Requirement of Bmp8b for the Generation of Primordial Germ Cells in the Mouse
Ying Ying,
Xiao-Ming Liu,
Amy Marble,
Kirstie A. Lawson and
Guang-Quan Zhao
Department of Pathobiology (Y.Y., X.-M.L., A.M., G.-Q.Z.)
University of Missouri College of Veterinary Medicine Columbia,
Missouri 65211
Hubrecht Laboratory (K.A.L.) Netherlands
Institute for Developmental Biology 3584 CT Utrecht, The
Netherlands
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ABSTRACT
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In the mouse embryo, the generation of primordial
germ cells (PGCs) from the epiblast requires a bone morphogenetic
protein-4 (BMP4) signal from the adjacent extraembryonic ectoderm. In
this study, we report that Bmp8b, a member of the
Gbb-60A class of the BMP superfamily, is expressed in the
extraembryonic ectoderm in pregastrula and gastrula stage mouse embryos
and is required for PGC generation. A mutation in Bmp8b on
a mixed genetic background results in the absence of PGCs in 43% null
mutant embryos and severe reduction in PGC number in the remainder. The
heterozygotes are unaffected. On a largely C57BL/6 background,
Bmp8b null mutants completely lack PGCs, and
Bmp8b heterozygotes have a reduced number of PGCs. In
addition, Bmp8b homozygous null embryos on both genetic
backgrounds have a short allantois, and this organ is missing in some
more severe mutants. Since Bmp4 heterozygote embryos have
reduced numbers of PGCs, we used a genetic approach to generate
double-mutant embryos to study interactions of Bmp8b and
Bmp4. Embryos that are double heterozygotes for the
Bmp8b and Bmp4 mutations have similar defects
in PGC number as Bmp4 heterozygotes, indicating that the
effects of the two BMPs are not additive. These findings suggest that
BMP4 and BMP8B function as heterodimers and homodimers in PGC
specification in the mouse.
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INTRODUCTION
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Primordial germ cells (PGCs) progress through a series of
developmental stages to give rise to mature sperm or oocytes (1 ). In
the mouse, PGCs can be first seen at the midgastrula stage
(E7.25-E7.75; noon on the day of the copulation plug is defined as
E0.5) and detected as a cluster of alkaline phosphatase-positive cells
located in the extraembryonic mesoderm posterior to the primitive
streak (2 ). These PGCs subsequently migrate through the base of the
allantois, the endoderm of the hindgut, and the mesenchyme of the
mesentery to reach the genital ridges.
Unlike many other organisms, there is no evidence for a determined germ
cell lineage in preimplantation mouse embryos. Lawson and Hage (3 ),
using lineage-tracing techniques, found that only cells of the proximal
region of the epiblast (close to the extraembryonic ectoderm) at
E6.0E6.5 contribute to PGCs in the later embryo. Moreover, the
descendants of a given cell in the proximal epiblast can be found in
both germ cells and other lineages, mainly in extraembryonic mesoderm,
and no labeled cells give rise only to germ cells. This indicates that
before early gastrulation, the fate of PGCs is not completely fixed.
Tam and Zhou (4 ), using epiblast transplantation techniques,
demonstrated that cells of the distal epiblast (normally precursors of
the neuroectoderm and surface ectoderm) are able to generate PGCs if
they are transplanted in close proximity with extraembryonic ectoderm
before E6.5. However, cells of the proximal epiblast never give rise to
PGCs if they are transplanted into the distal region (far away from the
extraembryonic ectoderm). Therefore, before E6.5, epiblast cells at
different locations are able to generate PGCs only if they are placed
adjacent to the extraembryonic ectoderm, suggesting that signals from
this tissue are critical for PGC fate specification.
Recently, Lawson et al. (5 ) showed that bone morphogenetic
protein 4 (BMP4), a member of the transforming growth factor-ß
superfamily, is required for PGC generation in the mouse. Moreover,
Bmp4 is expressed in the extraembryonic ectoderm before and
during gastrulation and later in the extraembryonic mesoderm in mid- to
late-primitive streak embryos. On several genetic backgrounds, all of
the Bmp4 null (homozygous) mutants fail to generate PGCs,
and Bmp4 heterozygous embryos have a reduced number of PGCs
(
50% of wild-type) at various developmental stages. This suggests
that Bmp4 is not only absolutely required for PGC
generation, but its activity is dose dependent. Chimeric embryos were
generated by injecting wild-type ES cells, which contribute to the
epiblast but not the extraembryonic ectoderm or visceral endoderm, into
Bmp4 homozygous null embryos. No PGCs were produced in these
chimeras, although extraembryonic mesoderm, derived from the injected
wild-type ES cells, expressed normal levels of Bmp4. These
data clearly reveal that the extraembryonic ectoderm-derived BMP4
protein is required for PGC generation.
