The role of Trp53 in the transcriptional response to ionizing radiation in the developing brain.

Brain formation results from a series of well-timed consecutive waves of cellular proliferation, migration and differentiation. Acute irradiation during pregnancy selectively interferes with these events to result in malformations such as microcephaly, reduced cortical thickness and mental retardation. In the present study we performed a straight-through cDNA-microarray analysis of the developing mouse brain at embryonic day E13, 3 h after in utero exposure to 50 cGy X-radiation. This dataset was used as an indication of genes involved in different pathways that are activated upon early radiation exposure, and for further evaluation using quantitative PCR (qPCR). Microarray and qPCR data revealed that the main activated pathways in irradiated wild-type embryos are involved in the regulation of a p53-mediated pathway that may lead to cell cycle delay/arrest and increased levels of apoptosis. To define whether the transcriptional radiation response was solely p53 mediated, we analysed the expression of cell cycle regulating genes in a Trp53 null mutant. The modulated expression of cell cycle regulating genes such as cyclins and Cdk genes indicated the induction of a cell cycle arrest, without evidence for the onset of apoptosis. Additional gene-expression studies have shown that various E2F transcription factors may be involved in this event. Together, these results provide a detailed view of the different p53-related mechanisms that are triggered in response to ionizing radiation in the developing brain.

Expression of Frzb-1 during chick development.

We cloned the chick homolog of Xenopus and mouse Frzb-1, a secreted Wnt antagonist and performed in situ hybridizations to determine the pattern of cFrzb-1 expression in the developing chick embryo. At early stages, cFrzb-1 transcripts are located exclusively in the ectodermal layer corresponding to the neural plate. The labelling continues in the neural tube, but is always excluded from the floor plate. cFrzb-1 mRNA is expressed by migrating cephalic and truncal neural crest cells. Later, cFrzb-1 transcripts are found in a subset of neural crest derivatives such as cephalic cartilage, nerves and spinal ganglia. In addition to ectodermal derivatives, cFrzb-1 transcripts were also observed in mesodermal derivatives such as vertebral and limb cartilage, the adrenal cortex, the gonads, and a subpopulation of blood cells.

Transplacental delivery of the Wnt antagonist Frzb1 inhibits development of caudal paraxial mesoderm and skeletal myogenesis in mouse embryos.

Axial structures (neural tube/notochord) and surface ectoderm activate myogenesis in the mouse embryo; their action can be reproduced, at least in part, by several molecules such as Sonic hedgehog and Wnts. Recently, soluble Wnt antagonists have been identified. Among those examined only Frzb1 was found to be expressed in the presomitic mesoderm and newly formed somites and thus its possible role in regulating myogenesis was investigated in detail. When presomitic mesoderm or newly formed somites were cultured with axial structures and surface ectoderm on a feeder layer of C3H10T1/2 cells expressing Frzb1, myogenesis was abolished or severely reduced in presomitic mesoderm and the three most recently formed somites. In contrast, no effect was observed on more mature somites. Inhibition of myogenesis did not appear to be associated with increased cell death since the final number of cells in the explants grown in the presence of Frzb1 was only slightly reduced in comparison with controls. In order to examine the possible function of Frzb1 in vivo, we developed a method based on the overexpression of the soluble antagonist by transient transfection of WOP cells with a Frzb1 expression vector and injection of transfected cells into the placenta of pregnant females before the onset of maternofoetal circulation. Frzb1, secreted by WOP cells, accumulated in the embryo and caused a marked reduction in size of caudal structures. Myogenesis was strongly reduced and, in the most severe cases, abolished. This was not due to a generalized toxic effect since only several genes downstream of the Wnt signaling pathway such as En1, Noggin and Myf5 were downregulated; in contrast, Pax3 and Mox1 expression levels were not affected even in embryos exhibiting the most severe phenotypes. Taken together, these results suggest that Wnt signals may act by regulating both myogenic commitment and expansion of committed cells in the mouse mesoderm.

The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals.

Embryological and genetic evidence indicates that the vertebrate head is induced by a different set of signals from those that organize trunk-tail development. The gene cerberus encodes a secreted protein that is expressed in anterior endoderm and has the unique property of inducing ectopic heads in the absence of trunk structures. Here we show that the cerberus protein functions as a multivalent growth-factor antagonist in the extracellular space: it binds to Nodal, BMP and Wnt proteins via independent sites. The expression of cerberus during gastrulation is activated by earlier nodal-related signals in endoderm and by Spemann-organizer factors that repress signalling by BMP and Wnt. In order for the head territory to form, we propose that signals involved in trunk development, such as those involving BMP, Wnt and Nodal proteins, must be inhibited in rostral regions.

The prechordal midline of the chondrocranium is defective in Goosecoid-1 mouse mutants.

Gsc-1 expression marks cells with Spemann organizer, or axis-inducing, activity in the vertebrate gastrula. Gsc-1 knockouts, however, did not display phenotypes related to the early phase of expression. In this paper, additional phenotypes for the Gsc-1 mouse mutant are presented. Examination of the base of the cranium in the dorsal view revealed fusions and deletions in the midline of the prechordal chondrocranium. These defects were correlated with the sites of expression of Gsc-1 in the prechordal plate/foregut endoderm in the day 7.5/8.5 embryo. Gsc-1 expression in proximal limb buds was correlated with malformations of the shoulder and hip articulations. In addition, ribs in the seventh cervical vertebra were observed with low penetrance. The role of Gsc-1 during gastrulation and axial development is discussed in relation to possible compensatory interactions with other genes such as HNF-3beta and the recently identified Gsc-2 and Gsc-3 genes.

