Functional analysis of secreted and transmembrane proteins critical to mouse development.

We describe the successful application of a modified gene-trap approach, the secretory trap, to systematically analyze the functions in vivo of large numbers of genes encoding secreted and membrane proteins. Secretory-trap insertions in embryonic stem cells can be transmitted to the germ line of mice with high efficiency and effectively mutate the target gene. Of 60 insertions analyzed in mice, one-third cause recessive lethal phenotypes affecting various stages of embryonic and postnatal development. Thus, secretory-trap mutagenesis can be used for a genome-wide functional analysis of cell signaling pathways that are critical for normal mammalian development and physiology.

Agrin isoforms with distinct amino termini: differential expression, localization, and function.

The proteoglycan agrin is required for postsynaptic differentiation at the skeletal neuromuscular junction, but is also associated with basal laminae in numerous other tissues, and with the surfaces of some neurons. Little is known about its roles at sites other than the neuromuscular junction, or about how its expression and subcellular localization are regulated in any tissue. Here we demonstrate that the murine agrin gene generates two proteins with different NH(2) termini, and present evidence that these isoforms differ in subcellular localization, tissue distribution, and function. The two isoforms share approximately 1,900 amino acids (aa) of common sequence following unique NH(2) termini of 49 or 150 aa; we therefore call them short NH(2)-terminal (SN) and long NH(2)-terminal (LN) isoforms. In the mouse genome, LN-specific exons are upstream of an SN-specific exon, which is in turn upstream of common exons. LN-agrin is expressed in both neural and nonneural tissues. In spinal cord it is expressed in discrete subsets of cells, including motoneurons. In contrast, SN-agrin is selectively expressed in the nervous system but is widely distributed in many neuronal cell types. Both isoforms are externalized from cells but LN-agrin assembles into basal laminae whereas SN-agrin remains cell associated. Differential expression of the two isoforms appears to be transcriptionally regulated, whereas the unique SN and LN sequences direct their distinct subcellular localizations. Insertion of a "gene trap" construct into the mouse genome between the LN and SN exons abolished expression of LN-agrin with no detectable effect on expression levels of SN-agrin or on SN-agrin bioactivity in vitro. Agrin protein was absent from all basal laminae in mice lacking LN-agrin transcripts. The formation of the neuromuscular junctions was as drastically impaired in these mutants as in mice lacking all forms of agrin. Thus, basal lamina-associated LN-agrin is required for neuromuscular synaptogenesis, whereas cell-associated SN-agrin may play distinct roles in the central nervous system.

Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes.

A strategy was devised for identifying regions of the mouse genome that are transcriptionally active in a temporally and spatially restricted manner during development. The approach is based on the introduction into embryonic stem cells of two types of lacZ reporter constructs that can be activated by flanking mouse genomic sequences. Embryonic stem cells containing the lacZ constructs were used to produce chimaeric mice. Developmental regulation of lacZ expression occurred at a high frequency. Molecular cloning of the flanking endogenous genes and introduction of these potential insertional mutations into the mouse germ line should provide an efficient means of identifying and mutating novel genes important for the control of mammalian development.

Neuropilin-2 regulates the development of selective cranial and sensory nerves and hippocampal mossy fiber projections.

Neuropilin-1 and neuropilin-2 bind differentially to different class 3 semaphorins and are thought to provide the ligand-binding moieties in receptor complexes mediating repulsive responses to these semaphorins. Here, we have studied the function of neuropilin-2 through analysis of a neuropilin-2 mutant mouse, which is viable and fertile. Repulsive responses of sympathetic and hippocampal neurons to Sema3F but not to Sema3A are abolished in the mutant. Marked defects are observed in the development of several cranial nerves, in the initial central projections of spinal sensory axons, and in the anterior commissure, habenulo-interpeduncular tract, and the projections of hippocampal mossyfiber axons in the infrapyramidal bundle. Our results show that neuropilin-2 is an essential component of the Sema3F receptor and identify key roles for neuropilin-2 in axon guidance in the PNS and CNS.

Mutation and analysis of Dan, the founding member of the Dan family of transforming growth factor beta antagonists.