Among Bmp superfamily members, the closely related and
linked Bmp8a and Bmp8b, members of the
Gbb-60A subfamily (6 7 8 ), are expressed in male germ cells
and play a role in spermatogenesis by supporting germ cell
proliferation and survival (9 10 ). Homozygosity for a null mutation in
Bmp8a does not affect the initiation of spermatogenesis, but
half of the Bmp8a mutants show varying degrees of germ cell
degeneration (10 ). In contrast, absence of a functional
Bmp8b gene causes defects both in the initiation and
maintenance of spermatogenesis. Although they develop to adulthood with
no obvious abnormality in other systems, a high proportion of
Bmp8b null mutant males have small testes, and some
Bmp8b null mutant adults are infertile (9 ). These findings
prompted us to examine whether germ cell deficiency phenotypes were
present during embryogenesis in Bmp8a and Bmp8b
null mutants. We report here that Bmp8b is expressed in the
extraembryonic ectoderm of pregastrula and gastrula stage embryos and
is also required for PGC generation.
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RESULTS
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Bmp8b Mutant Embryos Have Defects in PGCs
Histological examination of PGCs after staining for alkaline
phosphatase in sectioned and whole-mount embryos fails to detect any
germ cell deficiency in Bmp8a null mutants (data not shown).
However, a null mutation in Bmp8b significantly affects PGC
development. Figure 1
compares PGCs in
histological sections of Bmp8b heterozygous and homozygous
mutants. On a mixed genetic background (129/Sv x Black Swiss),
there was no significant difference between Bmp8b
heterozygotes and wild-type embryos in either the number of PGCs or
their physical distribution (data not shown). However, in all of
Bmp8b homozygous mutant embryos, few or no PGCs were
observed in serial sections (Fig. 1
, B, C, E, F, H, I, and L). In about
40% homozygotes, PGCs were not found in any section throughout the
entire embryo. Our observations further reveal that there were no
differences between Bmp8b heterozygotes and those
homozygotes that have PGCs, in terms of the location and distribution
of the PGCs in the hindgut, dorsal mesentery, and genital ridges (data
not shown). This suggests that Bmp8b does not play a major
role in PGC migration but rather affects PGC number.

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Figure 1. Histological Comparison of PGCs in
Bmp8b Heterozygous and Homozygous Embryos at E8.5E11.5
Embryos were collected from crosses of Bmp8b
heterozygotes on a mixed genetic background (129/Sv x Black
Swiss). All sections were stained for alkaline phosphatase as
described. AC, Cross-sections of E8.5 embryos. DF, Sagittal
sections of E9.5 embryos. G and H, Frontal sections of E10.5 embryos.
I, Cross-section through genital ridges of a E10.5 embryo. Only
background staining is detected. JL, Cross- sections through
urogenital ridges of E11.5 embryos. Arrows indicate PGCs
in hindgut (AH) and genital ridges (J and K). * Marks the hindgut, @
marks the aorta, and arrowheads mark the genital ridges.
Scale bar, 60 µm in AC, K, and L; 120 µm in DF,
I, and J; 240 µm in G and H.
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Bmp8b Is Required for Generation of PGCs
Histological observations clearly indicate that Bmp8b
plays a role in PGC development. To further pinpoint the function of
Bmp8b, we used whole-mount alkaline phosphatase staining to
quantify PGCs in embryos at different developmental stages. There was
no significant difference in the number of PGCs between wild-type
embryos and Bmp8b heterozygotes on the mixed genetic
background. At E7.75E8.0, the mean numbers of PGCs are 75 and 65 in
the wild-type and Bmp8b heterozygous embryos, respectively
(data from the embryos at the headfold stages). By E8.25E8.5 (data
summarized from embryos at the 812 somite stages), the average number
of PGCs increases to 144 and 155 in the wild-type and Bmp8b
heterozygotes, respectively, and increases further to approximately 269
and 237 by E8.75E9.0 (data summarized from embryos at the 1822
somite stages). The regression analysis (Fig. 2B
) of PGC number
vs. somite number matches these data, indicating that the
regression lines of these two groups are not significantly different.
However, the number of PGCs in the Bmp8b homozygous null
embryos is significantly lower than those in the wild-type and
Bmp8b heterozygous embryos. As shown in Fig. 2B
, 43% of
Bmp8b homozygous null embryos (n = 47) have no PGCs,
while the remainder (57%) have less than 40 PGCs before the 24-somite
stage. There are no significant differences between the slopes of the
regression lines of heterozygotes and Bmp8b homozygotes that
have PGCs, but the elevation of the regression line is significantly
reduced in the latter. This suggests that BMP8B plays a role in PGC
generation, but not in PGC proliferation, and/or survival.

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Figure 2. PGC Numbers in Wild-Type and Bmp8b
Mutant Embryos at Different Developmental Stages
PGCs were counted in whole-mount embryos after alkaline phosphatase
staining. Embryos were collected from a mixed genetic background
(129/Sv x Black Swiss). A, PGC numbers at the headfold stage were
expressed as means with SEs. The number in
parentheses is the number of embryos in each group. * Indicates
significant difference of the Bmp8b homozygotes compared
with wild-type (P < 0.001) and
Bmp8b heterozygote groups (P <
0.001). B, Regression analysis of log PGC number (Y) vs.
somite number (X) for wild-type, Bmp8b heterozygotes,
and Bmp8b homozygotes (PGC values > 0). There was
no significant difference between wild-type (solid circles and
heavy line, Y = 0.029X + 1.827, n = 46) and
Bmp8b heterozygotes (open circles and light
line, Y = 0.03X + 1.766, n = 84). However, the number
of PGCs in the Bmp8b homozygous mutant group
(triangles and dashed line, Y = 0.03X + 0.74,
n = 39) is significantly reduced compared with those in the
wild-type and Bmp8b heterozygote groups
(P < 0.001).
