Michael Nehls

Eomesodermin is required for mouse trophoblast development and mesoderm formation.

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior–posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.

Two genetically separable steps in the differentiation of thymic epithelium

The development of the thymus depends initially on epithelial-mesenchymal and subsequently on reciprocal lympho-stromal interactions. The genetic steps governing development and differentiation of the thymic microenvironment are unknown. With the use of a targeted disruption of the whn gene, which recapitulates the phenotype of the athymic nude mouse, the WHN transcription factor was shown to be the product of the nude locus. Formation of the thymic epithelial primordium before the entry of lymphocyte progenitors did not require the activity of WHN. However, subsequent differentiation of primitive precursor cells into subcapsular, cortical, and medullary epithelial cells of the postnatal thymus did depend on activity of the whn gene. These results define the first genetically separable steps during thymic epithelial differentiation.

An EF hand mutation in Stim1 causes premature platelet activation and bleeding in mice

Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.

Isolation and biochemical characterization of LEAP-2, a novel blood peptide expressed in the liver.

The human genome contains numerous genes whose protein products are unknown in terms of structure, interaction partner, expression, and function. To unravel the function of these orphan genes, it is of particular value to isolate native forms of protein and peptide products derived from these genes. From human blood ultrafiltrate, we characterized a novel gene‐encoded, cysteine‐rich, and cationic peptide that we termed liver‐expressed antimicrobial peptide 2 (LEAP‐2). We identified several circulating forms of LEAP‐2 differing in their amino‐terminal length, all containing a core structure with two disulfide bonds formed by cysteine residues in relative 1–3 and 2–4 positions. Molecular cloning of the cDNA showed that LEAP‐2 is synthesized as a 77‐residue precursor, which is predominantly expressed in the liver and highly conserved among mammals. This makes it a unique peptide that does not exhibit similarity with any known human peptide regarding its primary structure, disulfide motif, and expression. Analysis of the LEAP‐2 gene resulted in the identification of an alternative promoter and at least four different splicing variants, with the two dominating transcripts being tissue‐specifically expressed. The largest native LEAP‐2 form of 40 amino acid residues is generated from the precursor at a putative cleavage site for a furin‐like endoprotease. In contrast to smaller LEAP‐2 variants, this peptide exhibited dose‐dependent antimicrobial activity against selected microbial model organisms. LEAP‐2 shares some characteristic properties with classic peptide hormones and it is expected that the isolation of this novel peptide will help to unravel its physiological role.

Abnormal bone marrow stroma in mice deficient for nemo‐like kinase, Nlk

The stromal compartment of the bone marrow is composed of various cell types that provide trophic and instructive signals for hematopoiesis. The mesenchymal stem cell is believed to give rise to all major cellular components of the bone marrow microenvironment. Nemo‐like kinase, Nlk, is a serine‐threonine kinase that connects MAP kinase and Wnt signaling pathways; its in vivo function in mouse is unknown. We have generated mice with a targeted disruption of Nlk and find that the complex phenotype significantly varies with the genetic background. Whereas C57BL/6 mice lacking Nlk die during the third trimester of pregnancy, the 129/Sv background supports survival into adolescence; such mice are growth retarded and suffer from various neurological abnormalities. We show here that the Nlk deficiency syndrome includes aberrant differentiation of bone marrow stromal cells. Varying degrees of morphological abnormality, such as increased numbers of adipocytes, large blood sinuses and absence of bone‐lining cells are observed in the bone marrow of mutant mice. Nlk deficient mice thus provide a novel model to study the genetic requirements for bone marrow stromal differentiation.

Random mutagenesis in the mouse as a tool in drug discovery

The flood of raw information generated by large-scale data acquisition technologies in genomics, microarrays and proteomics is changing the early stages of the drug discovery process. Although many more potential drug targets are now available compared with the pre-genomics era, knowledge about the physiological context in which these targets act--information crucial to both discovery and development--is scarce. Random mutagenesis strategies in the mouse provide scalable approaches for both the gene-driven validation of candidate targets in vivo and the discovery of new physiological pathways by phenotype-driven screens.

YAC/P1 contigs defining the location of 56 microsatellite markers and several genes across a 3.4-cM interval on mouse Chromosome 11

The characterization of three YAC/P1 contigs from adjacent segments of the central region of mouse Chromosome (Chr) 11 is described. These contigs are based upon 63 YACs and 40 P1 recombinants. From these clones, 185 end sequences were obtained, of which 147 sequences could be converted into sequencetagged sites and mapped within the three contigs. Deletions were detected in 16 out of 63 YACs; 19 of 63 YACs were found to be chimeric. No such aberrations were found in P1 recombinants. A total of 22 public and 34 newly developed microsatellite markers were unambiguously localized to and ordered in the contigs. In the cryb1/Nf1 interval of the central contig, several new genes have been identified by exon trapping and precisely localized with respect to known STS markers.

Transcription factors that control development of the thymic microenvironment

Abstract Very little is known about genetic determinants of thymic stromal cell differentiation. However, two genes involved in lineage commitment of the thymic stroma have recently been identified, representing a starting point in the genetic dissection of the lineage composition and phenotypic heterogeneity of the thymic microenvironment.

Deductive genomics: a functional approach to identify innovative drug targets in the post-genome era.

The sequencing of the human genome has generated a drug discovery process that is based on sequence analysis and hypothesis-driven (inductive) prediction of gene function. This approach, which we term inductive genomics, is currently dominating the efforts of the pharmaceutical industry to identify new drug targets.According to recent studies, this sequence-driven discovery process is paradoxically increasing the average cost of drug development, thus falling short of the promise of the Human Genome Project to simplify the creation of much needed novel therapeutics. In the early stages of discovery, the flurry of new gene sequences makes it difficult to pick and prioritize the most promising product candidates for product development, as with existing technologies important decisions have to be based on circumstantial evidence that does not strongly predict therapeutic potential. This is because the physiological function of a potential target cannot be predicted by gene sequence analysis and in vitro technologies alone.In contrast, deductive genomics, or large-scale forward genetics, bridges the gap between sequence and function by providing a function-driven in vivo screen of a highly orthologous mammalian model genome for medically relevant physiological functions and drug targets. This approach allows drug discovery to move beyond the focus on sequence-driven identification of new members of classical drug-able protein families towards the biology-driven identification of innovative targets and biological pathways.

Only one nemo-like kinase gene homologue in invertebrate and mammalian genomes

The nemo-like kinase (Nlk) connects the MAP kinase and Wnt signalling pathways. We have found that invertebrate (Caenorhabditis elegans, Drosophila melanogaster) and mammalian genomes (Mus musculus and Homo sapiens) each contain only a single functional Nlk gene. The mouse genome also harbours a transcriptionally silent processed Nlk pseudogene residing on chromosome 2. Thus, while genes encoding upstream (such as Wnts and frizzelds) and downstream (such as TCF/LEF) components of the Wnt signalling pathway have been extensively diversified during evolution, genes encoding components of the common core of the connecting signalling structure (such as beta-catenin, GSK beta and Nlk) have been maintained in single copies.