The Dan family of transforming growth factor beta antagonists is a large, evolutionarily conserved family of proteins. Little is known about either the specificity of these antagonists or the biological roles of these proteins. We have characterized Dan, the founding member of this family, with regard to both its biochemical specificity and its biological roles. Although DAN is not an efficient antagonist of BMP-2/4 class signals, we found that DAN was able to interact with GDF-5 in a frog embryo assay, suggesting that DAN may regulate signaling by the GDF-5/6/7 class of BMPs in vivo. Intriguingly, in developing neurons, Dan mRNA was localized to axons, suggesting a potential role for the DAN protein in axonal outgrowth or guidance. Mice lacking Dan activity were generated by gene targeting and displayed subtle, background-dependent defects.

Nerve growth factor regulates gene expression by several distinct mechanisms.

To help elucidate the mechanisms by which nerve growth factor (NGF) regulates gene expression, we have identified and studied four genes (a-2, d-2, d-4, and d-5) that are positively regulated by NGF in PC12 cells, including one (d-2) which has previously been identified as a putative transcription factor (NGF I-A). Three of these genes, including d-2, were induced very rapidly at the transcriptional level, but the relative time courses of transcription and mRNA accumulation of each of these three genes were distinct. The fourth gene (d-4) displayed no apparent increase in transcription that corresponded to the increase in its mRNA, suggesting that NGF may regulate its expression at a posttranscriptional level. Thus, NGF positively regulates gene expression by more than one mechanism. These genes could also be distinguished on the basis of their response to cyclic AMP. The expression of d-2 and a-2 was increased by cholera toxin and further augmented by NGF; however, cholera toxin not only failed to increase the levels of d-5 and d-4 mRNA but also actually inhibited the NGF-dependent increase. The expression of each of these genes, including d-2 (NGF I-A), was also increased by fibroblast growth factor, epidermal growth factor (EGF), phorbol myristate acetate, and in some cases insulin, showing that the regulation of these genes is not unique to NGF. Because each of these genes was expressed in response to phorbol myristate acetate and EGF, their expression may be necessary but is certainly not sufficient for neurite formation. The protein kinase inhibitor K-252a prevented the NGF-associated, but not the acidic FGF-associated, induction of d-2 and d-5 gene expression, suggesting that these two growth factors may regulate gene expression via different cellular pathways. The study of the regulation of the expression of these and other NGF-inducible genes should valuable new information concerning how NGF and other growth factors cause neural differentiation.

RNA polymerases stall and/or prematurely terminate nearby both early and late promoters on polyomavirus DNA.

Levels of transcription within the E and L strands of the five major PstI fragments of polyomavirus (strain AT3) were measured by pulse-labeling RNA both in infected cells and in isolated nuclei or viral transcription complexes during the late phase of infection. Quantification was assured by hybridization to single-stranded DNAs in solution followed by collection of hybrids on nitrocellulose filters and ribonuclease treatment. The level of in vivo transcription in the region of the early (E strand) promoter was two- to threefold higher than that in all other E-strand regions, suggesting that most RNA polymerases prematurely terminate transcription shortly downstream from this promoter during the late phase. In vitro transcription levels in this region were five- to tenfold higher than in the remainder of the E strand, suggesting that many RNA polymerases 'stall' shortly after initiation in vivo but can be reactivated and continue transcription in vitro upon exposure to detergents and high salt solution. Some premature termination nearby the late (L strand) promoter was also detected by the same method. Strikingly, many RNA polymerases also stalled on the L strand in the region of the early promoter, some 5 x 10(3) bases downstream from the late promoter. Treatment of cells with dichlororibofuranosylbenzimidazole did not affect polymerases that stalled or terminated prematurely, but strongly reduced the presence of polymerases that normally transcribed throughout the entire E or L strand. Examination of the size of RNA chains produced during in vitro incubations showed that many polymerases stalled in vivo within 50 to 100 nucleotides downstream from the initiation sites on both DNA strands. The number of polymerases active in vitro at the E strand promoter was similar to the number of polymerases at the L strand promoter. However, in contrast to L-strand transcription, most of the polymerases that initiated at the E-strand promoter were incapable of extended transcription in vivo. These results suggest that large T antigen-mediated repression of E-strand transcription is not simply due to the exclusion of RNA polymerases from the early promoter. Stalling and/or premature termination by RNA polymerases shortly downstream from the early promoter appears to be a mechanism by which temporal regulation of polyomavirus gene expression can be effected.

