Rudi Balling

Genome-wide, large-scale production of mutant mice by ENU mutagenesis

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).

Antagonistic Interactions between FGF and BMP Signaling Pathways: A Mechanism for Positioning the Sites of Tooth Formation

Vertebrate organogenesis is initiated at sites that are often morphologically indistinguishable from the surrounding region. Here we have identified Pax9 as a marker for prospective tooth mesenchyme prior to the first morphological manifestation of odontogenesis. We provide evidence that the sites of Pax9 expression in the mandibular arch are positioned by the combined activity of two signals, one (FGF8) that induces Pax9 expression and the other (BMP2 and BMP4) that prevents this induction. Thus it appears that the position of the teeth is determined by a combination of two different types of signaling molecules produced in wide but overlapping domains rather than by a single localized inducer. We suggest that a similar mechanism may be used for specifying the sites of development of other organs.

OCT-4 - A GERMLINE-SPECIFIC TRANSCRIPTION FACTOR MAPPING TO THE MOUSE T-COMPLEX

Oct‐4 is a maternally expressed octamer‐binding protein encoded by the murine Oct‐4 gene. It is present in unfertilized oocytes, but also in the inner cell mass and in primordial germ cells. Here we show that the ectopic expression of Oct‐4 in HeLa cells is sufficient for transcriptional activation from the octamer motif, indicating that Oct‐4 is a transcription factor. Therefore, Oct‐4 is the first transcription factor described that is specific for the early stages of mouse development. The spatial and temporal expression patterns were further determined using in situ hybridization. With this technique Oct‐4 expression is detected in the oocyte, in the blastocyst and before gastrulation in the embryonic ectoderm. After day 8 Oct‐4 expression decreases and is restricted to primordial germ cells from about day 8.5 onwards. Therefore Oct‐4 is a transcription factor that is specifically expressed in cells participating in the generation of the germline lineage. Linkage analysis using B X D recombinant inbred mouse strains demonstrates that Oct‐4 maps to chromosome 17 in or near the major histocompatibility complex. Several mouse mutants in the distal region of the mouse t‐complex affecting blastocyst and embryonic ectoderm formation also map to this region.

A family of octamer-specific proteins present during mouse embryogenesis: Evidence for germline-specific expression of an Oct factor.

We have analysed various adult organs and different developmental stages of mouse embryos for the presence of octamer‐binding proteins. A variety of new octamer‐binding proteins were identified in addition to the previously described Oct1 and Oct2. Oct1 is ubiquitously present in murine tissues, in agreement with cell culture data. Although Oct2 has been described as a B‐cell‐specific protein, similar complexes were also found with extracts from brain, kidney, embryo and sperm. In embryo and brain at least two other proteins, Oct3 and Oct7, are present. A new microextraction procedure allowed the detection of two maternally expressed octamer‐binding proteins, Oct4 and Oct5. Both proteins are present in unfertilized oocytes and embryonic stem cells, the latter containing an additional protein, Oct6. Whereas Oct4 was not found in sperm or testis, it is expressed in male and female primordial germ cells. Therefore Oct4 expression is specific for the female germline at later stages of germ cell development. Our results indicate that a family of octamer‐binding proteins is present during mouse development and is differentially expressed during early embryogenesis. Protease clipping experiments of Oct4 and Oct1 suggest that both proteins contain similar DNA‐binding domains.

Pax : a murine multigene family of paired box-containing genes

A murine multigene family has been identified that shares a conserved sequence motif, the paired box, with developmental control and tissue-specific genes of Drosophila. To date five murine paired box-containing genes (Pax genes) have been described and one, Pax-1, has been associated with the developmental mutant phenotype undulated. Here we describe the paired boxes of three novel Pax genes, Pax-4, Pax-5, and Pax-6. Comparison of the eight murine paired domains of the mouse, the five Drosophila paired domains, and the three human paired domains shows that they fall into six distinct classes: class I comprises Pox meso, Pax-1, and HuP48; class II paired, gooseberry-proximal, gooseberry-distal, Pax-3, Pax-7, HuP1, and HuP2; class III Pax-2, Pax-5, and Pax-8; class IV Pax-4; class V Pox neuro; and class VI Pax-6. Pax-1 and the human gene HuP48 have identical paired domains, as do Pax-3 and HuP2 as well as Pax-7 and HuP1, and are likely to represent homologous genes in mouse and man. Identical intron-exon structure and extensive sequence homology of their paired boxes suggest that several Pax genes represent paralogs. The chromosomal location of all novel Pax genes and of Pax-3 and Pax-7 has been determined and reveals that they are not clustered.

Teeth. Where and how to make them.

