showed no severe
also linked showed 5-somite
the cephalic develop-ing overt phenotypes
to glial or anterior stage
region has neural tissue abnormaliti than purely
CNS fates and (category 4).
rarely been in the dorsal es, whereas in-bred
and retinal posterior The third
targeted portion of low to strains. We
patterning, NT de- category
experimenta the NT. middle collected
neurogenes fects, (cate-
lly (Mackay contributio litters at
is, and failure to
et al., 2006; n chimeras E9.5 and, as
astroglioge turn, and
Soo et al., showed expected,
nesis, as reduced
112 CASTRANIO AND MISHINA
gory 3) is defined by phenotypes in-cluding
reduced overall size, heart-looping defects,
an open NT in the cephalic region, as well
as a failure of the embryo to turn for 14%
of het-erozygous and 43% of homozygous
null embryos, respectively. The sec-ond
category contains only heterozy-gous mice
with a partially open ce-phalic NT that is
usually found in the forebrain region and
occasionally in the hindbrain region with
closure oc-curring normally at closure
point 2. The remaining heterozygotes were
considered normal in nature and ac-counted
for 21% of all collected het-erozygous
embryos. A high incidence of abnormalities
found in the het-erozygous embryos
reflects a skew in the Mendelian ratio
versus weaning stage ratios (WT:Het:mut
58:81:0 from Het x Het 129/B6
background, WT/Het is 1.4 instead of 2.0).
Simply, Bmp2 homozygous null,
heterozygous and chimeric embryos
showed compa-rable defects in cephalic
neural tube closure. For clarification, we
will focus for the remainder of this report
on cephalic neural tube development pre-
viously categorized in Table 1.
BMP2 Plays a Role in the
Development of Dorsal
Neural Tissue
Histological sections of these embryos
magnify the defects previously de-scribed
and illuminated other unex-
pected abnormalities in tissue devel-
opment (Figs. 2 4). A pattern emerged for
exclusion of Bmp2 null ES cells from
certain tissues in devel-oping chimeric
embryos. Based on available WT cells, null
cells were ex-cluded from the dorsal
portion of the developing neural tube, the
dorsal head mesenchyme, and the surface
ec-toderm as well as the developing limb
buds, atria, posterior embryo, lateral plate
mesoderm, primitive gut, and second
branchial arch (Fig. 3AD). With greater
incorporation of Bmp2 null cells, as in
higher contribution chimeras, those tissues
either failed to develop or were severely
impaired (Figs. 2D,E, 3EG). Null cells
could be excluded from limb buds, head
mesen-chyme, and the dorsal portion of the
NT until null cell incorporation reached the
high level. At the high and very high
contribution levels, there was not a pattern
of exclusion and the few WT cells available
were randomly distributed within the em-
bryo and development was stalled (Figs.
1D, 2CE). Exclusion from the dorsal
portion of the NT was through-out the
embryo even where the NT was properly
closed in the trunk re-gion of the embryo
(Fig. 3AD).
Sections from abnormal heterozy-gous
embryos (category 2) showed ab-normal
neural tissue in the dorsal por-tion of the
developing NT in the cephalic region
including the develop-ing eye (Fig. 2F, H).
Abnormalities ap-
peared in the most anterior sections (Fig.
4D) along with disorganization of the
forebrain region (Fig. 4, arrow-heads).
Sections within the cephalic region showed
an increasing amount of disorder from
anterior to posterior and, finally, an
inability to close the NT in the forebrain
region. The mes-enchyme appeared greatly
reduced surrounding the dorsal portions of
the NT (Fig. 4D, E, arrows). The develop-
ing eye, although formed, showed some
irregular neural tissue but all other
developing tissues including the surface
ectoderm appeared normal. Middle-high
contribution chimeras showed the same
anterior to posterior degeneration (Fig. 3E
G). Note that WT cells (pink) nearly
exclusively pop-ulated the dorsal portion of
the NT and dorsal head mesenchyme and
still the neuroectoderm appears abnormal
in size and shape (Fig. 3EG, arrow-heads)
relative to WT (Fig. 3HJ, ar-rowheads).
As with the heterozygous embryos,
disorganization occurs in op-tic and otic
vesicles and cells are ab-normal in
appearance (Fig. 3E, F). Ho-mozygous
nulls showed open neural tube defects with
abnormal neural tis-sue (Fig. 2I) or a
complete failure to develop neural tissue
(data not shown) similar to very high
contribu-tion chimeras (Fig. 2D, E, respec-
tively) as well as a lack of organization and
delayed NT development. The ventral
portion of the NT for most em-bryos
appeared relatively normal.

