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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 E6.0–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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1Go 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. 1Go, 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 E8.5–E11.5

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. A–C, Cross-sections of E8.5 embryos. D–F, 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. J–L, Cross- sections through urogenital ridges of E11.5 embryos. Arrows indicate PGCs in hindgut (A–H) and genital ridges (J and K). * Marks the hindgut, @ marks the aorta, and arrowheads mark the genital ridges. Scale bar, 60 µm in A–C, K, and L; 120 µm in D–F, I, and J; 240 µm in G and H.

 
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 E7.75–E8.0, 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 E8.25–E8.5 (data summarized from embryos at the 8–12 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 E8.75–E9.0 (data summarized from embryos at the 18–22 somite stages). The regression analysis (Fig. 2BGo) 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. 2BGo, 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).

 
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. 1Go and 2Go), 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. 3Go, 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. 3CGo). 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. 3Go, D–F) but not in the endoderm or extraembryonic mesoderm (Fig. 3Go, 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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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 E6.0–E6.5 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 A–F, 50 µm in panels G and H.

 
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. 2Go, 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. 4AGo). However, most of the Bmp8b homozygous null embryos are developmentally retarded at E7.5–8.25 stages (Fig. 4BGo). Moreover, many Bmp8b null mutants have a shorter allantois when compared with wild-type and heterozygotes with the same number of somites (Fig. 4CGo). 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. 5AGo). 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

A–C, 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.

 
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. 2Go, 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. 6AGo, 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. 6BGo, 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. 6Go, 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).

 
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. 5BGo, P < 0.001). Morphological observation reveals that 25% of the Bmp8b homozygous null embryos completely lacked an allantois and showed delayed turning (Fig. 4DGo). As summarized in Table 1Go, 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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Table 1. Embryonic Lethality of Bmp8b Mutants at Different Generation Backcross onto the C57BL/6 Background

 
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. 6CGo, 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 5Go and 6Go); 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. 2Go and 5Go); 4) complete absence of PGCs and a relatively short or sometimes absent allantois in Bmp8b homozygotes on a largely C57BL/6 background ( Figs. 4–6GoGoGo); 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. 6BGo 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. 6CGo). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PGC Staining and Counting
For staining PGCs in sections, E8.5–E11.5 embryos were collected and fixed in 4% paraformaldehyde in PBS for 2–3 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, E7.5–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 {alpha}-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 [{alpha}-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 {chi}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 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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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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