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The conclusion that BMP8B affects PGC generation is supported by data
from embryos at the late streak stage. During normal development, PGCs
can first be recognized within and surrounding a cluster of cells with
strong alkaline phosphatase activity in the posterior midline in the
region from which the allantois will elongate (2 ). The alkaline
phosphatase-positive cluster and recognizable PGCs were present in all
wild-type (n = 12) and heterozygous (n = 25) embryos at the
late streak stage. In contrast, neither PGCs nor an
alkaline-phosphatase positive cluster were found in any
Bmp8b homozygous null embryos (n = 8) at this
stage. The small number of PGCs found at later stages was in the
expected position for the stage (i.e. associated with the
endoderm, at the lip of the hindgut, or within the hindgut). These
results suggest that not only are fewer PGCs formed in the mutant
embryos, but also their generation is delayed.
Bmp8b Is Expressed in the Extraembryonic Ectoderm in
Pregastrula and Gastrula Stage Mouse Embryos
Since both Bmp4 and Bmp8b are critical
for PGC formation (Ref. 5 and Figs. 1
and 2
), it is essential to
compare their spatiotemporal expression during early embryogenesis.
After whole-mount in situ hybridization at E6.25,
Bmp4 mRNA is detected in the proximal region of the
extraembryonic ectoderm adjacent to the epiblast and persists in this
location through E6.5 (Fig. 3
, A and B).
At E7.5, Bmp4 expression is detected not only in the
extraembryonic ectoderm and developing chorion, but also in derivatives
of the extraembryonic mesoderm, including amnion, yolk sac mesoderm,
and allantois (Fig. 3C
). This RNA expression pattern is consistent with
the LacZ expression reported in
Bmp4lacZ heterozygotes (5 ). To
precisely map the sites of Bmp8b expression, we performed
in situ hybridization on whole-mount embryos and on embryo
sections from E5.5E7.5 using Bmp8b antisense riboprobes.
Bmp8b signal is detected in the extraembryonic ectoderm in
E5.5 embryos (data not shown). At E6.0E7.5, there is strong signal
for Bmp8b throughout the entire extraembryonic ectoderm
(Fig. 3
, DF) but not in the endoderm or extraembryonic mesoderm (Fig. 3
, FH). These results indicate that Bmp4 is expressed in
both the extraembryonic ectoderm and extraembryonic mesoderm, whereas
Bmp8b expression is limited to the extraembryonic ectoderm
during gastrulation.

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Figure 3. Comparison of Bmp8b and
Bmp4 Expression Patterns in Mouse Embryos from E6.0 to
E7.5 by Whole-Mount in Situ Hybridization
A, At E6.25, Bmp4 mRNA is detected in the extraembryonic
ectoderm in a ring immediately adjacent to the epiblast. B, At E6.5,
Bmp4 expression persists in the extraembryonic region
near the epiblast. C, At E7.5, Bmp4 transcripts are
detected in both extraembryonic ectoderm and mesoderm, including amnion
and chorion. D and E, At E6.0E6.5 stage, Bmp8b is
expressed in the whole area of the extraembryonic ectoderm. F,
Bmp8b messages are detected only in the extraembryonic
ectoderm and ectoplacental cone, but not in the extraembryonic
mesoderm. G and H, Transverse sections through a E6.75 embryo (plane of
section indicated by red dashed line in panel E) in
darkfield and brightfield, respectively, displaying
Bmp8b expression in the extraembryonic ectoderm and the
invading trophoblast cells, but not in the endoderm. Black
arrows indicate boundaries between the extraembryonic ectoderm
and the epiblast; white arrowhead indicates
extraembryonic ectoderm; white arrow points to
trophoblast; asterisks indicate exocoelomic space
showing the extraembryonic mesoderm expression in panel C. en,
Embryonic endoderm; ep, epiblast; xe, extraembryonic ectoderm; xn,
extraembryonic endoderm. Scale bar, 40 µm in panels
AF, 50 µm in panels G and H.
