Ectopic lens induction in fish in response to the murine homeobox gene Six3

Recent findings show an unexpected conservation of genes involved in vertebrate and insect eye development. The Drosophila homeobox gene sine oculis is crucial for eye development. Its murine homologue, Six3 is expressed in the anterior neural plate, a region which is involved in lens induction in Xenopus. To examine whether Six3 participates in the process of eye formation, mouse Six3 was ectopically expressed in fish embryos. The results show that Six3 is sufficient to promote ectopic lens formation in the area of the otic vesicle and that retinal tissue is not a prerequisite for ectopic lens differentiation. Our findings suggest a conserved function for Six3 in metazoan eye development.     

The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos

The pattern of RNA expression of the murine Dlx-2 (Tes-1) homeobox gene is described in embryos ranging in age from E8.5 through E11.5. Dlx-2 is a vertebrate homologue of the Drosophila Distal-less (Dll) gene. Dll expression in the Drosophila embryo is principally limited to the primordia of the brain, head and limbs. Dlx-2 is also expressed principally in the primordia of the forebrain, head and limbs. Within these regions it is expressed in spatially restricted domains. These include two discontinuous regions of the forebrain (basal telencephalon and ventral diencephalon), the branchial arches, facial ectoderm, cranial ganglia and limb ectoderm. Several mouse and human disorders have phenotypes which potentially are the result of mutations in the Dlx genes.     

The ectodermal control in chick limb development: Wnt-7a, Shh, Bmp-2 and Bmp-4 expression and the effect of FGF-4 on gene expression

We have manipulated the chick limb bud by dorsoventrally inverting the ectoderm, by grafting the AER to the dorsal or ventral ectoderm and by insertion of an FGF-4 soaked heparin bead to the mesoderm. After dorso-ventral reversal of the ectoderm, Wnt-7a expression is autonomous from an early stage of limb development in the original dorsal ectoderm. Exogenous FGF-4 causes ectopic Wnt-7a expression and induces ectopic Shh. In addition, exogenous FGF-4 increases the thickness of cartilages and also shortens them, and both Bmp-2 and Bmp-4 may mediate this effect. The ectoderm outside the AER can regulate not only the dorso-ventral polarity of the underlying mesenchyme cells but also the cartilage formation, and both Bmp-2 and Bmp-4 may mediate this control.     

Combinatorial signalling by Xwnt-11 and Xnr3 in the organizer ephithelium

The epithelium of the Spemann organizer plays an important role in embryonic axis formation and transplantation experiments have shown that epithelial organizer cells have potent axis-inducing potential. Known axis-inducing molecules like noggin and chordin are not expressed in the epithelium and cannot account for its inductive properties. Xwnt-11 is expressed in the epithelium but has only poor dorsalizing activity. In an expression screen for genes that are able to functionally cooperate with Xwnt-11 we have identified a cDNA encoding Xenopus nodal-related 3 (Xnr3), a member of the TGF-β family, coexpressed with Xwnt-11 in the organizer epithelium. Xwnt-11 and Xnr3 act highly cooperatively in inducing secondary embryonic axes and dorsalizing ventral mesoderm. Xwnt-11/Xnr3 interfere with BMP signalling without themselves inducing chordin or noggin. The results indicate that induction by the organizer epithelium may result from the combinatorial action of instructive Xnr3 and permissive Xwnt-11 signalling.     

Molecular Analysis of the NF2 Tumor-Suppressor Gene in Schwannomatosis

Patients with multiple schwannomas without vestibular schwannomas have been postulated to compose a distinct subclass of neurofibromatosis (NF), termed "schwannomatosis." To compare the molecular-genetic basis of schwannomatosis with NF2, we examined the NF2 locus in 20 unrelated schwannomatosis patients and their affected relatives. Tumors from these patients frequently harbored typical truncating mutations of the NF2 gene and loss of heterozygosity of the surrounding region of chromosome 22. Surprisingly, unlike patients with NF2, no heterozygous NF2-gene changes were seen in normal tissues. Examination of multiple tumors from the same patient revealed that some schwannomatosis patients are somatic mosaics for NF2-gene changes. By contrast, other individuals, particularly those with a positive family history, appear to have an inherited predisposition to formation of tumors that carry somatic alterations of the NF2 gene. Further work is needed to define the pathogenetics of this unusual disease mechanism.     