Fig. 1. Abnormalities found in chimeras, heterozygous, and homozygous null embryos. BMP2 chimeras (AE) were generated from null ES cells
carrying the LacZ gene and wild type blastocysts and were harvested at E9.5. Shown are different levels of contribution of null ES cells to chimeric
development in increasing amounts from A to E. X-gal stains null ES cells blue. Note that defects appear at fairly low levels of contribution. Black
arrowheads show defects of the cephalic region and grey arrowheads show other stage-specific developmental defects. A: No contribution chimera
shows no apparent defects. B: Low contribution chimera has an abnormal-shaped head and fails to form telencephalic vesicles. C: Middle
contribution chimera shows postneurulation (closed neural tube) defects in the forebrain, midbrain, and hindbrain regions of the developing embryo.
The heart has not looped properly. D: High contribution chimera has open neural tube defects as well as stalled development in the anterior and
posterior portions of the embryo. There is also abnormal heart looping, a failure to develop limb buds, and failure of embryonic turning. E: Very high
contribution chimera fails to develop any normal structures including somites. BMP2 Heterozygous and Homozygous Null Embryos, E9.5. White
arrowheads show cephalic defects while grey arrowheads show other stage-specific developmental defects. FJ: Defects occur in some
heterozygous embryos where postneurulation defects show abnormal head shape and lack of telencephalic vesicles (F) or embryos have open NT
defects in the forebrain region (G, H) or forebrain-midbrain region, while some have completely open NT in the cephalic region (J). KO: For
homozygous nulls, defects in the developing embryo include failure to close the NT, abnormal heart development, lack of limb bud development, and
also failure to turn. Scale bar 1 mm.

Fig. 2. Sections from the cephalic region. Cephalic sections (AJ) show interesting patterns of development. Chimeric sections are paraffin sections
while heterozygous and null sections are frozen sections. Chimeras (BE) show an increase in severity of defects proportional to the incorporation of
ES cells. Blue cells indicate ES cell-derived Bmp2 null cells. B: Sections from a low contribution chimera show relatively normal development similar
to WT (A). C: Sections from a high contribution chimera show defects in the dorsal neural tube and head mesenchyme in the forebrain and hindbrain
regions. F: Sections from heterozygous mice show similar defects to high contribution chimeras. G,H: Magnified views show disorganization and
abnormal neural tissue in the dorsal portions of the neural tube. D,E: Sections from very high contribution chimeras show severe abnormalities
including an expanded and disorganized neural tube (D) or an absence of neural tissue altogether (E). I: Homozygous nulls show an abnormal neural
tube. J: Magnified view shows the neural mesenchymal-like mixture. Ch, chimera; het, heterozygous mouse; hb, hindbrain; fb, forebrain; wt, wildtype;
null, homozygous null. Scale bar 1 mm.
Fig. 1.

Fig. 2.
114 CASTRANIO AND MISHINA
 Scanning electron microscopy showed
abnormalities in the heterozygous and
homozygous null embryos. In the het-
erozygous embryo (category 2) (Fig. 5B),
closure points 1, 2, and 3 appeared closed
but tissue between those points (Fig. 5B,
blue arrow; Fig. 5E, arrowhead) was open
and neural tissue was exposed. The over-all
shape of the head was irregular in contrast
to the WT (Fig. 5A). The three regions of
the brain that define this stage were
unrecognizable. The null embryo (Fig. 5C)
had a completely open neural tube (blue
arrow) and although the otic vesicle (a
surface ectoderm-derived tis-sue) appeared
normal, there was no neu-ral tube closure
or brain segmentation oc-curring in the
cephalic region (Fig. 5C, F). Neural tissues
that appeared columnar in the WT control
(Fig. 5K, arrowheads) were round and
mesenchymal in homozy-gous null
embryos that showed closure defects (Fig.