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Bmp8b Homozygous Mutants Have Extraembryonic Mesoderm
Defects
Both PGCs and extraembryonic mesoderm are derived from common
precursors in proximal epiblast cells (3 ). Mutational inactivation of
Bmp4 ablates not only PGCs, but also the allantois (5 ), an
organ derived from the extraembryonic mesoderm. Inactivation of
Bmp8b results in a similar PGC-deficient phenotype, although
somewhat milder in that 57% of homozygous null embryos do have some
PGCs (Fig. 2
, A and B). Because Bmp4 and Bmp8b
are both expressed in the extraembryonic ectoderm, it is likely that
Bmp8b also plays a role in extraembryonic mesoderm
development, a possibility that was examined by morphological analysis
of the allantois. On the mixed genetic background, Bmp8b
heterozygotes are phenotypically normal. At E6.5, no obvious
morphological differences are observed between the wild-type,
Bmp8b heterozygous, and homozygous embryos (Fig. 4A
). However, most of the
Bmp8b homozygous null embryos are developmentally retarded
at E7.58.25 stages (Fig. 4B
). Moreover, many Bmp8b null
mutants have a shorter allantois when compared with wild-type and
heterozygotes with the same number of somites (Fig. 4C
). After a more
detailed examination of the initiation and growth of the allantois in
heterozygote crosses, it was found that 49% of the wild-type and
heterozygous embryos (n = 37) had an allantoic bud at the late
streak stage. However, no bud was present in any homozygous null
embryos (n = 8) at this stage. All wild-type and heterozygous
embryos had an elongated allantois at the neural plate stage (n =
25), but in 6/11 null mutant embryos, only a small bud was present at
this stage. This difference in the presence of an allantoic bud up to
the neural plate stage is statistically significant (P
< 0.01). The allantois in all null mutant embryos was more elongated
at the headfold stage, but it was consistently shorter than that of the
wild-type and heterozygotes at this same stage (Fig. 5A
). The diameters at the base and at
half-length in both frontal and lateral views increased slightly during
development and did not differ at equivalent developmental stages in
all three genotypes (data not shown). To show that the short allantois
was not due to a general growth defect in the null mutants, we
expressed allantois development as the ratio of allantois length to the
embryonic length. Embryonic length was measured from the most proximal
anterior part through the node to the posterior junction of amnion and
embryo. The ratio of allantois length to axis length remained
significantly smaller in the null mutants at all stages (data not
shown). Despite a relatively short allantois, fusion with the chorion
occurred in the mutants around the six-somite stage. These results
indicate that initiation of the allantois is delayed in
Bmp8b homozygous null embryos, but further development is
relatively normal, permitting fetal development to term on the mixed
genetic background.

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Figure 4. Developmental Delay and Defects of
Bmp8b Null Mutants
AC, Embryos were collected from Bmp8b heterozygous
crosses on a mixed genetic background (129/Sv x Black Swiss). A,
At E6.5, no obvious differences were observed between wild-type (+/+),
Bmp8b heterozygous (+/-), and Bmp8b
homozygous null (-/-) embryos. B, At E7.75, most Bmp8b
heterozygous embryos were morphologically normal, while
Bmp8b homozygous embryos were much smaller than their
wild-type and heterozygous littermates. C, A Bmp8b
heterozygote with normal allantois at the five-somite stage
(lower embryo) and a Bmp8b homozygous
embryo with a short allantois at five-somite stage
(upper) after alkaline phosphatase staining (note the
diffuse staining in the neuroepithelium at this stage). No PGCs are
seen in the posterior region of the Bmp8b homozygous
embryo. D, Bmp8b mutant embryos at E9.25 on a largely
C57BL/6 background. Bmp8b homozygous embryo (-/-)
lacked an allantois (right), showed a severe truncation
of posterior region, and delayed turning. Bmp8b
heterozygotes (+/-) had an intact allantois (middle).
Black arrows indicate junction between the epiblast and
extraembryonic ectoderm; white arrow indicates the
location of PGCs; white arrowhead indicates amnion. al,
Allantois; ps, posterior streak. Scale bars, 40 µm in
panel A, 67 µm in B, 100 µm in C, and 150 µm in D.
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Figure 5. Allantois Length of Bmp8b Mutant
Embryos
Allantois length (µm) was measured in embryos with intact allantois
at the headfold stage. A, Embryos collected from Bmp8b
heterozygous crosses on a mixed genetic background (129/Sv x
Black Swiss). B, Embryos collected from crosses of Bmp8b
heterozygote males with N6 generation onto C57BL/6 and females with N2
generation on C57BL/6 genetic background. Results were expressed as
means ± SEs. The number in parentheses
is the number of embryos in each group. a, P <
0.05, b, P < 0.01 compared with wild-type groups.
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A C57BL/6 Genetic Background Exacerbates the Bmp8b
Mutant Phenotype
The number of PGCs in Bmp8b heterozygous embryos on a
mixed genetic background is not significantly different from that in
wild-type embryos (Fig. 2
, A and B). Lawson et al. (5 )
demonstrated a more severe PGC-deficient phenotype in Bmp4
heterozygotes on a (C57BL/6 x CBA) background than on a
(129/Sv x Black Swiss) genetic background. Furthermore,
Bmp4 heterozygotes on a largely C57BL/6 background also
develop more severe defects such as cystic kidney, craniofacial
malformations, microphthalmia, and preaxial polydactyly of the right
hindlimb than on the outbred background (11 ). This suggests that the
C57BL/6 strain is more sensitive than outbred strains to BMP dosage for
germ cell development. To explore this possibility, we crossed the
Bmp8b mutation into the C57BL/6 strain by backcrossing to
C57BL/6 inbred mice. Embryos derived from a cross between
Bmp8b heterozygous males at the N6 backcross generation onto
C57BL/6 and Bmp8b heterozygous females at the N2-N5
backcross generation onto C57BL/6 show no gross morphological
differences between wild-type and Bmp8b heterozygotes (data
not shown). However, when PGCs were counted, it was found that the
number in the Bmp8b heterozygous group is significantly
reduced. At the headfold stage, the mean number of PGCs in the
Bmp8b heterozygotes is 35, approximately 60% of the
wild-type PGC level (Fig. 6A
, P
< 0.05). Regression analysis shows that although the slopes of the
regression lines are not significantly different between the
Bmp8b heterozygotes and the wild-type embryos, the mean
number of PGCs in the former is consistently lower than that of the
wild-type group (Fig. 6B
, P < 0.01). This suggests
that the major defect in the Bmp8b heterozygous embryos is a
reduction in the founding population of PGCs. In the Bmp8b
homozygous group, 46 of 50 (92%) embryos contained no PGCs (Fig. 6
, A
and B), further supporting the inference that BMP8B is essential for
PGC formation.

