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Bone morphogenetic protein-4 (BMP-4) is a member of the TGF-I~ superfamily of polypeptide signaling molecules, closely related to BMP-2 and to Drosophila decapentaplegic (DPP). To elucidate the role of BMP-4 in mouse development the gene has been inactivated by homologous recombination in ES cells. Homozygous mutant Bmp-4 t~lblh embryos die between 6.5 and 9.5 days p.c., with a variable phenotype. Most Bmp-4 t~Ibl~ embryos do not proceed beyond the egg cylinder stage, do not express the mesodermal marker 1~Brachyury), and show little or no mesodermal differentiation. Some homozygous mutants develop to the head fold or beating heart/early somite stage or beyond. However, they are developmentally retarded and have truncated or disorganized posterior structures and a reduction in extraembryonic mesoderm, including blood islands. These results provide direct genetic evidence that BMP-4 is essential for several different processes in early mouse development, beginning with gastrulation and mesoderm formation. Moreover, in the presumed absence of zygotic ligand, it appears that homozygous mutants can be rescued partially by related proteins or by maternal BMP-4.[Key Words: BMP-4; mouse embryo; targeted mutation; lethal embryonic phenotype] Received May 3, 1995; revised version accepted July 12, 1995.Bone morphogenetic proteins-2 and -4 (BMP-2 and BMP-4) are two closely related members of the transforming growth factor (TGF)-~ superfamily of secreted polypeptide signaling molecules (for review, see Kingsley 1994; Hogan 1995). The carboxy-terminal mature regions of the two proteins are 92% identical at the amino acid level. Moreover, they have been highly conserved during evolution; in Drosophila a single gene, decapentaplegic (dpp), encodes a protein with -75% sequence identity to BMP-2 and BMP-4 in the carboxy-terminal mature region. At the functional level, human BMP-4 can rescue the dorsal-ventral pattern defects of dpp null mutants, and Drosophila DPP protein can induce ectopic bone in mice (Padgett et al. 1993;Sampath et al. 1993). Genetic analysis has shown that DPP is required at several different stages of Drosophila development. It acts as a dorsalizing morphogen in the dorsal-ventral patterning of the blastoderm embryo, mediates an inductive interaction between mesoderm and endoderm in the larval midgut, and plays a role in the proximal-distal patterning of the leg and wing imaginal discs (for review, see Campbell et al. 1993;Diaz-Benjumea and Cohen 1993;Wall and Hogan 1994).The striking evolutionary conservation of BMP-4 and 1These authors contributed equally to this work. 2Present address:

Stem cells are currently in the news for two reasons: the successful cultivation of human embryonic stem cell lines and reports that adult stem cells can differentiate into developmentally unrelated cell types, such as nerve cells into blood cells. Both intrinsic and extrinsic signals regulate stem cell fate and some of these signals have now been identified. Certain aspects of the stem cell microenvironment, or niche, are conserved between tissues, and this can be exploited in the application of stem cells to tissue replacement therapy.

In many organisms the allocation of primordial germ cells (PGCs) is determined by the inheritance of maternal factors deposited in the egg. However, in mammals, inductive cell interactions are required around gastrulation to establish the germ line. Here, we show that Bmp4 homozygous null embryos contain no PGCs. They also lack an allantois, an extraembryonic mesodermal tissue derived, like the PGCs, from precursors in the proximal epiblast. Heterozygotes have fewer PGCs than normal, due to a reduction in the size of the founding population and not to an effect on its subsequent expansion. Analysis of ␤-galactosidase activity in Bmp4 lacZneo embryos reveals that prior to gastrulation, Bmp4 is expressed in the extraembryonic ectoderm. Later, Bmp4 is expressed in the extraembryonic mesoderm, but not in PGCs. Chimera analysis indicates that it is the Bmp4 expression in the extraembryonic ectoderm that regulates the formation of allantois and primordial germ cell precursors, and the size of the founding population of PGCs. The initiation of the germ line in the mouse therefore depends on a secreted signal from the previously segregated, extraembryonic, trophectoderm lineage.

The mammalian respiratory system—the trachea and the lungs—arises from the anterior foregut through a sequence of morphogenetic events involving reciprocal endodermal-mesodermal interactions. The lung itself consists of two highly branched, tree-like systems—the airways and the vasculature—that develop in a coordinated way from the primary bud stage to the generation of millions of alveolar gas exchange units. We are beginning to understand some of the molecular and cellular mechanisms that underlie critical processes such as branching morphogenesis, vascular development, and the differentiation of multipotent progenitor populations. Nevertheless, many gaps remain in our knowledge, the filling of which is essential for understanding respiratory disorders, congenital defects in human neonates, and how the disruption of morphogenetic programs early in lung development can lead to deficiencies that persist throughout life.

Summary To directly test the contribution of Scgb1a1+ Clara cells to postnatal growth, homeostasis and repair of lung epithelium, we generated a Scgb1a1-CreERTM “knock-in” mouse line for lineage tracing these cells. Under all conditions tested the majority of Clara cells in the bronchioles both self-renew and generate ciliated cells. In the trachea, Clara cells give rise to ciliated cells but do not self-renew extensively. Nevertheless, they can contribute to tracheal repair. In the postnatal mouse lung it has been proposed that bronchioalveolar stem cells (BASCs) which co-express Scgb1a1 (Secretoglobin1a1) and SftpC (Surfactant Protein C), contribute descendants to both bronchioles and alveoli. The putative BASCs were lineage labeled in our studies. However, we find no evidence for the function of a special BASC population during postnatal growth, adult homeostasis or repair. Rather, our results support a model in which the trachea, bronchioles and alveoli are maintained by distinct populations of epithelial progenitor cells.