Ulrich Rüther

Serum response factor is essential for mesoderm formation during mouse embryogenesis

The transcription factor serum response factor (SRF), a phylogenetically conserved nuclear protein, mediates the rapid transcriptional response to extracellular stimuli, e.g. growth and differentiation signals. DNA–protein complexes containing SRF or its homologues function as nuclear targets of the Ras/MAPK signalling network, thereby directing gene activities associated with processes as diverse as pheromone signalling, cell‐cycle progression (transitions G0–G1 and G2–M), neuronal synaptic transmission and muscle cell differentiation. So far, the activity of mammalian SRF has been studied exclusively in cultured cells. To study SRF function in a multicellular organism we generated an Srf null allele in mice. SRF‐deficient embryos (Srf−/−) have a severe gastrulation defect and do not develop to term. They consist of misfolded ectodermal and endodermal cell layers, do not form a primitive streak or any detectable mesodermal cells and fail to express the developmental marker genes Bra (T), Bmp‐2/4 and Shh. Activation of the SRF‐regulated immediate early genes Egr‐1 and c‐fos, as well as the α‐Actin gene, is severely impaired. Our study identifies SRF as a new and essential regulator of mammalian mesoderm formation. We therefore suggest that in mammals Ras/MAPK signalling contributes to mesoderm induction, as is the case in amphibia.

Evidence for genetic control of Sonic hedgehog by Gli3 in mouse limb development

Sonic hedgehog (Shh) expression in the developing limb is associated with the zone of polarising activity (ZPA), and both are restricted to the posterior part of the limb bud. We show that the expression patterns of Shh and Gli3, a member of the Gli-family believed to function in transcriptional control, appear to be mutually exclusive in limb buds of mouse embryos. In the polydactyly mouse mutant extra toes (Xt), possessing a null mutation of Gli3, Shh is additionally expressed in the anterior region of the limb bud. The transcript of Ptc, the putative receptor for Shh protein, can be detected anteriorly as well. Other genes known to be involved in limb outgrowth and patterning, like Fibroblast growth factor (Fgf), Bone morphogenetic protein (Bmp), and Hoxd are misexpressed in relation to the ectopic Shh expression domain in Xt limb buds. This data suggest that Gli3 is a regulator of Shh expression in mouse limb development.

Cloning of Fatso (Fto), a novel gene deleted by the Fused toes (Ft) mouse mutation

Abstract. The Fused toes (Ft) mouse mutation was created by insertional mutagenesis, resulting in the deletion of several hundred kb of genomic sequences of mouse Chromosome (Chr) 8. Mice heterozygous for the Ft mutation are characterized by partial syndactyly of forelimbs and massive thymic hyperplasia indicating that programmed cell death is affected. Homozygous Ft/Ft embryos die at midgestation and show severe malformations of craniofacial structures. Furthermore, establishment of left-right asymmetry is random. Here we report on the positional cloning of a novel gene by exon trap analysis of a genomic clone encoding wild-type sequences corresponding to parts of the deletion in Ft mutants. RT-PCR experiments demonstrated that the newly identified gene, Fatso (Fto), is expressed throughout embryonic development. Wide expression was also found in tissues of adult mice. We show that expression of Fto is completely absent in mouse embryonic fibroblasts homozygous for the Ft mutation. In addition, we isolated the full-length cDNA which encodes a putative 58-kDa protein showing no similarities to known proteins or protein motifs. The expression data of Fto define it as a candidate gene involved in processes such as programmed cell death, craniofacial development, and establishment of left-right asymmetry.

Mouse Dac, a novel nuclear factor with homology to Drosophila dachshund shows a dynamic expression in the neural crest, the eye, the neocortex, and the limb bud

Dac is a novel nuclear factor in mouse and humans that shares homology with Drosophila dachshund (dac). Alignment with available sequences defines a conserved box of 117 amino acids that shares weak homology with the proto‐oncogene Ski and Sno. Dac expression is found in various neuroectodermal and mesenchymal tissues. At early developmental stages Dac is expressed in lateral mesoderm and in neural crest cells. In the neural plate/tube Dac expression is initially seen in the prosencephalon and gets gradually restricted to the presumptive neocortex and the distal portion of the outgrowing optic vesicle. Furthermore, Dac transcripts are detected in the mesenchyme underlying the Apical Ectodermal Ridge (AER) of the extending limb bud, the dorsal root ganglia and chain ganglia, and the mesenchyme of the growing genitalia. Dac expression in the Gli 3 mutant extra toes (Xt/Xt) shows little difference compared to the expression in wild‐type limb buds. In contrast, a significant expansion of Dac expression are observed in the anterior mesenchyme of the limb buds of hemimelic extra toes (Hx/+) mice. FISH analysis reveals that human DAC maps to chromosome 13q22.3–23 and further fine‐mapping defined a position of the DAC gene at 54cM or 13q21.1, a locus that associates with mental retardation and skeletal abnormalities. Dev Dyn 1999;214:66–80.

Insertional mutagenesis in transgenic mice

Increasing numbers of transgenic mouse lines have resulted in several dozens of mutants created by insertional mutagenesis. The advantages of different vector systems and the problems associated with the analysis of mutations and the cloning of the affected genes are discussed in this review.