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Figure 6. PGC numbers in Bmp8b Mutant Embryos
on a Largely C57BL/6 Background and in Bmp4/Bmp8b Double
Heterozygotes on a Mixed Genetic Background at Different Developmental
Stages
A, PGC numbers at the headfold stage were collected from crosses of
males with N6 generation onto C57BL/6 and females with N2 generation
onto C57BL/6 and expressed as means with SEs. The
number in parentheses is the number of embryos. *,
P < 0.05 (Bmp8b heterozygote was
compared with wild-type group). B, Regression analysis of log PGC
number (Y) vs. somite number (X) for wild-type,
Bmp8b heterozygotes, and Bmp8b
homozygotes on the same genetic background as in panel A. Regression
equation of wild-type (solid circles and heavy line,
n = 52) and Bmp8b heterozygotes (open
circles and light line, n = 101) are Y = 0.027X +
1.749 and Y = 0.028X + 1.442, respectively. In
Bmp8b homozygous null embryos, only four had PGCs
(triangles, n = 45). C, Linear regression analysis
of log PGC number of Bmp4/Bmp8b double heterozygotes
vs. somite number. Embryos were collected from
intercrosses of Bmp8b and Bmp4
heterozygotes on a mixed (129/Sv x Black Swiss) genetic
background. There was no significant difference in PGC number between
the wild-type and Bmp8b heterozygotes. The PGC numbers
in Bmp4 heterozygotes and Bmp8b/Bmp4
double heterozygotes were significantly less than in
Bmp8b heterozygotes (P = 0.01).
However, no significant difference was observed between
Bmp4 heterozygotes and Bmp8b/Bmp4 double
heterozygotes (P > 0.05). Squares and upper
light line for wild-type embryos (n = 21), solid
circles and upper heavy line for Bmp8b
heterozygotes (n = 30), triangle and lower light
line for Bmp4 heterozygotes (n = 26), and
open circle and lower heavy line for
Bmp8b/Bmp4 double heterozygotes (n =
38).
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On the largely C57BL/6 background, the mean allantois length in
Bmp8b homozygous embryos at the headfold stage is
significantly shorter than that of wild-type and Bmp8b
heterozygotes (Fig. 5B
, P < 0.001). Morphological
observation reveals that 25% of the Bmp8b homozygous null
embryos completely lacked an allantois and showed delayed turning (Fig. 4D
). As summarized in Table 1
, at
E8.75E9.25, there are no significant changes in the expected ratio of
genotypes among wild-type, Bmp8b heterozygotes, and
homozygotes on this genetic background at the N5 backcross. However, at
the N6 backcross generation onto C57BL/6, the proportion of viable
Bmp8b homozygous embryos is greatly reduced at E9.25. At
E8.75E9.25, 3 of 31 embryos were Bmp8b homozygous mutants,
and they were severely retarded in development, two being at the
headfold stage with no allantois. On this genetic background, only 2 of
38 mice examined after birth were Bmp8b homozygous mutant,
suggesting that most Bmp8b homozygous mutant embryos on a
largely C57BL/6 background die prenatally.
Bmp8b and Bmp4 Mutations Do Not Have An
Additive Effect on PGC Generation
Lawson et al. (5 ) demonstrated that Bmp4 is
expressed in the extraembryonic ectoderm and is required for PGC fate
specification. In this study, we show that Bmp8b is also
expressed in the extraembryonic ectoderm and plays a role in PGC
generation. To determine the genetic interaction of the two genes in
PGC formation, we intercrossed Bmp4 and Bmp8b
heterozygotes to generate double heterozygotes on a mixed genetic
background. The PGC number in Bmp8b heterozygotes is not
significantly different from that in the wild type. However, the number
of PGCs in Bmp4 heterozygotes and Bmp8b/Bmp4
double heterozygotes is consistently lower than that in
Bmp8b heterozygotes (Fig. 6C
, P < 0.01).
There is no significant difference in the elevation or slope of the
regression line for PGC number vs. somite number between
Bmp4 heterozygotes and Bmp8b/Bmp4 double
heterozygotes. These results suggest that PGC generation is more
sensitive to BMP4 levels than BMP8B levels and that heterozygous
mutations in both Bmp4 and Bmp8b do not have an
additive effect.
