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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 E6.0–E6.5 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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) 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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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 E5.5–E7.5 using Bmp8b antisense riboprobes.
Bmp8b signal is detected in the extraembryonic ectoderm in
E5.5 embryos (data not shown). At E6.0–E7.5, there is strong signal
for Bmp8b throughout the entire extraembryonic ectoderm
(Fig. 3, D–F) but not in the endoderm or extraembryonic mesoderm (Fig. 3, F–H). 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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, 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 E7.5–8.25 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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, 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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, 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
E8.75–E9.25, 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
E8.75–E9.25, 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.
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, 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. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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. 4–6); 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 (E6.0–E7.5) 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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For whole mount staining, E7.5–E9.5 embryos were dissected from the
uterus and fixed in freshly prepared 4% paraformaldehyde in PBS for
1–2 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 1–2 h. After washing three times with distilled
water, they were stained with freshly prepared 
-naphthyl
phosphate/fast red TR (Sigma, St. Louis, MO) for 15–20
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 E6.0–E7.5. 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 6–7 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 60–65 C for 16–20 h.
High-stringency washes were performed with 2 x SSC, 50%
formamide at 60–65 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 O’Connor Starter Scholar Research Award to G.-Q. Zhao.
Received for publication January 26, 2000.
Revision received March 13, 2000.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 C. G. M. Extavour Evolution of the bilaterian germ line: lineage origin and modulation of specification mechanisms Integr. Comp. Biol., November 1, 2007; 47(5): 770 - 785. [Abstract] [Full Text] [PDF]
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 J. Bowles and P. Koopman Retinoic acid, meiosis and germ cell fate in mammals Development, October 1, 2007; 134(19): 3401 - 3411. [Abstract] [Full Text] [PDF]
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 K. Hayashi, S. M. C. de Sousa Lopes, and M. A. Surani Germ Cell Specification in Mice Science, April 20, 2007; 316(5823): 394 - 396. [Abstract] [Full Text] [PDF]
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 Y. Yabuta, K. Kurimoto, Y. Ohinata, Y. Seki, and M. Saitou Gene Expression Dynamics During Germline Specification in Mice Identified by Quantitative Single-Cell Gene Expression Profiling Biol Reprod, November 1, 2006; 75(5): 705 - 716. [Abstract] [Full Text] [PDF]
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S-Y Lin, J R Morrison, M M Matzuk, and D M de Kretser Spermatogenesis does not require the local production of follistatin. Reproduction, October 1, 2006; 132(4): 601 - 605. [Abstract] [Full Text] [PDF]
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K.R. Barnett, C. Schilling, C.R. Greenfeld, D. Tomic, and J.A. Flaws Ovarian follicle development and transgenic mouse models Hum. Reprod. Update, September 1, 2006; 12(5): 537 - 555. [Abstract] [Full Text] [PDF]
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E. A. Bosman, K. A. Lawson, J. Debruyn, L. Beek, A. Francis, L. Schoonjans, D. Huylebroeck, and A. Zwijsen Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels Development, September 1, 2006; 133(17): 3399 - 3409. [Abstract] [Full Text] [PDF]
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D. M. Maatouk, L. D. Kellam, M. R. W. Mann, H. Lei, E. Li, M. S. Bartolomei, and J. L. Resnick DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell lineages Development, September 1, 2006; 133(17): 3411 - 3418. [Abstract] [Full Text] [PDF]
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R. L Jones, C. Stoikos, J. K Findlay, and L. A Salamonsen TGF-{beta} superfamily expression and actions in the endometrium and placenta. Reproduction, August 1, 2006; 132(2): 217 - 232. [Abstract] [Full Text] [PDF]
