The type I activin receptor ActRIB is required for egg cylinder organization and gastrulation in the mouse - PubMed (original) (raw)

The type I activin receptor ActRIB is required for egg cylinder organization and gastrulation in the mouse

Z Gu et al. Genes Dev. 1998.

Abstract

ActRIB is a type I transmembrane serine/threonine kinase receptor that has been shown to form heteromeric complexes with the type II activin receptors to mediate activin signal. To investigate the function of ActRIB in mammalian development, we generated ActRIB-deficient ES cell lines and mice by gene targeting. Analysis of the ActRIB-/- embryos showed that the epiblast and the extraembryonic ectoderm were disorganized, resulting in disruption and developmental arrest of the egg cylinder before gastrulation. To assess the function of ActRIB in mesoderm formation and gastrulation, chimera analysis was conducted. We found that ActRIB-/- ES cells injected into wild-type blastocysts were able to contribute to the mesoderm in chimeric embryos, suggesting that ActRIB is not required for mesoderm formation. Primitive streak formation, however, was impaired in chimeras when ActRIB-/- cells contributed highly to the epiblast. Further, chimeras generated by injection of wild-type ES cells into ActRIB-/- blastocysts formed relatively normal extraembryonic tissues, but the embryo proper developed poorly probably resulting from severe gastrulation defect. These results provide genetic evidence that ActRIB functions in both epiblast and extraembryonic cells to mediate signals that are required for egg cylinder organization and gastrulation.

PubMed Disclaimer

Figures

Figure 1

Expression of ActRIB in early postimplantation mouse embryos. In situ hybridization on sections of wild-type embryos from E5.5 to E7.5. Sagittal section of an E5.5 embryo (A), an E6.0 embryo (B), and sections of E6.5 embryos (C–E). (C) Sagittal section; (D) transverse section in the extraembryonic region, (E) or embryonic region. (F,G) Transverse sections in the extraembryonic (F) or embryonic (G) regions of an E7.5 embryo. (am) amniotic fold; (ee) embryonic ectoderm; (en) endoderm; (me) mesoderm; (ps) primitive streak; (ve) visceral endoderm; (xe) extraembryonic ectoderm.

Figure 2

Disruption of ActRIB by homologous recombination results in early embryonic lethality. (A) (Top) Wild-type ActRIB locus shows the exon encoding the kinase subdomain VI (▪). (Middle) The targeting vector contains a 1.8-kb neo cassette (□ box), inserted at the _Nde_I site inside the kinase subdomain VI of the ActRIB gene. (Bottom) The mutated ActRIB locus. The 5′ external probe, a 1.6-kb genomic fragment, for Southern analysis is shown. (S) _Sal_I; (R) _Eco_RI; (N) _Nde_I; (B) _Bst_EII. (B) Genotype analysis of a litter of newborn mice from heterozygous mating. Tail genomic DNA was digested with _Eco_RI and blotted with the 5′ external probe. The genotypes are marked as +/+ for the wild-type and +/− for the heterozygote. No homozygous live-born mice were found. Arrowheads indicate the 13-kb wild-type and 10-kb mutant bands. (C,D) Gross morphology of normal embryos (left) and ActRIB−/− mutant littermates (right) at E7.5 (C) and late E8.5 (D). In the wild-type, anterior is marked as A and posterior as P. In the mutant embryos, no apparent anterior–posterior polarity was observed, whereas the parietal endoderm (PE) is present. (E). Analysis of T expression in E7.5 embryos by whole-mount in situ bybridization. Wild-type embryo (left); _ActRIB_−/− littermate (right). (ps) Primitive streak.

Figure 3

Histological analysis of _ActRIB_−/− embryos. Sagittal sections of the wild-type (A,C,I) and mutant embryos (B,D–H, J–M) at E5.5 (A,B), E6.5 (C–H), E7.5 (I–K) and E8.5 (L,M). In each panel, the ectoplacental cone (or the proximal region) is positioned towards the top. The distal visceral endoderm in the E5.5 mutant embryo is detached from the epiblast (B). The E6.5 mutant embryos contain only vacuolated columnar visceral endoderm cells (D–H). Germ layers or cell clusters that are stained darker are identified as epiblast through analysis of whole series of embryo sections. (M) An enlarged section of an E8.5 mutant embryo shown in L. Cell death of the epiblast in this mutant is apparent. (al) allantois; (am) amnion; (ch) chorion; (de) dead cells; (ee) embryonic ectoderm; (me) mesoderm; (pe) parietal endoderm; (ve) visceral endoderm; (xe) extraembryonic ectoderm. Bar, 50 μm.

Figure 4

Analysis of expression of lineage-specific genes in the _ActRIB_−/− embryos. Hemotoxylin and eosin (H/E) stained sections (A,E) and immunostaining with SSEA-1 (B,F), Troma-1 (C,G) and anti-laminin (D,H) antibodies of the wild-type (A–D) and mutant (E–H) embryo sections at E6.5. The section in A is adjacent to that in B; the section in E is adjacent to that in F. Other sections are from different embryos. In each panel, the ectoplacental cone (or the proximal region) is positioned toward the top. The two clusters of stained epiblast cells in E are marked with arrowheads.