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DISCUSSION
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The pathways governing PGC formation in different species are
remarkably different, even among vertebrates (12 13 14 15 ). In
Drosophila, pole cells in the blastula stage embryo are
already determined to become germ cells as a result of cytoplasmic
localization of maternally inherited molecules (1 16 17 ). In
Caenorhabditis elegans, germ cells are determined by the
asymmetrical segregation of P granules, the cytoplasmic determinants of
germ cells (18 19 ). In zebrafish, the PGCs are specified by asymmetric
localization of cytoplasmic factors in random positions relative to the
future embryonic axis (15 ). Although it has been well established that
the germ cells in the mouse originate from the epiblast (3 4 ),
maternally inherited or asymmetrically segregated germ cell
determinants have not been identified.
Lineage analysis in prestreak and early primitive streak stage mouse
embryos has identified a population of epiblast cells near the junction
with the extraembryonic ectoderm that gives rise to both PGCs and
components of the extraembryonic mesoderm, including the allantois (3 20 ). Recent experiments have identified a requirement for BMP4 produced
in the extraembryonic ectoderm in establishing the PGC lineage in the
mouse (5 ). Several models have been proposed for how Bmp4
functions. In a one-signal model, BMP4, secreted by the extraembryonic
ectoderm, acts on the proximal epiblast cells to induce PGC/allantois
precursors. The precursors that receive the highest dose of BMP4 over a
given time become allocated to the PGC lineage, a process that is
thought to occur normally around the time the cells enter or have
passed through the primitive streak. The precursors exposed to a lower
dose of BMP4 give rise to the allantois and other components of the
extraembryonic mesoderm. In a two-signal model, BMP4, secreted by the
extraembryonic ectoderm, first induces PGC/allantois precursors, which
then receive a second signal or signals in the extraembryonic mesoderm,
resulting in the allocation of some of the descendants of these
precursors to the PGC or to the allantois lineage. The nature of the
putative second signal is unknown. Regardless of which model is
correct, the data presented here suggest a more complex situation in
which both BMP8B and BMP4, secreted by the extraembryonic ectoderm,
regulate PGCs and allantois cell fate.
Reduction in BMP signal from the extraembryonic ectoderm leads to a
range of phenotypes. In increasing order of severity, these are 1)
about 40% reduction in PGC number and a normal allantois in
Bmp8b heterozygotes on a largely C57BL/6 background (Figs. 5
and 6
); 2) more than 50% reduction in PGC number and 9% embryos
without PGCs, and no defect in the allantois in Bmp4
heterozygotes on all backgrounds tested (5 ); 3) about 90% reduction in
PGC number, 43% embryos with no PGCs, and a delay in the initiation of
the allantois in Bmp8b homozygotes on a mixed genetic
background (Figs. 2
and 5
); 4) complete absence of PGCs and a
relatively short or sometimes absent allantois in Bmp8b
homozygotes on a largely C57BL/6 background (
Figs. 46

); and 5)
complete absence of both PGCs and allantois in Bmp4
homozygous null embryos on all backgrounds tested (5 ). The elevation of
the regression line of PGC number on somite number is lowered in both
Bm8b and Bmp4 heterozygotes, but the slope of the
regression line is not significantly different (Fig. 6B
and Ref. 5 ),
suggesting that BMP8B, as well as BMP4, influences the size of the
founding population of PGCs rather than affecting PGC proliferation
and/or survival.
The different effect of Bmp8b dosage on PGCs and allantois
is consistent with the notion that allocation to the PGC lineage is
more sensitive to the level of BMPs than allocation to the allantois
lineage. The size of both founding populations may be reduced, but that
of the allantois is reduced to a lesser extent than that of the PGCs.
This explanation is in agreement with the fact that the allantois is
derived both from cells in the most proximal epiblast and epiblast
cells further away from the junction, where lower BMP8B concentrations
are expected (3 ). Alternatively, the rapidly proliferating allantois
may be able to compensate considerably in its growth after its initial
formation (21 ), whereas the more slowly dividing PGC population can not
(14 ).
Once PGCs and allantois have been initiated, the further development of
both is independent of BMP8B. The expansion of the PGC population after
the headfold stage is unaffected by the absence of Bmp8b or
by Bmp4 heterozygosity. The further development of the
allantoic bud is also relatively normal in Bmp8b mutants,
but may well be regulated by other BMPs: Bmp4 is expressed
in the extraembryonic mesoderm including the allantois (5 ), as are
Bmp5 and Bmp7, two members of the Gbb-60A class
of the BMP superfamily (6 7 8 ). Allantois development and fusion with
the chorion are defective in Bmp5/Bmp7 double mutants (22 ).
Thus while BMPs from the extraembryonic ectoderm are required to
establish the germline and initiate an allantoic bud, further
development of the allantois may be controlled by a variety of BMPs, or
other proteins, produced by the extraembryonic mesoderm.
Even in wild-type embryos, the number of PGCs at any stage is lower on
the C57BL/6 background than on an outbred genetic background. Moreover,
PGC number is affected by Bmp8b heterozygosity on the
C57BL/6 background, but not on the outbred genetic background. Taken
together, these observations suggest that BMP signaling is affected by
strain-specific alleles.