5H, arrowhead). Where clo-sure was
occurring, the surface ectoderm appeared
overwhelmed by abnormal neu-ral tissue as
closure point 3 attempted to zip toward
closure point 2 (Fig. 5H, star) while in the
WT the surface ectoderm contains the
neuroectoderm within its boundaries as the
NT and surface ecto-derm zip shut (star,
Fig. 5K). Near the midbrain-forebrain
junction close to clo-sure point 2 (green
box, Fig.5 I. L), abnor-mal tissue remained
but the surface ecto-derm appeared normal
and distinct. The surface ectoderm still met
the leading edge of the NT, but appeared to
be sepa-rate and distinct (Fig. 5I, arrow)
whereas in the WT the two remain
integrated (Fig. 5L, arrow).
forebrain regions (Guo et al., 2007) where
Bmp2 heterozygous embryos showed
abnormalities. Expression of Wnt1 in
homozygous null embryos was normal
even when the entire ce-phalic NT was
open (data not shown; Correia et al., 2007).
To determine the role of BMP2 sig-
naling in the control of cell death ver-sus
proliferation, phosphohistone 3 staining
and TUNEL analysis were performed. As
shown in Figure 7AC, phosphohistone 3
(pH3) immunohisto-chemistry revealed no
overt differ-ences in proliferation in
mesenchymal and neural tissue among wild
type and Bmp2 heterozygous and null em-
bryos. However, TUNEL assay dem-
onstrated that heterozygous (category 2, 3)
and homozygous null (category 3, 4)
embryos displayed unique patterns of cell
death in comparison to wild type embryos.
Heterozygous embryos with
postneurulation defects (cate-gory 2)
showed cell death in the dorsal
mesenchyme as well as within the neural
tube and surface ectoderm in the hindbrain
region (Fig. 8DF). Ap-optosis occurred
mostly in the head mesenchyme (n 3) and
in the surface ectoderm directly adjacent to
the clos-ing NT in category 3 heterozygous
em-bryos with open NT defects (Fig. 8G
I). In category 3 Bmp2 null embryos, cell
death occurred in the otic vesicles, surface
ectoderm, and head mesen-chyme (Fig.
8K) but none was seen in the most anterior
region of the head where no forebrain
neuroectoderm
was observed whereas the hindbrain
contained abnormal neuroectoderm (Fig.
8J, n 3). For category 3 het-erozygous
embryos and category 4 null embryos
where dorsal mesen-chyme was greatly
reduced, apoptosis (Fig. 8L and data not
shown) occurred mostly in the remaining
mesenchyme along the surface ectoderm.
These re-sults, together with histological
obser-vations, suggest that BMP2 plays
unique and critical roles in maintain-ing
proper identity of the neuroecto-derm and
prevention of apoptosis in the mesenchyme
and surface ecto-derm, especially in the
dorsal portions of the cephalic region.
Does p53-Dependent
Apoptosis Affect Neural
Tube Closure in BMP2
Embryos?
Because of the increase in apoptosis in
BMP2 embryos, we considered whether it
played a role in the failure of the NT to
close. Recently, it has been reported (Jones
et al., 2008; Pani et al., 2002) that p53
affects apoptosis during neural tube
development. Enhanced apoptosis in the
neuroectoderm is found in the Pax3
(Phelan et al., 1997) and Tcof1 (Dixon et
al., 2006) mutants and these were rescued
(Jones et al., 2008; Pani et al., 2002) by
pifithrin- , an inhibitor of p53. We
considered whether we could also rescue
apoptosis and ultimately NT defects found
in Bmp2 embryos by us-ing pifithrin- . We
treated pregnant fe-

Expression of Pax3 Is
Missing in the Developing
Cephalic Neural Tube
Whole mount in situ hybridization (WMIS)
using Bmp2 homozygous null embryos
showed an absence of Pax3 (n 3), which
was normally expressed in the dorsal-most
portion of the neu-ral tube in wild type
embryos (Fig. 6AE). However, we found
normal ex-pression patterns in the cephalic
re-gion for Pax6, Wnt1, Shh, Otx2 (data not
shown, n 3 respectively) as well as Noggin
and Bmp4 (see Supplemen-tary Fig. S1,
which is available online; n 3,
respectively). Although Wnt1 is considered
a dorsal NT marker, in the cephalic region
it is restricted to the caudal mesencephalon
and not the
Fig. 3. Exclusion of Bmp2 null cells in chimeric embryos. Sections from the head trunk and tail
portion of various embryos (AD) show a consistent pattern of exclusion of ES cell-derived Bmp2
null cells (blue) from these tissues in the developing embryo: the head mesenchyme, body wall,
dorsal portion of the NT, the lateral plate mesoderm, ventricular chambers of the heart, dorsal
portion of otic vesicles, limb buds, second branchial arch, and extraembryonic tissues. WT cells
from host blastocysts stain pink. A moderate contribution chimera serial sectioned through the
cephalic region (EG) shows increased severity of neural tube defects in the hindbrain and the
forebrain regions (arrowheads) when compared to the WT (HJ). The neural tube becomes disor-
ganized and tissue appears abnormal. Exclusion of null cells is consistent. Mesenchyme appears
normal and condensed in the forebrain regions but less dense in the hindbrain region (arrows in E,
H). The otic vesicle (a surface ectoderm derived tissue) is abnormal as is the optic vesicle (a NT-
derived tissue). Embryos shown before sectioning in the left boxes with approximate positions of
sections (for EJ). Embryo left of AD is only representing sectioning positions. Scale bar 1 mm.