Figure 5

Chimeric embryos derived by injection of _ActRIB_−/− cells into wild-type blastocysts. The gross morphology of E9.5 embryos with low (A) or moderate (B) contribution of _lacZ_-labeled homozygous mutant cells appears normal. Sagittal (C) and transverse (D) sections of late E7.5 embryos show moderate contribution of mutant cells in allantois (al), mesoderm at posterior of the primitive streak (ps), and lateral mesoderm (lm). (E) An E7.0 chimeric embryo shows no blue staining of ActRIB−/− cells in mesoderm layer (me), even though the contribution in the adjacent embryonic ectoderm (ee) is relatively high. (F) A late E7.5 chimeric embryo section with very high contribution of mutant cells. No primitive streak or embryonic mesodermal cells were found, although some extraembryonic mesoderm (xm) cells were observed in an amniotic fold-like structure. Anterior and posterior of the embryo in C are marked as A and P, respectively. Bar, 50 μm.

Figure 6

Teratomas derived from _ActRIB_−/− ES cells. Histology sections of teratomas derived from _ActRIB_−/− ES cells (A–D). Tissues such as cartilage (ca), fat cells (fc), keratinized epithelium (ke), and smooth muscle (sm) are shown.

Figure 7

Expression of ALK4 transgene in _ActRIB_−/− ES cells. (A) A diagram of ALK4 transgene expression vector. The human ALK4 cDNA tagged with a triple HA epitope at the 3′ end of the coding sequence was inserted between the EF-1α promoter and SV40 polyadenylation signal followed by a hygromycin cassette. (B) Expression of HA–ALK4 protein in the transgenic ES cell lines was analyzed by immunoblotting the ES cell protein extracts with 12CA5, the monoclonal antibody against the HA epitope. The arrow head indicates the HA–ALK4 bands. The expression is higher in cell lines 20 and 42 than in lines 21 and 7. (C) Genotype of ALK4 transgenic ES cell lines and wild-type control cells. Genomic DNA from wild-type and the transgenic cell lines was digested with _Eco_RI and analyzed by Southern hybridization by use of a 1.6-kb _Hin_dIII–_Eco_RI ALK4 cDNA fragment as a probe. Cell lines 7, 21, and 42 each contain one copy and line 20 contains two copies of the transgene (data not shown). The bands representing the wild-type allele, the ActRIB mutant allele, and the ALK4 transgene are marked on the right. Arrowheads indicate two DNA fragments shared by both wild-type and _ActRIB_−/− mutant alleles.

Figure 8

Rescuing _ActRIB_−/− ES cell defect with an ALK4 transgene in chimeras. The chimeric embryos were derived by injection of the _ActRIB_−/− (ALK4+) cells (line 20) into the wild-type blastocysts. X-gal-stained E8.5 (A) and E9.5 (B) embryos appear normal. (C,D) Histological sections of an E8.5 embryo (C) and an E9.5 (D) show high contribution of transgenic cells in almost all mesodermal cell types (C) or a salt and pepper pattern of intermingle of transgenic cells with the wild-type cells derived from the blastocyst (D). (E) An enlarged part of the embryo in C shows the notochord (nc) and definitive endoderm (de), completely derived from the transgenic cells. (F,G) Sections from an E9.5 embryo show the mesenchyme (me), neuroepithelium (ne), neural tube (nt), and somite (s). Bar, 67 μm.

Figure 9

Chimeric embryos derived by injection of wild-type ES cells into the _ActRIB_−/−/ blastocysts. (A) Two E9.5 embryos were dissected and their parietal yolk sac DNA was genotyped as _ActRIB_−/−. The embryonic tissues (em) in the chimeras were very small. (B) E9.5 _ActRIB_−/− chimeric embryo was stained with X-gal and sectioned to show the visceral yolk sac mesoderm (ym) and blood cells (bl). The yolk sac mesoderm consists almost entirely of _lacZ_-marked wild-type cells; the yolk sac endoderm (ye) is derived from _ActRIB_−/− blastocyst. (C) Control E7.5 embryo generated by injecting the same wild-type ES cells into a wild-type or heterozygous blastocyst shows normal embryonic structures. (D) Transverse section of a late E7.5 abnormal chimera. Mesodermal cells were formed inside a yolk sac-like structure, but no obvious embryonic tissues were found. (bl) Blood cells; (ye) yolk sac endoderm; (ym) yolk sac mesoderm; (ys) yolk sac. Bar, 67 μm.

References

1. Albano RM, Arkell R, Beddington RSP, Smith JC. Expression of inhibin subunits and follistatin during postimplantation mouse development: Decidual expression of activin and expression of follistatin in primitive streak, somites and hindbrain. Development. 1994;120:803–813. - PubMed
1. Armes NA, Smith JC. The ALK-2 and ALK-4 activin receptors transduce distinct mesoderm-inducing signals during early Xenopus development but do not co-operate to establish thresholds. Development. 1997;124:3797–3804. - PubMed
1. Asashima M, Nakano H, Shimada K, Kinoshita K, Ishii K, Shibai H, Ueno N. Wilhelm Roux’s Arch Dev Biol. 1990;198:330–335. - PubMed
1. Attisano L, Wrana JL, Cheifetz S, Massagué J. Novel activin receptors: Distinct genes and alternative mRNA splicing generate a repertoire of serine/threonine kinase receptors. Cell. 1992;68:97–108. - PubMed
1. Attisano L, Carcamo J, Ventura F, Weis FMB, Massagué J, Wrana JL. Identification of human activin and TGF-beta type I receptor that form heteromeric kinase complexes with type II receptors. Cell. 1993;75:671–680. - PubMed

The type I activin receptor ActRIB is required for egg cylinder organization and gastrulation in the mouse - PubMed (original) (raw)