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C. Itman, S. Mendis, B. Barakat, and K. L. Loveland All in the family: TGF-{beta} family action in testis development. Reproduction, August 1, 2006; 132(2): 233 - 246. [Abstract] [Full Text] [PDF]
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A. Roy and M. M Matzuk Deconstructing mammalian reproduction: using knockouts to define fertility pathways Reproduction, February 1, 2006; 131(2): 207 - 219. [Abstract] [Full Text] [PDF]
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K. L. Loveland, C. Hogarth, A. Szczepny, S. M. Prabhu, and D. A. Jans Expression of Nuclear Transport Importins beta 1 and beta 3 Is Regulated During Rodent Spermatogenesis Biol Reprod, January 1, 2006; 74(1): 67 - 74. [Abstract] [Full Text] [PDF]
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M. Donnison, A. Beaton, H. W. Davey, R. Broadhurst, P. L'Huillier, and P. L. Pfeffer Loss of the extraembryonic ectoderm in Elf5 mutants leads to defects in embryonic patterning Development, May 15, 2005; 132(10): 2299 - 2308. [Abstract] [Full Text] [PDF]
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T. P. Zwaka and J. A. Thomson A germ cell origin of embryonic stem cells? Development, January 15, 2005; 132(2): 227 - 233. [Abstract] [Full Text] [PDF]
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 R. M. Monteiro, S. M. C. de Sousa Lopes, O. Korchynskyi, P. t. Dijke, and C. L. Mummery Spatio-temporal activation of Smad1 and Smad5 in vivo: monitoring transcriptional activity of Smad proteins J. Cell Sci., September 15, 2004; 117(20): 4653 - 4663. [Abstract] [Full Text] [PDF]
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S. M. C. de Sousa Lopes, B. A.J. Roelen, R. M. Monteiro, R. Emmens, H. Y. Lin, E. Li, K. A. Lawson, and C. L. Mummery BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo Genes & Dev., August 1, 2004; 18(15): 1838 - 1849. [Abstract] [Full Text] [PDF]
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L. S. Campos, D. P. Leone, J. B. Relvas, C. Brakebusch, R. Fassler, U. Suter, and C. ffrench-Constant {beta}1 integrins activate a MAPK signalling pathway in neural stem cells that contributes to their maintenance Development, July 15, 2004; 131(14): 3433 - 3444. [Abstract] [Full Text] [PDF]
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J. Aubin, A. Davy, and P. Soriano In vivo convergence of BMP and MAPK signaling pathways: impact of differential Smad1 phosphorylation on development and homeostasis Genes & Dev., June 15, 2004; 18(12): 1482 - 1494. [Abstract] [Full Text] [PDF]
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M. De Felici, M.L. Scaldaferri, M. Lobascio, S. Iona, V. Nazzicone, F.G. Klinger, and D. Farini Experimental approaches to the study of primordial germ cell lineage and proliferation Hum. Reprod. Update, May 1, 2004; 10(3): 197 - 206. [Abstract] [Full Text] [PDF]
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H. Ohta, T. Wakayama, and Y. Nishimune Commitment of Fetal Male Germ Cells to Spermatogonial Stem Cells During Mouse Embryonic Development Biol Reprod, May 1, 2004; 70(5): 1286 - 1291. [Abstract] [Full Text] [PDF]
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 X. Qi, T.-G. Li, J. Hao, J. Hu, J. Wang, H. Simmons, S. Miura, Y. Mishina, and G.-Q. Zhao BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways PNAS, April 20, 2004; 101(16): 6027 - 6032. [Abstract] [Full Text] [PDF]
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S. Shimasaki, R. K. Moore, F. Otsuka, and G. F. Erickson The Bone Morphogenetic Protein System In Mammalian Reproduction Endocr. Rev., February 1, 2004; 25(1): 72 - 101. [Abstract] [Full Text] [PDF]
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D. Okamura, T. Kimura, T. Nakano, and Y. Matsui Cadherin-mediated cell interaction regulates germ cell determination in mice Development, December 29, 2003; 130(26): 6423 - 6430. [Abstract] [Full Text] [PDF]
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C. G. Extavour and M. Akam Mechanisms of germ cell specification across the metazoans: epigenesis and preformation Development, December 15, 2003; 130(24): 5869 - 5884. [Abstract] [Full Text] [PDF]
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E. E. Nilsson and M. K. Skinner Bone Morphogenetic Protein-4 Acts as an Ovarian Follicle Survival Factor and Promotes Primordial Follicle Development Biol Reprod, October 1, 2003; 69(4): 1265 - 1272. [Abstract] [Full Text] [PDF]
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Y. Toyooka, N. Tsunekawa, R. Akasu, and T. Noce Embryonic stem cells can form germ cells in vitro PNAS, September 30, 2003; 100(20): 11457 - 11462. [Abstract] [Full Text] [PDF]