Fig. 4. Serial sections of wild type and abnormal heterozygous mouse embryos. Transverse frozen
sections were made serially through the cephalic region. Serial sections through a heterozygous
mouse show increasing disorganization especially in the forebrain region (DF, arrowheads).
Abnormal neural tissue and disorganization (star) pervade the anterior-most regions of the head.
Mesenchyme appears evenly distributed and not condensed (DF, arrows). The forebrain region is
open (E, F) compared to WT (AC) and neural tissue is disorganized. Blue boxes (in B, E)
highlight areas of interest. Magnified images in corresponding blue boxes show disorganization
and expan-sion of neuroectoderm and the lack of mesenchymal condensation in the forebrain
region. Scale bar 1 mm.
 Bmp2 AND NEURAL TUBE DEFECTS 115

Fig. 3.
Fig. 4.
116 CASTRANIO AND MISHINA
 heterozygo phalic ion and is 1.5 for penetrance heterozygo
us and region or dissolu-tion, 129SvEv may be tes, we
males at homozygo fail to close as do gous and inbred, 1.4 subject to have et al., 2008).
stages us null the NT al- abnormal homozygo for Swiss the genetic confidence Embryos
appropriate embryos together. heterozygou us mutant Web-ster, background. her with
to NT clo- show Chimeras s and em-bryos 0.84 for contributio genotype
sure. comparabl did not homozygous for Bmp2 C57BL6/J n affects fn/- show
Overall, we e show ce- null varied so congenic). embryonic closure
found no phenotypes phalic NT embryos. much These facts developme defects in
rescue in the closure Higher con- (Table 1). suggest that Another nt. the ce-
(Table 2) for develop- defects tribution Since these genetic potential Recently, phalic
homozygous ing neural specifically chimeras mice come mod-ifiers explanation we neural tube
null tube and in in the fail to from a would for generated a similar to
embryos progressive forebrain or exclude and mixed influence phenotypic mouse with the ones
regard-less se-verity midbrain have backgroun frequency variation is a described in
of treatment based on region as cephalic d, we and severity maternal conditional this study,
with most the het- regions that cannot of the con- allele for and its fre-
classified as available erozygous contain ei- exclude the phenotypes tribution Bmp2. The quency
category 4. amount of mice did ther reduced influence described in since, as floxed varies
We found BMP2 probably mesenchym of genetic this study. stated allele in- depending
partial signal. because e and modifiers We also above, sur- cludes a on the geno-
rescue for Chimeras they have disorga- as one of experienced vivability of neomycin type of
heterozygou show not the ability to nized neural the a reduced the selection mother,
s embryos only a exclude null tissue or possible number of heterozygou cassette in more
with fewer predisposit cells and, increased reasons. As heterozygou s pups was the Bmp2 frequent if
cate-gory 2 ion to therefore, mesen- men-tioned s pups from lower when locus mother is
as well as exclude prevent chyme and in the WT (male) mothers (floxed- fn/-
category 3 null cells those de- the absence Results heterozygou were het- neo allele, resulting in
in the from fects. Those of neural section, we s (fe-male) erozygous abbreviate lower ex-
pifithrin- specific chimeras tissue, the expe- breeding. for Bmp2. d fn) that pression
-treated tissues of that show same as rienced a For the We also can levels of
embryos the post- homozygous lower mixed back- discov-ered transcribe Bmp2 in the
versus the developing neurulation null number of ground we that among only 10% decidua and
DMSO- em-bryo defects embryos. heterozy- used in this heterozygou of the uterus
treated but they demonstrate gous pups study, the s inter- transcript (Singh et al.,
embryos. also show that surface It is at weaning pro-portion crosses when com- 2008).
