We study the molecular genetics of patterning, axis formation, and organogenesis in the developing mouse embryo. Our current work examines key control elements of these processes and shows the emergence of a scenario of protein-protein interactions that controls every aspect of the body plan. We have used a loss-of-function approach to identify basic components of protein complexes that activate target genes during the course of development.

Transcriptional Cofactors
Chen, Grinberg, Huang, Mukhopadhyay, Schindler, Teufel, Westphal; in collaboration with Agulnick, Bayarsaihan, Dawid, Dorward, Downs, Hukriede, Kennison, Ogryzko, Podtelejnikov, Segal, Yamashita
Over the years, our laboratory has carried out extensive studies on LIM-homeodomain proteins. These Lhx gene products are important transcriptional regulators of invertebrate and vertebrate embryonic development. They are involved in early patterning events, the development of the nervous system, and organogenesis. Their action is facilitated by co-factors that were identified by their ability to dimerize and to bind to the LIM domain, a specialized zinc-finger structure present in Lhx gene products and in a number of other nuclear proteins. In vertebrates, these LIM-binding co-factors are known as Ldb, Nli, or Clim; in Drosophila as Chip; and in C. elegans as Ldb1. Protein-protein interactions involving Ldb/Nli/Clim (henceforth referred to as Ldb) and Chip are not restricted to LIM domain–containing factors but can involve a host of other transcriptional regulators as well. Ample evidence supports the notion that the Ldb and Chip co-factors are essential components of developmental programs controlled by trans-criptional regulators. More recently, the Rlim cofactor was identified and shown to control transcription factors negatively by targeting Ldb proteins for degradation. Furthermore, competition of transcription factors for binding to Chip or Ldb can alter developmental cell fates.

The mammalian Ldb1 protein is found in multi-protein complexes containing various combinations of LIM-homeodomain, LIM-only, bHLH, GATA, and Otx transcription factors. These proteins exert key functions during embryogenesis. We have established that targeted deletion of the Ldb1 gene in mice results in a pleiotropic phenotype, revealing fundamental roles of Ldb1 in important developmental decisions. First, there is no heart anlage in the Ldb1-null mutant embryo. Our data suggest that Ldb1 gene function is essential for proper allocation of cardiac mesoderm to the heart field in the gastrulating embryo. Abnormal migration of the heart mesoderm in the mutants may abolish inductive interactions of the tissue with anterior endoderm and thus prevent heart development. In addition, head development is severely curtailed in the mutant, and head structures are truncated anterior to the hindbrain. In about 40 percent of the mutants, posterior axis duplication is observed. The expression of several Wnt inhibitors is curtailed in the mutant, suggesting that Wnt pathways may be involved in axial patterning events regulated by Ldb1. Abnormal organizer gene expression during gastrulation may account for the observed axis defects in the Ldb1 mutant embryos. Finally, we observed in the Ldb1-null mutant conceptus severe defects in mesoderm-derived extraembryonic structures, including the allantois, blood islands of the yolk sack, primordial germ cells, and the amnion, attesting to a pivotal role of Ldb1 in mesoderm formation.

To identify additional components of Ldb/Chip-containing nuclear protein complexes, we generated HeLa cells that express FLAG and HA epitope–tagged mouse Ldb1, purified nuclear complexes with the aid of the tags, and identified constituent proteins by mass spectrometry of tryptic peptides. We identified Ssdp proteins (previously described as sequence-specific, single-stranded-DNA-binding proteins) as components of Ldb1-associated nuclear complexes in these cells. Ssdp proteins are associated with Ldb1 in a variety of additional mammalian cell types. The association is specific, does not depend on the presence of nucleic acids, and is functionally significant. Genes encoding Ssdp proteins are well conserved in evolution from Drosophila to humans. Whereas the vertebrate Ssdp gene family has several closely related members, the Drosophila Ssdp gene is unique. In Xenopus, Ssdp encoded by Drosophila Ssdp or mouse Ssdp1 mRNA enhances axis induction by Ldb1 in conjunction with the LIM-homeobox gene Xlim1. Furthermore, we were able to demonstrate an interaction between Ssdp and Chip (the fly homolog of Ldb1) in Drosophila wing development. These findings indicate functional conservation of Ssdp as a co-factor of Ldb1 in transcriptional regulation during invertebrate and vertebrate development.

Novel Genes
Hasuike, Malik, Miyamoto, Mukhopadhyay, Teufel, Westphal, Zhao; in collaboration with Dawid, Fiorenza, Kawahara
Our laboratory has identified a number of novel genes involved in the development of the mouse embryo. We detected the first two, Thg-1pit and MPPa2, in a screen for genes that are differentially expressed in the developing pituitary of wild-type and Lhx3-null mutant embryos. The genes are active at early stages of pituitary development. A third gene, Mbx, encodes a homeodomain transcription factor that is initially restricted to the midbrain region of the mouse embryo but later extends to the developing forebrain. Finally, we cloned Frcp1 and Frcp2, two genes that are also primarily expressed in the brain. These genes contain an FNIII motif characteristic for a variety of receptor and cell adhesion proteins.