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M. Tsuda, Y. Sasaoka, M. Kiso, K. Abe, S. Haraguchi, S. Kobayashi, and Y. Saga Conserved Role of nanos Proteins in Germ Cell Development Science, August 29, 2003; 301(5637): 1239 - 1241. [Abstract] [Full Text] [PDF]
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M. Pellegrini, P. Grimaldi, P. Rossi, R. Geremia, and S. Dolci Developmental expression of BMP4/ALK3/SMAD5 signaling pathway in the mouse testis: a potential role of BMP4 in spermatogonia differentiation J. Cell Sci., August 15, 2003; 116(16): 3363 - 3372. [Abstract] [Full Text] [PDF]
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B. Lu and C. E. Bishop Late Onset of Spermatogenesis and Gain of Fertility in POG-Deficient Mice Indicate that POG Is Not Necessary for the Proliferation of Spermatogonia Biol Reprod, July 1, 2003; 69(1): 161 - 168. [Abstract] [Full Text] [PDF]
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L. A. Naiche and V. E. Papaioannou Loss of Tbx4 blocks hindlimb development and affects vascularization and fusion of the allantois Development, June 15, 2003; 130(12): 2681 - 2693. [Abstract] [Full Text] [PDF]
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T. Ara, Y. Nakamura, T. Egawa, T. Sugiyama, K. Abe, T. Kishimoto, Y. Matsui, and T. Nagasawa Impaired colonization of the gonads by primordial germ cells in mice lacking a chemokine, stromal cell-derived factor-1 (SDF-1) PNAS, April 29, 2003; 100(9): 5319 - 5323. [Abstract] [Full Text] [PDF]
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 B. Lu and C. E. Bishop Mouse GGN1 and GGN3, Two Germ Cell-specific Proteins from the Single Gene Ggn, Interact with Mouse POG and Play a Role in Spermatogenesis J. Biol. Chem., April 25, 2003; 278(18): 16289 - 16296. [Abstract] [Full Text] [PDF]
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M. Mukhopadhyay, A. Teufel, T. Yamashita, A. D. Agulnick, L. Chen, K. M. Downs, A. Schindler, A. Grinberg, S.-P. Huang, D. Dorward, et al. Functional ablation of the mouse Ldb1 gene results in severe patterning defects during gastrulation Development, February 1, 2003; 130(3): 495 - 505. [Abstract] [Full Text] [PDF]
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H. Chang, C. W. Brown, and M. M. Matzuk Genetic Analysis of the Mammalian Transforming Growth Factor-{beta} Superfamily Endocr. Rev., December 1, 2002; 23(6): 787 - 823. [Abstract] [Full Text] [PDF]
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 A. I. Agoulnik, B. Lu, Q. Zhu, C. Truong, M. T. Ty, N. Arango, K. K. Chada, and C. E. Bishop A novel gene, Pog, is necessary for primordial germ cell proliferation in the mouse and underlies the germ cell deficient mutation, gcd Hum. Mol. Genet., November 15, 2002; 11(24): 3047 - 3053. [Abstract] [Full Text] [PDF]
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T. Fujiwara, N. R. Dunn, and B. L. M. Hogan Bone morphogenetic protein 4 in the extraembryonic mesoderm is required for allantois development and the localization and survival of primordial germ cells in the mouse PNAS, November 9, 2001; (2001) 241508898. [Abstract] [Full Text] [PDF]
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K. D. Tremblay, N. R. Dunn, and E. J. Robertson Mouse embryos lacking Smad1 signals display defects in extra-embryonic tissues and germ cell formation Development, September 15, 2001; 128(18): 3609 - 3621. [Abstract] [Full Text] [PDF]
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Y. Ying, X. Qi, and G.-Q. Zhao Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways PNAS, June 20, 2001; (2001) 151242798. [Abstract] [Full Text] [PDF]
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 J. S. Richards Perspective: The Ovarian Follicle--A Perspective in 2001 Endocrinology, June 1, 2001; 142(6): 2184 - 2193. [Full Text] [PDF]
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Y. Ying and G.-Q. Zhao Detection of Multiple Bone Morphogenetic Protein Messenger Ribonucleic Acids and Their Signal Transducer, Smad1, During Mouse Decidualization Biol Reprod, December 1, 2000; 63(6): 1781 - 1786. [Abstract] [Full Text]
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Y. Ying, X. Qi, and G.-Q. Zhao Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways PNAS, July 3, 2001; 98(14): 7858 - 7862. [Abstract] [Full Text] [PDF]
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T. Fujiwara, N. R. Dunn, and B. L. M. Hogan Bone morphogenetic protein 4 in the extraembryonic mesoderm is required for allantois development and the localization and survival of primordial germ cells in the mouse PNAS, November 20, 2001; 98(24): 13739 - 13744. [Abstract] [Full Text] [PDF]
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