BMP8B belongs to the Gbb-60A class of BMP ligands, while BMP4 belongs
to the Decapentaplegic (DPP) class (6 8 23 ). The two genes encoding
these proteins are coexpressed in the right tissue (the extraembryonic
ectoderm) and at the right time for inducing PGC/allantois precursors
in the epiblast and for regulating the allocation of these cells to the
PGC or allantois fate. Theoretically, there are several possible ways
in which the genes and their products might interact. First,
Bmp4 may regulate Bmp8b expression or vice versa.
To test this possibility we performed whole-mount in situ
hybridization using Bmp4 and Bmp8b probes on
embryos (E6.0E7.5) collected from Bmp8b and
Bmp4 heterozygous crosses on a mixed genetic background,
respectively. Results showed no obvious difference in Bmp4
expression levels among wild-type, Bmp8b heterozygous, and
Bmp8b homozygous embryos (data not shown). The same was true
for Bmp8b expression among Bmp4 mutant embryos
(data not shown). Thus, Bmp4 does not control the expression
of Bmp8b in the extraembryonic ectoderm and vice versa.
Another possible model that we have considered is that BMP4 and BMP8B,
although synthesized in the same extraembryonic cells, can form only
homodimers (obligate homodimer model) and that they bind in the
adjacent epiblast to different receptor complexes and activate
different downstream pathways that synergize with each other. This
model is similar to one proposed for the interaction between DPP and
GBB-60A in wing patterning and growth in Drosophila (7 24 ).
However, a simple synergism model would not explain the finding that
there is no significant difference between PGC number in double
Bmp4/Bmp8b heterozygotes compared with Bmp4
single heterozygotes on a mixed genetic background (Fig. 6C
). It would
also require that different Type I receptors are expressed in the
responding cells (see below). Another possibility is that BMP4 and
BMP8B form only heterodimers in the extraembryonic ectoderm close to
the junction with the epiblast (obligate heterodimer model). However,
this simple model is also unlikely because it predicts that the
phenotypes of Bmp4 and Bmp8b null mutants should
be identical, on the same genetic background. However, Bmp4
null mutants always lack both PGCs and an allantois, even on the
(129/Sv x Black Swiss) mixed background, whereas 57% of the
Bmp8b null mutants have PGCs under these circumstances. Such
a difference could result from the relative levels of BMP4 and BMP8B in
the extraembryonic ectoderm. A third possibility, representing one of
several more complex scenarios, is that extraembryonic ectoderm cells
secrete a mixture of BMP4/BMP8B heterodimers and BMP4 and BMP8B
homodimers, with the biological activity of these proteins in terms of
PGC specification being BMP4/BMP8B heterodimer > BMP4
homodimer > BMP8B homodimer (mixed heterodimer/homodimer model).
Thus, in Bmp4 null mutants, only BMP8B homodimers can be
made, and their activity is insufficient to generate either PGCs or an
allantois. In Bmp8b null mutants, on the other hand, BMP4
homodimers are made but have a higher biological activity, compared
with BMP8B, and induce the formation of some PGCs and a smaller than
normal allantois. In this model, the fact that there is no significant
difference in PGC number in Bmp4/Bmp8b double heterozygotes
compared with Bmp4 heterozygotes might be because
heterozygosity for Bmp8b tips the balance toward making more
BMP4 homodimers. In its simplest form this model postulates that
homodimers and heterodimers signal through the same type I receptor but
bind with different affinities or activate the receptor to different
extents. However, it is theoretically possible to combine this model
with one in which homodimers and heterodimers bind to receptors
containing different type I subunits. At present it appears that only
one BMP type I receptor, ALK3, is expressed in epiblast cells and
extraembryonic ectoderm of the pregastrula and early gastrula mouse
embryos (25 ). However, genes for two type I receptors, Alk2
and Alk3, are expressed in the visceral endoderm (26 27 ).
This raises the possibility, which still must be explored, that BMP4
and BMP8B act indirectly through the extraembryonic endoderm, rather
than directly on the epiblast.
 |
MATERIALS AND METHODS
|
---|
PGC Staining and Counting
For staining PGCs in sections, E8.5E11.5 embryos were
collected and fixed in 4% paraformaldehyde in PBS for 23 h, followed
by dehydration through increasing concentrations of cold ethanol. The
embryos were cleared in xylene and embedded in low melting wax
(Fisher Scientific, Pittsburgh, PA). Samples were serially
sectioned at 7 µm, mounted on glass slides, dewaxed, and hydrated in
descending concentrations of ethanol. Alkaline phosphatase activity was
detected by incubation in NBT/BCIP substrate solution for 15 min at
room temperature according to instructions of the manufacturer
(Roche Molecular Biochemicals, Indianapolis, IN). The
sections were counterstained with eosin using standard procedures.
For whole mount staining, E7.5E9.5 embryos were dissected from the
uterus and fixed in freshly prepared 4% paraformaldehyde in PBS for
12 h. They were further treated as in Ref. 5 , or the procedure was
slightly modified as follows: After washing three times with PBS, they
were further dissected to remove the trophoblast, but both the amnion
and yolk sac were left attached. The embryos were then treated with
70% ethanol for 12 h. After washing three times with distilled
water, they were stained with freshly prepared
-naphthyl
phosphate/fast red TR (Sigma, St. Louis, MO) for 1520
min at room temperature (2 ), washed, and retained in PBS. To count the
PGCs, the yolk sacs without PGCs were removed, somite number was
counted, and the length of the allantois was measured. Then, the
embryos were cut to give anterior and posterior halves. The stained
posterior portions of young embryos (before E8.25) were directly
flattened on a slide in 70% glycerol under a coverslip. For the older
embryos, the hindgut was isolated for PGC counting under the microscope
(400x magnification). The anterior portion of the embryo was used for
DNA purification and genotyping.