Very early and transient vegetal-plate expression of SpKrox1, a Krüppel/Krox gene from Strongylocentrotus purpuratus

All endodermal and mesenchymal cells of the sea urchin embryo descend from the vegetal plate, a thickened epithelium of approximately 50 cells arising at the early blastula stage. Cell types that derive from the vegetal plate are specified conditionally by inductive interactions with underlying micromeres, but the molecular details of vegetal-plate specification remain unresolved. In a search for regulatory proteins that have roles in vegetal-plate specification, a screen was performed to clone Krüppel/Krox-related genes from a Strongylocentrotus purpuratus embryo cDNA library. One newly identified clone, named SpKrox1, contained four zinc fingers and a leucine zipper domain. SpKrox1 expression was low in unfertilized eggs, increased severalfold to the early blastula stage and decreased between the early gastrula and pluteus stages. SpKrox1 mRNA was first seen in macromeres of 16-cell stage embryos and was restricted to cells of the developing vegetal plate thereafter. Vegetal-plate expression corresponded to a ring of cells around the blastopore and overlapped the expression patterns of other genes with potential roles in vegetal plate-specification. As the vegetal-plate cells invaginated into the blastopore, SpKrox1 expression was lost, suggesting that its role was not in endoderm differentiation per se but rather in the initial establishment of the vegetal plate.     

Distinct structural domains in the planarian brain defined by the expression of evolutionarily conserved homeobox genes

 Homeobox genes such as orthodenticle in Drosophila and its mouse homologues, Otx1 and Otx2, are known to be essential for rostral brain development. To investigate the molecular basis of brain evolution, we searched for otd/Otx-related homeobox genes in the planarian Dugesia japonica, and identified two genes, DjotxA and B, whose expression appears to be restricted to the cephalic ganglion (brain). DjotxA was expressed more medially, in the region containing the termini of the visual axons, and in the visual cells, suggesting involvement in establishment of the visual system. DjotxB was expressed in a discrete region just lateral to the DjotxA-positive domain, but not in the more lateral branch structures, which in turn are characterized by the expression of Djotp, a planarian homeobox gene related to mouse Orthopedia (Otp). In transverse sections of planarians, DjotxA and B expression were observed only at the anterior ends of the stumps, corresponding to the regional pattern of the regenerating brain. Our findings suggest that the planarian brain is composed of structurally distinct and functionally diverse domains which are defined by the discrete expression of the three evolutionarily conserved homeobox genes. Received: 17 June 1998 / Accepted: 20 August 1998     

Morphological changes in transgenic poplar induced by expression of the rice homeobox gene OSH1

Genetically transformed lombardy poplar (Po-pulus nigra L. var. italica Koehne) plants were regenerated after co-cultivation of stem segments with Agrobacterium tumefaciens strain LBA4404 that harbored a binary vector which included the rice gene for a homeodomain protein (OSH1) and a gene for neomycin phosphotransferase. The expression of the OSH1 gene under control of the cauliflower mosaic virus 35S promoter induced morphological abnormalities in the leaves and stems of the newly generated transgenic poplar plants. This result suggests that OSH1 can function as a regulator of morphogenesis in transgenic poplar, as it does in transgenic rice, Arabidopsis, and tobacco plants. Received: 16 October 1998 / Revision received: 27 November 1998 / Accepted: 12 December 1998     

Effect of F-2 and T-2 fusariotoxins on experimental Cryptosporidium baileyi infection in chickens