The identification of new genes: gene trapping in transgenic mice.

Ongoing efforts to clone, sequence and map genes in the mouse have far exceeded our ability to define their functional role. The generation of mutations is an important first step towards understanding the function of genes in normal mouse development and physiology. Gene trapping in embryonic stem cells provides an efficient method to identify, clone and mutate genes at random, permitting the functional analysis of new genes in mice.

Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function.

The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection.

Entrapment vectors: a new tool for mammalian genetics.

The use of mouse embryonic stem (ES) cells has facilitated the creation of mouse strains carrying desired genetic alterations. Recently, "entrapment" vectors have been developed to identify and mutate genes active during embryogenesis. These vectors in combination with ES cells may provide an efficient screening strategy to find genes that control mouse development.

RNA polymerase II pauses in vitro, but does not terminate, at discrete sites in promoter-proximal regions on polyomavirus transcription complexes.

Many RNA polymerases stall and/or prematurely terminate transcription nearby the early and late promoters of polyomavirus in vivo during the late phase of productive infection. In this paper we analyzed the RNAs made when these promoter-proximal RNA polymerases were allowed to elongate their nascent chains in vitro on viral transcription complexes isolated from infected cells. RNA was labeled in the presence of a high specific activity of one [alpha-32P]-ribonucleoside triphosphate (rNTP) (less than 1 microM final concentration) and saturating concentrations of the other three rNTPs. Under these conditions, promoter-proximal RNAs of discrete sizes were produced. We show that these discrete RNA species are produced by pausing of RNA polymerase II due to limiting concentrations of one of the rNTPs, and that the positions of the pause sites depend on which rNTP is limiting. This pausing does not result in release of the RNA polymerase or the nascent RNA chain from the transcription complex, as these chains can be further extended when high concentrations of all four rNTPs are supplied. Our results conflict with the interpretations of other investigators who suggest that formation of discrete RNA products, under similar in vitro conditions, reflects authentic termination by RNA polymerase II.

Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells.

A full understanding of the function of genes that control developmental events can be obtained only by a combination of molecular and mutational analysis. One putative developmental gene is the mouse engrailed-like gene En-2, which was isolated by virtue of its extensive homology to Drosophila engrailed, which contributes to the control of segmentation in the developing insect. Our hybridization analysis in situ has revealed that expression of En-2 is restricted to a specific domain of the developing central nervous system from 8 days of development on, indicating a role for the gene in establishing spatial domains in the brain. Unfortunately no En-2 mutations are available to elucidate further its function in development. To this end, we report here the isolation of three pluripotent embryonic stem cell lines in which one copy of the homoeobox-containing gene, En-2, has been altered by homologous recombination.

A gene trap approach in mouse embryonic stem cells: the lacZ reported is activated by splicing, reflects endogenous gene expression, and is mutagenic in mice.

We have confirmed that the gene trap vector pGT4.5 creates spliced fusion transcripts with endogenous genes and prevents the synthesis of normal transcripts at the site of integration. cDNA was prepared to the lacZ fusion transcript in three ES cell lines to recover endogenous exon sequences upstream of lacZ. Each of the clones detected a unique-sized endogenous transcript, as well as the fusion transcript in the ES cell line from which the clone was derived. Sequence analysis of these clones and larger clones isolated from a random-primed cDNA library showed that the splice acceptor was used properly. For two insertions, the expression patterns of the lacZ reporter and the associated endogenous gene were compared in situ at three embryonic stages and were found to be similar. Three gene trap insertions were transmitted into the germ line, and abnormalities were observed with two of the three insertions in the homozygous state. RNA obtained from mice homozygous for the two mutant gene trap insertions was analyzed for normal endogenous transcripts and negligible amounts were detected, indicating that little splicing around the gene trap insertion occurred. This work demonstrates the capacity of the gene trap vector to generate lacZ fusion transcripts, to accurately report endogenous gene expression, and to mutate the endogenous gene at the site of integration.

The gene trap approach in embryonic stem cells: the potential for genetic screens in mice.