In Situ Hybridization
RNA in situ hybridization using probes for
Bmp8b and Bmp4 was performed on whole-mount or
sectioned embryos at E6.0E7.5. Whole-mount hybridization was
essentially the same as described previously (20 28 ), except that a
higher temperature (70 C) was used during hybridization and washing and
a blocking reagent (Roche Molecular Biochemicals) was used
during antibody incubation. Digoxigenin-labeled antisense and sense
riboprobes were prepared using full-length Bmp8b and
Bmp4 cDNA as templates and a RNA transcription kit according
to instructions of the manufacturer (Roche Molecular Biochemicals).
In situ hybridization on sections was performed as
previously described with slight modifications (29 ). Briefly, freshly
dissected uteri with embryos were rinsed in PBS and fixed in freshly
prepared 4% paraformaldehyde in PBS for 12 h at 4 C, followed by
dehydration via a series of increasing concentrations of ethanol. The
tissues were cleared in xylene and embedded in paraplast (Fisher Scientific, Pittsburgh, PA). Samples were sectioned at 67 µm
and collected on superfrost plus glass slides (Fisher Scientific). Antisense and sense RNA probes of Bmp8b
labeled with [
-35S]UTP were generated for
hybridization. Hybridization was carried out at 6065 C for 1620 h.
High-stringency washes were performed with 2 x SSC, 50%
formamide at 6065 C. Autoradiography was carried out using NTB-2
emulsion (Eastman Kodak , Rochester, NY), and the slides
were exposed for 2 weeks at 4 C. Photomicrographs were taken using both
light- and darkfield optics.
Genotyping by PCR
The genotypes of the embryos were determined by PCR analysis.
The yolk sac or part of the embryo tissue was collected and digested
overnight at 55 C in 100 µl of lysis buffer containing 50
mM Tris·HCl, 20 mM EDTA, 10 mM
NaCl, 0.5% SDS, and 0.5 mg/ml proteinase K. Genomic DNA was purified
by phenol-chloroform (1:1) extraction, precipitated by isoproponal, and
washed with 70% ethanol. DNA was then dissolved in distilled water.
The three primers for Bmp8b used in each reaction were 4S:
5'-CCA ACA AAC ACC TAG GAA TCC-3'derived from the sense strand of exon
4, 5A: 5'-GCA AAC TTC TCT GCC GTG A-3' derived from the antisense
strand of exon 5, and Neo2: 5'-CCT TCT TGA CGA GTT CTT CTG
AGG-3'derived from a neomycin-resistant gene. The PCR products were
resolved on a 1% gel. The sizes of the amplified fragments are mutant
band, Neo2+5A = 300 bp; and wild-type band, 4S+5A = 500 bp.
The primers for Bmp4 were the same as previously described
(30 ).
Statistical Analysis
The incidence of embryos with an allantoic bud was analyzed by
the
2 test. ANOVA was used for analysis of PGC
number and allantois length. Regression lines were compared using F
test. P < 0.05 was considered a statistically
significant difference.
Experimental Animals
Most Bmp8b (Bmp8btm)
and Bmp4 (Bmp4tm1) mutant
mice used for PGC analysis were maintained on a mixed genetic
background (129/Sv x Black Swiss) (9 30 ). To obtain
Bmp8b mutants on a largely C57BL/6 background,
Bmp8b heterozygous mutant males were mated with C57BL/6
inbred females. The F1 heterozygous males were
backcrossed with C57BL/6 females to obtain N2 female Bmp8b
heterozygotes. The same strategy was used to obtain N3, N4, N5, and N6
male and female Bmp8b heterozygotes. Bmp8b/Bmp4
double mutants were generated by crossing Bmp8b
heterozygotes and Bmp4 heterozygotes on a mixed genetic
background. Analysis of Bmp8b and Bmp4 expression
patterns was conducted using wild-type ICR outbred embryos.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Dr. Brigid L. M. Hogan for generously
providing Bmp4 mutant mice and for critical comments and
suggestions on the manuscript. We thank Rui-An Wang and
Yaxiong Chen for excellent help in embryo dissection and alkaline
phosphatase staining during the early phase of this project, and Don
Carpenter for excellent assistance in mouse colony maintenance and
mouse genotyping. We also thank Drs. Lilana Solnica-Krezel, Mary Ann
Handel, and Richard Behringer for critical comments on the
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Guang-Quan Zhao, M.D. Ph.D., 209C Connaway Hall, Department of Pathobiology University of Missouri, College of Veterinary Medicine, Columbia, Missouri 65211.
This work is supported by a University of Missouri Research Board
grant, a grant from the National Institute of Child Health (HD-36218),
and Basil OConnor Starter Scholar Research Award to G.-Q. Zhao.
Received for publication January 26, 2000.
Revision received March 13, 2000.
 |
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