There were abnormal ectoderm interesting stage from of when severe pared with These facts
also more phe- closure to consider heterozygo heterozygot phenotypes the wild further
category 1 notypes at takes prece- why us es for Bmp2 were found type locus support the
(normal) a fairly low dence over phenotypes intercross at weaning in (Singh idea that a
heterozy- level of NT closure (WT/Het is stage is homozygous maternal
found in
gous contribu- itself. We 1.4 instead nearly 50% null influence
both
embryos tion. never of 2.0, as ex- embryos, including
heterozy-
recovered. Beginning observed proportion pected. heterozygou expres-sion
with embryos of Het is However, s littermates levels of
abnormal with a 58% this ratio usually had Bmp2 may
neural closed instead of varies de- NT defects affect the
DISCUSS 67%). With se-verity of
ION tissue and neural tube pending on as well.
the failure while the over 5 their genetic When the
The to surface years of background, homozygous embryonic
Severity of invaginate ectoderm re- breeding, 38% for nulls were phenotypes
Defects in tel- mained we 129SvEv less severe, shown in
BMP2 encephalic open. experience inbred, 42% heterozygou Table 1
vesicles, Chimeric d sim-ilar for Swiss s embryos especially
Null and
chimeras embryos phenomena Webster, from the when ex-
Heterozyg , but rates pression
with in- with NT and 35% for same
ous of levels in
creasing closure C57BL6/J mother had
Embryos reduction Bmp2
contributio defects also congenic. no
Is n of null show were These embryos are
phenotype
Dose cells then defects in varied numbers or only mild decreased.
Dependen fail to dorsal based on again sug- postneurula- Maternal
t maintain neural tissue their gest that the tion defects. influence,
neural developmen genetic severity of Since all ge-netic
Chimeric backgroun background,
embryos tissue in t, in-cluding phenotypes pregnant fe-
the ce- disorganizat d (WT/Het and males were and other
and
unknown Since other presence of
genetic BMPs, wild type
modifiers especially cells for
may all BMP4 and middle to
contribute to BMP7, are high con-
phenotypic also tribution
variation in expressed chimeras
Bmp2 het- in the and for
erozygous developing heterozy-
and cephalic gous
homozygous region embryos
null em- (Furuta et (with one
bryos, al., 1997) functional
which is of the copy of
ultimately E8.5E9.5 Bmp2)
controlled embryo, it demonstrate
by the is not only a
available important dosage
amount of to note that requirement
BMP2. the pattern of BMP2
Clearly, a of but also a
minimal expression failure of
reduction of for each other BMPs
BMP2 BMP is to rescue
signaling unique nor-mal NT
will with developmen
negatively potentially t and tissue
affect overlappin iden-tity.
embry-onic g func- While
developmen tions. Our exclusion of
t at this histologica Bmp2 nulls
stage. We, l data show ES cells
therefore, that from
believe that chimeras specific
the severity exclude tissues
of defects Bmp2 null including
among cells when the surface
chimeras, possible. ectoderm
heterozygou Specificall implies a
s and y, in the ce- cell-
homozygous phalic
null region,
embryos is exclusion
in-versely of Bmp2
proportional null cells
to the may
amount of illuminate
BMP2 the
signaling. requiremen
t of BMP2
for NT
closure in
the
BMP2 cephalic
May Play regions of
a Role in heterozygo
us and
Dorsal homozy-
Identity of gous null
the embryos.
Cephalic These NT
Neural closure
Tube defects
occurring
in the
 Bmp2 AND NEURAL TUBE DEFECTS 117

autonomous function for BMP2 (Morin-

Kensicki et al., 2001), exclu-sion from
tissues that do not express BMP2, such as
the dorsal neuroecto-derm, suggests a non-
cell-autonomous function for BMP2 in
those tissues. Since Bmp2 is a signaling
molecule, availability of BMP2 from
neighbor-ing WT cells may be a
determining factor for contribution to those
tissues that do not express BMP2. These
ac-tivities suggest that BMP2 has unique
and non-overlapping functions in the
cephalic region for NT development. Since
dorsal identity issues have been
investigated mainly in the trunk re-gion
(Dietrich et al., 1993; Goulding et al.,
1993b; Meyer and Roelink, 2003), the
cephalic region may be regulated

Fig. 5. Scanning electron microscopy analyses

of cephalic regions. Scanning electron micros-
copy (SEM) of WT (A, D), heterozygous (B, E),
and homozygous null embryos (C, F). Compar-
ison of forebrain region for each embryo (white
arrowheads) shows overt open neural tube de-
fects (blue arrows). SEM analysis at higher
magnification shows open neural tube defects.