EXPRESSION PROFILE OF GLI FAMILY MEMBERS AND SHH IN NORMAL AND MUTANT MOUSE LIMB DEVELOPMENT

Gli genes represent a small family, encoding zinc‐finger proteins of the Krüppel‐type. The family consists of Gli(1), Gli2, and Gli3, all of which are expressed in the developing mouse limb bud. To assess the role of the Gli family and Sonic hedgehog (Shh) in mouse limb development, we compared the expression domains of all three Gli genes and of Shh. Although each Gli gene has its own distinct expression pattern in limb buds, at 10.5–11.5 dpc all three genes were found not to be expressed in the posterior region, the presumptive Shh expression domain. This transient mutually exclusive expression suggested a potential interaction between Gli genes and Shh. To address this matter, we analysed the expression of Gli genes and Shh in two polydactyly mouse mutants, Extra toes (Xt) and Hemimelic‐extra toes (Hx) which express Shh ectopically in the anterior region of the limb field. Since Xt mice lack Gli3 expression, the ectopic Shh expression is genetically linked to the absence of Gli3. In Hx mice we found a down‐regulation of Gli3 in the anterior region of the limb bud. In both mutants Gli2 expression pattern was not altered, whereas Gli1 expression was anteriorly up‐regulated adjacent to the ectopic Shh domain. These results strongly suggest a positive regulation of Gli1 by Shh and a negative interaction between Shh and Gli3. Dev. Dyn. 1998;211:88–96.© 1998 Wiley‐Liss, Inc.

The expression pattern of nodal and lefty in the mouse mutant Ft suggests a function in the establishment of handedness

We have investigated the expression of left/right (L/R) asymmetry markers, nodal and lefty, in the situs inversus mouse mutant Fused toes (Ft). Both genes exhibited bilateral expression in the lateral plate mesoderm (LPM) at developmental stages whereas in wildtype embryos these genes were found to be expressed exclusively in the left LPM. Inspection of tail location and primitive heart tube looping, structures known to be handed in their orientation, documented a random orientation of these structures. Crossing of the Ft mutation into a different genetic background resulted in a strong reduction of this random orientation. Although the major fraction of these individuals still displayed nodal and lefty on both sides of the LPM, expression was almost always found to be weaker in the right LPM. These results suggest that the establishment of asymmetry is independent of nodal or lefty signals. However, handed asymmetry, which means consistent L/R differences, such as the dextral looping of the primitive heart tube or the right-oriented tail, is directed by differences in the L/R expression pattern of these two genes.

Gli genes and limb development.

Abstract During development of the limb Shh plays a key role as a mediator of zone of polarizing activity (ZPA). However, the molecular mechanisms by which Shh directs anterior/posterior patterning in the limb remain unknown. Members of the Gli gene family encode zinc-finger transcription factors and represent likely candidates for being regulators of Shh target genes. In this review we would like to summarize the current knowledge on expression and function of Gli genes in limb development.

Ftl, a novel gene related to ubiquitin-conjugating enzymes, is deleted in the Fused toes mouse mutation

The dominant mouse mutation Fused toes is characterized by partial syndactyly of the limbs and thymic hyperplasia. Both morphological abnormalities were shown to be related to impaired regulation of programmed cell death. Ft/Ft embryos die in midgestation showing severe malformations of fore- and mid-brain as well as randomized situs. In Ft mice a large chromosomal deletion (about 300 kb) occurred after insertional mutagenesis. In this report we describe the identification of the first gene that has been mutated by Fused toes. The expression of the novel gene Ftl is reduced in Ft/+ mice and completely absent in Ft/Ft embryos. Analysis of the Ftl cDNA revealed an open reading frame that could code for a 32-kDa protein with similarities to ubiquitin-conjugating enzymes. Ftl transcripts with alternative 5′ UTR sequences as well as differential usage of polyadenylation sites were found. Interestingly, the 3’ parts of the longest Ftl transcripts are identical to the reverse complement of the 3′-most sequences of the Rb-related pl30 gene. Both genes are transcribed in opposite directions and overlap in their 3′ UTRs. Despite the close linkage, pl30 expression appeared not to be affected by the Ft mutation. In wild type mice, Ftl expression levels were found to be high in brain, kidney, and testes and detectable in all other adult organs and throughout embryonic development. Finally, we show that Ftl is conserved among mammals and identify the human homolog.

The mouse mutation Pdn (Polydactyly Nagoya) is caused by the integration of a retrotransposon into the Gli3 gene

Abstract. Mutations in the Gli3 gene are associated with a preaxial polydactyly in several mouse mutants such as extra-toes (Xt). The semidominant mouse mutant Pdn (Polydactyly Nagoya) is characterized by a mild polydactyly on the anterior side of the hind limbs. Homozygous Pdn mice show a more severe polydactyly, additional skeletal malformations, and abnormal brain development. Herein, we report the molecular basis of Pdn, being the integration of an Early Transposon (ETn) into the Gli3 gene. As a consequence, several novel Gli3 mRNAs are generated by alternatively spliced transcripts.