The gene trap approach in embryonic stem cells was developed as a means to screen for genes expressed during early postimplantation development in the mouse. We have validated the approach by showing that lacZ from the integrated vector is activated by splicing to endogenous exons and expressed in embryos in patterns that mimic those of the endogenous genes. These insertions can produce developmental defects in homozygous mice. The results indicate that a large screen of gene trap cell lines on the basis of embryonic lacZ expression is feasible and should provide a new source of genes, mouse mutants and mouse strains that express lacZ in particular domains and lineages. The gene trap approach could be extended to a smaller screen for genes based on mutant phenotypes.

Computational prediction of membrane-tethered transcription factors.

BACKGROUND: Sequestration of transcription factors in the membrane is emerging as an important mechanism for the regulation of gene expression. A handful of membrane-spanning transcription factors has been previously identified whose access to the nucleus is regulated by proteolytic cleavage from the membrane. To investigate the existence of other transmembrane transcription factors, we analyzed computationally all proteins in SWISS-PROT/TrEMBL for the combined presence of a DNA-binding domain and a transmembrane segment. RESULTS: Using Pfam hidden Markov models and four transmembrane-prediction programs, we identified with high confidence 76 membrane-spanning transcription factors in SWISS-PROT/TrEMBL. Analysis of the distribution of two proteins predicted by our method, MTJ1 and DMRT2, confirmed their localization to intracellular membrane compartments. Furthermore, elimination of the predicted transmembrane segment led to nuclear localization for each of these proteins. CONCLUSIONS: Our analysis uncovered a wealth of predicted membrane-spanning transcription factors that are structurally and taxonomically diverse, 56 of which lack experimental annotation. Seventy-five of the proteins are modular in structure, suggesting that a single proteolysis may be sufficient to liberate a DNA-binding domain from the membrane. This study provides grounds for investigations into the stimuli and mechanisms that release this intriguing class of transcription factors from membranes.

Rapid sequence analysis of gene trap integrations to generate a resource of insertional mutations in mice.

Gene trapping in murine embryonic stem cells is a proven method for the simultaneous identification and mutation of genes in the mouse. Gene trap vectors are designed to detect insertions within genes through the production of a fusion mRNA transcript, making the identification of the endogenous gene possible by 5' rapid amplification of cDNA ends (RACE). Although the amplification of specific cDNAs can be achieved rapidly, cloning and screening of informative-sized cDNAs has proven to be time consuming. To eliminate the need for cloning, we have developed a method for solid-phase sequencing of 5' RACE products. More than 150 independent gene trap cell lines were analyzed, and sequence information was obtained for every line successfully amplified by RACE. With the vector used in this study, 40% of the cell lines were found to contain properly spliced gene trap events. The remaining lines were either spliced inefficiently or contained deletions of the vector. These results highlight the advantage of sequencing gene trap integrations before further characterization. This work now paves the way for large-scale gene trap screens in mice and should greatly accelerate the functional analysis of the mammalian genome.

Capturing novel mouse genes encoding chromosomal and other nuclear proteins.

The burgeoning wealth of gene sequences contrasts with our ignorance of gene function. One route to assigning function is by determining the sub-cellular location of proteins. We describe the identification of mouse genes encoding proteins that are confined to nuclear compartments by splicing endogeneous gene sequences to a promoterless betageo reporter, using a gene trap approach. Mouse ES (embryonic stem) cell lines were identified that express betageo fusions located within sub-nuclear compartments, including chromosomes, the nucleolus and foci containing splicing factors. The sequences of 11 trapped genes were ascertained, and characterisation of endogenous protein distribution in two cases confirmed the validity of the approach. Three novel proteins concentrated within distinct chromosomal domains were identified, one of which appears to be a serine/threonine kinase. The sequence of a gene whose product co-localises with splicesome components suggests that this protein may be an E3 ubiquitin-protein ligase. The majority of the other genes isolated represent novel genes. This approach is shown to be a powerful tool for identifying genes encoding novel proteins with specific sub-nuclear localisations and exposes our ignorance of the protein composition of the nucleus. Motifs in two of the isolated genes suggest new links between cellular regulatory mechanisms (ubiquitination and phosphorylation) and mRNA splicing and chromosome structure/function.