Note the abnormal shape of the neural tissue
(red arrowhead in H) compared with stage-
matched WT (red arrowheads in K) and the
failure to close at any point of the developing
neural tube. There is invagination at the optic
pit, a medial/ventral NT derivative tissue. The
surface ectoderm appears normal and aligned
with the prospective dorsal portion of the NT
(white arrow, I) but the border becomes ob-
scure towards the forebrain region (star, H).
Images of corresponding regions from a WT
embryo before NT closure (E9.0) are shown for
comparison (JL). G: Dorso-lateral view, ante-
rior is left. J: Dorsal view. Fig. 5.

Fig. 6. Expression of Pax3. WMIS for Pax3 expression is shown in AE. WT embryos (A) show normal expression of Pax3 in the dorsal portion of the
NT throughout the embryo. Heterozygous embryos with open NT defects (B) show normal expression in the trunk region (black arrows) but lack
expression in the cephalic region (red arrows), as do homozygous null embryos ( CE). D is a dorsal view of the same embryo shown in C to further
clarify the absence of Pax3 in the edge of unclosed neural tube (red arrows).
118 CASTRANIO AND MISHINA
Fig. 7. Analysis of proliferation. Analysis using phosphohistone 3 (pH3) immunohistochemistry of cephalic sections for proliferation show no overt
differences among WT (A), heterozygous (B) and homozygous (C) nulls.
differently. The loss of Pax3 expres-sion in
the dorsal cephalic NT of these embryos
implies that BMP2 is neces-sary for dorsal
identity of the cephalic NT. And although
the loss of a single marker does not
definitively eliminate other possibilities,
the lack of Pax3 expression and the failure
of other BMPs to rescue the NT defect
strongly supports the idea that BMP2 plays
a role in dorsal identity of the cephalic NT.
BMP2 Is Required for
Normal Cephalic Neural Tube
Closure
Another important aspect of neural tube
closure is physical kinetics in-cluding cell
movement, cell prolifera-tion, and cell
shaping (Padmanabhan, 2006). Closure
defects in the cephalic region for
heterozygous embryos for Bmp2 ranges
from completely open to specific opening
in the midbrain and forebrain, and then, the
least severe case, open only in the forebrain
region of the NT. These openings may
reflect the faulty physical kinetics of NT
clo-sure in embryos lacking BMP2 expres-
sion. It is important to note that for the
cephalic region, the forebrain is the most
expansive region required for closure. It
would require the most pro-liferation of
mesenchyme, neural ecto-derm, and
surface ectoderm to close. Closure point 2
in the mouse is found near the midbrain
region and from this point the NT zips in
opposite di-rections toward the forebrain to
meet closure point 3 or toward the hind-
brain to meet closure point 1 (Copp et al.,
2003). Curly tail (ct) mutation causes a NT
closure defect in the cau-dal region (Copp
et al., 1988). It is speculated that alteration
in the phys-ical torque of the tail would be
a cause of the NT defect because of a lack
of proliferation in the posterior portion of
the embryo. It is notable that none of the
Bmp2 heterozygous embryos show open
NT defects in the hindbrain, if the forebrain
region is closed. These facts suggest the
possibility that the forebrain region because
of its physical size and shape requires more
BMP2 to support the proliferation of
mesenchy-mal tissues and surface
ectoderm.
It is believed that morphological changes
of the neural plate to form paired
dorsolateral hinge points (DLHPs) are
necessary for NT closure at the spinal level
(Copp et al., 2003). Recently, it has been
reported that the antagonism of BMP2
stimulates forma-tion of DLHPs in the
spinal region of the NT (Ybot-Gonzalez et
al., 2007). However, in the cephalic region,
forma-tion of DLHPs is not required
(Copp, 2005; Ybot-Gonzalez et al., 2002).
These reports support the role of BMP2 in
neural tube closure including the need for
regulated and organized expression for
normal neural tube closure.