Vertebrate head induction by anterior primitive endoderm.

In vertebrates the antero-posterior organization of the embryonic body axis is thought to result from the activity of two separate centers, the head organizer and the trunk organizer, as operationally defined by Spemann in the 1920s. Current molecular studies have supported the existence of a trunk organizer activity while the presence of a distinct head inducing center has remained elusive. Mainly based on analyses of headless mutants in mice, it has been proposed that the anterior axial mesoderm plays a determining role in head induction. Recent gain- and loss-of-function studies in various organisms, however, provide compelling evidence that a largely ignored region, the anterior primitive endoderm, specifies rostral identity. In this review we discuss the emerging concept that the anterior primitive endoderm, rather than the prechordal plate mesoderm, induces head development in the vertebrate embryo.

Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann organizer.

Frzb-1 is a secreted protein containing a domain similar to the putative Wnt-binding region of the frizzled family of transmembrane receptors. Frzb-1 is widely expressed in adult mammalian tissues. In the Xenopus gastrula, it is expressed and regulated as a typical Spemann organizer component. Injection of frzb-1 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. Frzb-1 antagonizes the effects of Xwnt-8 ectopic expression in a non-cell-autonomous manner. Cultured cells transfected with a membrane-tethered form of Wnt-1 bind epitope-tagged Frzb-1 in the 10(-10) M range. The results strengthen the view that the Spemann organizer is a source of secreted inhibitory factors.

Cerberus-like is a secreted factor with neutralizing activity expressed in the anterior primitive endoderm of the mouse gastrula.

We report the isolation of mouse cerberus-like (cer-l), a gene encoding a novel secreted protein that is specifically expressed in the anterior visceral endoderm during early gastrulation. Expression in the primitive endoderm starts before the appearance of the primitive streak and lasts until the head-fold stage. In later stages, a second region of expression is found in newly formed somites. Mouse cer-l shares some sequence similarity with Xenopus cerberus (Xcer). In Xenopus assays cer-l, like Xcer, mRNA acts as a potent neuralizing factor that induces forebrain markers and endoderm, but is unable to induce ectopic head-like structures as Xcer does. In addition to cer-l, anterior visceral endoderm was found to express the transcription factors Lim1, goosecoid and HNF-3beta that are also present in trunk organizer cells. A model of how head and trunk development might be regulated is discussed. Given its neuralizing activity, the secreted protein Cer-l is a candidate for mediating inductive activities of anterior visceral endoderm.

A family of novel myb-related genes from the resurrection plant Craterostigma plantagineum are specifically expressed in callus and roots in response to ABA or desiccation.

A cDNA and two genomic clones comprising highly similar genes that encode a protein with a Myb-related DNA-binding domain were isolated from the resurrection plant Craterostigma plantagineum. The structure of cpm5 and cpm10 (Craterostigma plantagineum myb) genes consists of three putative exons encoding a protein of 36.6 kDa. The cDNA of cpm7 encodes a closely related protein of 36.8 kDa. The canonical Myb domain present in transcriptional activators of yeast, animals and plants was localized in the amino terminus of deduced Cpm5, Cpm7 and Cpm10 proteins and corresponds to the two Myb repeats found in plants. The Myb domain of Cpm deduced proteins and a short stretch of amino acids adjacent to this region are closely related to a myb gene from Arabidopsis thaliana which is expressed in response to osmotic stress and ABA. The rest of the deduced protein has no similarity to other reported sequences. The myb-related genes in the Craterostigma genome comprise a small gene family of 6-8 members as estimated by hybridization with a bona fide Myb domain probe. Northern blot experiments showed specific expression of cpm10 in undifferentiated callus tissue up-modulated by ABA and expression of cpm7 mRNA in roots up-regulated by dehydration.

iroquois: a prepattern gene that controls the formation of bristles on the thorax of Drosophila.

We have isolated a Drosophila mutant where the lateral parts of the notum are completely naked, leaving unaffected a median stripe of hairs. This mutation, iroquois (iro), defines a new gene which maps at 69D. We show that, in the presumptive lateral notum of mutant discs, sense organ precursor cells fail to form and the proneural gene scute is not expressed. The expression of a reporter gene inserted near iro suggests that iro itself is massively expressed in this region of the disc. We propose that iro is a prepattern gene essential to activate the expression of scute in the regions of the disc that will form the lateral notum.

The determination of sense organs in Drosophila: a search for interacting genes.

The determination of sense organs in Drosophila requires the concerted action of a battery of genes, several of which have been identified. Previous experiments revealed that flies doubly heterozygous for mutations in two of these genes have a reduced number of sense organs, suggesting the existence of a direct interaction between the corresponding genes and/or their products. We have now used this observation to search for mutations in additional genes that would show similar interactions. We have detected 10 recessive mutations that show a dominant reduction in the number of bristles when simultaneously heterozygous for either Df(2)J27 or Df(4)M62f. Among these mutations, 3 are homozygous viable and show striking defects in their bristle patterns, confirming that the genes thus identified play a role in the patterning of sense organs. We conclude that the "gene dose titration" method (Botas et al., 1982) is an efficient method for identifying interacting genes involved in a common process, provided one can identify a well-defined phenotype to look at, and at least one mutation that alters the process. Our experience suggests that its efficiency should be substantially improved by the use of insertional mutagenesis.