The course of Cryptosporidium baileyi infection in chickens fed with different doses of fusariotoxins was compared with that of control groups. F-2 toxin levels of 0.187–1.5 mg kg⁻¹ and T-2 toxin levels of 0.187–6.0 mg kg⁻¹ were investigated. The experimental amimals were orally infected with 6 × 10⁵ C. baileyi oocysts at 1 week of age. Total daily oocyst output was monitored by a quantitative method. Acquired immunity was tested at the age of 4 weeks, by ELISA and by a challenge infection with an equal number of oocysts, upon recovery from the primary infection. The results show that in chickens kept on the lower doses of F-2 and T-2 toxins, the parasite infection ran a similar course to that in the control groups, and the animals became resistant to re-infection. However, when higher doses (2.0–6.0 mg kg⁻¹) of T-2 toxin were used, a depression of weight gain was observed with some other physiological parameters (PCV, weight of bursa, weight of thymus, skin thickness in PHA-P skin test) also indicating toxic effect and, simultaneously, the oocyst output decreased significantly and the patent period was slightly prolonged. Although certain modifications of the immune response could be revealed, the chickens became resistant to re-infection. Only early (1 week of age) parasite infection and 6 mg kg⁻¹ T-2 toxin in the feed significantly depressed body weight gain and immunity.     

A Xenopus c-kit-related receptor tyrosine kinase expressed in migrating stem cells of the lateral line system

The mammalian c-kit receptor tyrosine kinase gene is required during embryogenesis for the survival and/or proliferation of three migrating stem cell populations: primordial germ cells, haematopoietic stem cells and neural crest-derived melanoblasts. We have cloned a Xenopus gene, XKrkl, whose closest relative is c-kit. Differences in the expression pattern suggest that XKrkl is not the Xenopus homologue of c-kit; however, it is expressed in a migrating stem cell population, the precursor cells for the mechanosensory lateral line system. XKrkl is the first reported marker for lateral line stem cells.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

Effect of N-terminal deletions on the activity of pokeweed antiviral protein expressed in E. coli

Pokeweed antiviral protein (PAP) from Phytolacca americana is a highly specific N-glycosidase removing adenine residues (A⁴³²⁴ in 28S rRNA and A²⁶⁶⁰ in 23S rRNA) from intact ribosomes of both eukaryotes and prokaryotes. Due to the ribosome impairing activity the gene coding for mature PAP has not been expressed so far in bacteria whereas the full-length gene (coding for the mature 262 amino acids plus two signal peptides of 22 and 29 amino acids at both N- and C-termini, respectively) has been expressed in Escherichia coli. In order to determine: 1) the size of the N-terminal region of PAP which is required for toxicity to E. coli; and 2) the location of the putative enzymatic active site of PAP, 5′-terminal progressive deletion of the PAP full-length gene was carried out and the truncated forms of the gene were cloned in a vector containing a strong constitutive promoter and a consensus Shine-Dalgarno ribosome binding site. The ribosome inactivation or toxicity of the PAP is used as a phenotype characterized by the absence of E. coli colonies, while the mutation of PAP open reading frames in the small number of survived clones is used as an indicator of the toxicity to E. coli cells. Results showed that the native full-length PAP gene was highly expressed and was not toxic to E. coli cells although in vitro ribosome inactivating activity assay indicated it was active. However, all of the N-terminal truncated forms (removal of seven to 107 codons) of the PAP gene were toxic to E. coli cells and were mutated into either out of frame, early termination codon or inactive form of PAP (i.e., clone PAPΔ107). Deletion of more than 123 codons restored the correct gene sequence but resulted in the loss of the antiviral and ribosome inactivating activities and by the formation of a large number of clones. These results suggest that full-length PAP (with N- and C-terminal extensions) might be an inactive form of the enzyme in vivo presumably by inclusion body formation or other unknown mechanisms and is not toxic to E. coli cells. However, it is activated by at least seven codon deletions at the N-terminus. Deletions from seven through to 107 amino acids were lethal to the cells and only mutated forms (inactive) of the gene were obtained. But deletion of more than 123 amino acids resulted in the loss of enzymatic activity and made it possible to express the correct PAP gene in E. coli. Because deletion of Tyr94 and Va195, which are involved in the binding of the target adenine base, did not abolish the activity of PAP, it is concluded that the location previously proposed for PAP enzymatic active site should be reassessed.