Apoptosis plays a role in NT defects for
many mouse models (Padmanab-han, 2006;
Pani et al., 2002). Pax-3-deficient embryos
(Phelan et al., 1997) suffer from NTDs and
apoptosis. How-ever, these embryos were
rescued (Pani et al., 2002) by down-
regulating p53 protein using pifithrin- treat-
ment or germline mutation. Since we also
found a lack of Pax3 expression along with
NTDs in the Bmp2 mutant embryos, it was
reasonable to specu-late that the loss of
Pax3 would have induced p53-dependent
apoptosis and subsequent NTD. However,
our re-sults show a failure to rescue the
NTDs in Bmp2 homozygous null em-bryos.
These facts suggest that p53-dependent
apoptosis is not directly in-volved in NT
closure defects for BMP2 embryos, and the
loss of Pax3 may not be a direct cause of
those NTDs, but rather a result of the
NTDs. Future experiments to elucidate
mechanistic insight of apoptosis in the
mesen-chyme may help to understand the
di-rect cause(s) of the NTD in the ce-phalic
region. It would be interesting to include
colony development using this treatment to
show a possible in-crease in survival for
heterozygous mice, and a potentially
important fu-ture clinical application.
Cephalic Mesenchyme
STRUGGLES Without BMP2
Signaling
Null embryos for Bmp2 that survive
beyond gastrulation stages still suffer in the
next stage of development. From E8.5 to
E9.5, the anterior neural ridge needs to
proliferate, close, and differentiate into 3
distinct regions: forebrain, midbrain, and
hindbrain. Mesenchymal tissue in the head
that surrounds the developing NT shows an
interesting pattern of development with
condensation occurring over time
Fig. 8. Analysis of apoptosis. TUNEL assay as performed on WT (AC), Category 2 Heterozygous (DF), Category 3 heterozygous (GI) and
homozygous (JL) null embryos. Sections from left to right are from anterior to posterior within the cephalic region for each set. Apoptosis (green) was
increased in heterozygous mice with both open NT defects (yellow boxes GI) and postneurulation defects (red boxes, DF) when compared to WT
(respective boxes, AC). Homozygous nulls with abnormal neural tissue (arrowheads K,L) show an increase in apoptosis but nulls with no
recognizable neural tissue (J) in the forebrain region show no apoptosis in the cephalic region. Nuclei were stained with DAPI (blue). Red box
emphasizes the hindbrain region, while the yellow box emphasizes the forebrain region.
120 CASTRANIO AND MISHINA

TABLE 2. Pifithrin Treatment Does Not Rescue Abnormal Phenotypesa

Heterozygotes (%) Homozygous Nulls (%)

Categories PFT treated DMSO treated PFT treated DMSO treated
1. Normal 27 (79) 18 (46) 0 (0) 0 (0)
2. Postneurulation partial open NT 6 (18) 15 (38) 0 (0) 0 (0)
3. Hypomorphism, heart defects, open NT, 1 (3) 5 (13) 1 (14) 0 (0)
failure to turn
4. No development beyond E8.5 stage 0 (0) 1 (3) 6 (86) 7 (100)

a
Using pifithrin- treatment on pregnant females, we were able to find a partial rescue for heterozygous mice but none for homozygous nulls.
 in ectoderm head me for fixed in Environmen
proliferatio itself. mesenchy proper 4% tal Health
n and an Overall the me are all condensatio paraformal Sci-ences,
from the increase in mesenchym neural tube. crucial for n and the Bradley, dehyde, NIH.
center of the apoptosis e within Since neural neural tube pre-vention 1996). dehy-
head near in head these em- crest cells closure. As of apoptosis These mice drated, and
the ventral mesenchy bryos was migrate we allowing for were embedded
portions of me (Soo et reduced. In through the revealed, nor-mal NT maintained in paraffin
the NT al., 2002; very high head mesen- the closure. in a mixed (Kishigami
outward Zhao et al., con- chyme mesenchy background et al.,
1996). The tribution me does 2004).