Organs have to develop at precisely determined sites to ensure functionality of the whole organism. Organogenesis is typically regulated by a series of interactions between morphologically distinct tissues. The developing tooth of the mouse is an excellent model to study these processes and we are beginning to understand the networks regulating reciprocal tissue interactions at the molecular level. Synergistic and antagonistic effects of signaling molecules including FGFs and BMPs are recursively used to induce localized responses in the adjacent tissue layer (mesenchyme or epithelium). However, at different phases of odontogenesis these secreted growth factors have distinct effects and at the same time they are regulated by different upstream factors. The mesenchymal transcription factors Msx1 and Pax9 are initially regulated by epithelial FGFs and BMPs, but subsequently they function upstream of these signaling molecules. This cascade provides a molecular model by which reciprocal tissue interactions are controlled.

VARIATIONS OF CERVICAL-VERTEBRAE AFTER EXPRESSION OF A HOX-1.1 TRANSGENE IN MICE

To understand the function of murine homeobox genes, a genetic analysis is mandatory. We generated gain-of-function mutants by introducing genomic sequences of the Hox-1.1 gene under the control of a chicken beta-actin promoter into mice. Our previous data had shown that these transgenic mice are nonviable after birth and are born with craniofacial abnormalities. In a subsequent detailed analysis of severely affected animals, malformations of the basioccipital bone, the atlas, and the axis were observed. Manifestation of an additional vertebra, a proatlas, occurred at the craniocervical transition. The dominant interference of the Hox-1.1 transgene with developmental programs seems to occur around day 9 of gestation, the time of neural crest migration and somite differentiation. We discuss the resulting phenotype with respect to a developmental control function of Hox-1.1.

Octamer binding proteins confer transcriptional activity in early mouse embryogenesis.

Oct4 and Oct5 are two mouse maternally expressed proteins binding to the octamer motif. Both are found in unfertilized oocytes and embryonic stem cells, whereas Oct4 is also found in primordial germ cells. In this study, the activity of the octamer motif was analysed in two embryonic stem cell lines containing Oct4 and Oct5, the teratocarcinoma‐derived cell line F9 and the blastocyst‐derived cell line D3. It is known that oligomerization of the octamer motif creates a powerful B‐cell specific enhancer. As shown here, this oligomerized transcriptional element is also a very strong enhancer in F9 and D3 embryonic stem cells. After differentiation of the stem cells, both enhancer activity and the amount of the octamer binding proteins decrease. An intact octamer stimulates heterologous promoters in embryonic stem cells, whereas mutations in the octamer motif abolish transcriptional stimulation and binding of the octamer factors. The use of transgenic embryos demonstrates transcriptional activation in the inner cell mass but not in the trophoblast of blastocysts. The results indicate that Oct4 and Oct5 are active early in mouse development.

Impaired insulin secretory capacity in mice lacking a functional vitamin D receptor

It was the aim of this study to further explore the functional role of vitamin D in the endocrine pancreas. By gene targeting, we have recently generated mice in which a lacZ reporter gene is driven by the endogenous vitamin D receptor (VDR) promoter. These mice express a functionally inactive mutant VDR. Pancreatic islets but not exocrine pancreas cells showed strong lacZ reporter gene expression in mutant mice. To rule out possible influences of hypocalcemia on pancreatic endocrine function, a rescue diet enriched with calcium, phosphorus, and lactose was fed to wild‐type (WT) and VDR mutant mice. The rescue diet normalized body weight and mineral homeostasis in VDR mutants. In glucose tolerance tests, baseline blood glucose levels were unchanged in fasting VDR mutants. However, blood glucose was elevated after oral or subcutaneous glucose loading, and maximum serum insulin levels were reduced by ~60% in VDR mutants vs. WT mice on either diet. In addition, insulin mRNA levels were decreased in VDR mutant mice on both diets, whereas pancreatic β cell mass, islet architecture, and islet neogenesis were normal. These findings clearly establish a molecular role of the VDR in pancreatic insulin synthesis and secretion in vivo.

Undulated, a mutation affecting the development of the mouse skeleton, has a point mutation in the paired box of Pax 1.

undulated (un) homozygous mice exhibit vertebral malformations along the entire rostro-caudal axis. Pax 1, a murine paired box-containing gene, is expressed in ventral sclerotome cells and later in intervertebral disks along the entire vertebral column. We localized the Pax 1 gene on chromosome 2 between beta 2-microglobulin and the agouti locus to an area where un maps. DNA analysis of the un mutant revealed a point mutation in a highly conserved part of the paired box of Pax 1, leading to a Gly-Ser replacement. The chromosomal location and the mutation in the paired box of un mice in conjunction with Pax 1 gene expression in wild-type mice implicate a causative role of Pax 1 in generation of the vertebral column.