In Situ
toward the during this of C57Bl/6 Hybridiza
surface opm chimeras stage, it is not and Seven-
mutants and severely condense micro- tion
ectoderm impor-tant EXPERI 129SvEv.
and the show no af-fected to recognize or is poorly ES cell lines meter-thick Embryos
change in homozygous condensed MENTAL sections
dorsal their were created were
these nulls, the surroundin PROCED were harvested at
portions of migration from
the NT. Of parameters cephalic may be g the URES blastocysts counter- E9.5 and
the nu- but suggest region is affected by dorsal Generatio of crosses stained fixed in 4%
merous an actual either abnormal portion of n and between with Eosin paraformald
mouse movement entirely mesenchym the NT in Bmp2 Y such that ehyde in
Analysis
models with of neural tissue al Bmp2 null heterozygou blasto- PBS for 2 hr
of
neural tube mesenchy or entirely developmen embryos s males cyst- and then
me to mesenchym and in Bmp2 derived
defects t and neural containing dehydrated
support the al-like chimeric Chimeras cells and stored at
(Brook et tube closure. one copy of
growing tissue, embryos. and Bmp2 stained 20C in
al., 1991; However, a ROSA26
Ju-riloff and forebrain implying Correia et In Mutant reporter pink and 100%
Harris, region at that without al. reported abnormal Embryos after Cre ES-derived methanol.
2000; Soo et the BMP2 that BMP2 heterozygo recombi- cells Whole
Bmp2
al., 2002; expense of signal-ing to is important us mice, nation and stained mount in
heterozygou
Stumpo et the the for neural the mes- Bmp2 blue. Bmp2 situ
s mice were
al., 1995; midbrain proliferating crest cell enchyme heterozygou heterozygo hybridizatio
region and mesenchym condenses kindly us mice in
Zohn et al., migration s fe-males. n was
NT closure e the relatively provided by a mixed
2005), the but not Wild type performed
(Ybot- boundary nor-mally Allan back-
impact of induction blastocysts as described
Gonzalez between in the Bradley ground
normal (Cor-reia et from CD1 (Wilkin-son
et al., mesenchym midbrain (Zhang and were and Nieto,
develop- al., 2007). It females
ment of the 2002; Zohn e and would be an and were intercrosse 1993) using
head et al., neuroepithel im-portant hindbrain, injected d to obtain the follow-
mesenchym 2007). ium is lost future but less so with 5 to 15 ho- ing probes:
e on neu-ral Both Bmp2 resulting in endeavor to in the Bmp2 / ES mozygous Shh
tube closure heterozygo disorganizat address the forebrain cells and and (Echelard et
is highly us and ion, loss of function of in mice reim-planted heterozygo al., 1993)
relevant homozygo identity and, BMP2 in with open into us embryos
(Soo et al., us null ultimately, migrating NT defects. pseudopregn for Bmp2.
2002; Zhao embryos, failure to neural crest Moreover, ant females Plugged
et al., 1996; however, close the cells and we never to generate females
Zohn et al., show whether or observed Bmp2 / 7 / were
2007). normal not it embryos chime-ras. scored as
Changes in pro- impacts with a Chimeras 0.5 at noon
proliferation liferation neural tube closed were of plug
and with an closure. neural tube harvested at date. All
apoptosis increase in while the E9.5 and animal
have also apopto-sis surface stained for procedures
impacted targeted Our ecto-derm -galactosida were
the head specifically results show remained se activ-ity approved
mesenchym to the through open. (X-gal by the
e of mouse mesen- SEM and Therefore, staining Institutiona
models but chyme near histological we believe hereafter) as l Animal
in different the surface analyses that BMP2 de-scribed Care and
ways. ectoderm that the from the previously Use
Homozygou and dorsal develop- surface (Kishigami Committee
s mice for portion of ment, ectoderm et al., 2004). at the
Twist or the NT, organization signals to Stained National
Cart1 show and within , and the embryos Insti-tute
a de-crease the surface amount of mesenchy were post- of
Bmp2 AND NEURAL TUBE DEFECTS 121
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chemistry day 8 and 9. Liechtenstei Richard NIEHS/NIH Mouse
and Wnt1 using a Dispatche Copp AJ,
was Em-bryos n). Em- Harland, (ES071003- d
(Wilkinson Hummer X Greene
performed were bryos were Andrew 10) and a homolog1
et al., 1987) Sputter ND,
as harvested at mounted on Mc-Mahon, gift from is Murdoch
pro-vided described day E10.5. aluminum Coater Janet RIKEN required JN. 2003.
by Andrew (Anatech for long-
(Caspary et (Correia et SEM stubs Rossant, Brain range, but
The genetic
McMahon, al., 2002). al., 2007; with Ltd., Yasuhide Science basis of
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