Fine-tuning of cell signaling by glypicans.

Signaling peptides of the extracellular environment regulate cell biological processes underlying embryonic development, tissue homeostasis, and pathophysiology. The heparan sulphate proteoglycans, glypicans, have evolved as essential modulators of key regulatory proteins such as Wnt, Bmp, Fgf, and Shh. By acting on signal spreading and receptor activation, glypicans can control signal read-out and fate in targeted cells. Genetic and embryological studies have highlighted that glypicans act in a temporal and spatially regulated manner to modulate distinct cellular events. However, alterations of glypican function underlie human congenital malformations and cancer. Recent reports are starting to reveal their mechanism of action and how they can ensure tight modulation of cell signaling.

Expression of cripto, a novel gene of the epidermal growth factor gene family, leads to in vitro transformation of a normal mouse mammary epithelial cell line.

cripto is a gene encoding an epidermal growth factor-related protein that is expressed in undifferentiated embryonal carcinoma cells. To ascertain if cripto is capable of functioning as a transforming gene, a full-length human cripto complementary DNA under the transcriptional control of the Rous sarcoma virus long terminal repeat has been cotransfected with the selectable pSV2neo marker plasmid into immortalized mouse NOG-8 mammary epithelial cells. Several neomycin-resistant clones were isolated that express high levels of a specific cripto 4.5-kilobase mRNA transcript and possess multiple copies of cripto plasmid DNA. NOG-8 cells that express cripto are able to clone in soft agar and exhibit an approximately 3-fold increase in their anchorage-dependent growth in serum-free medium as compared to the neo-transfected NOG-8 cells. However, none of the cripto-expressing NOG-8 clones are able to form tumors in nude mice.

A GR-motif functions in nuclear accumulation of the large FGF-2 isoforms and interferes with mitogenic signalling.

Nuclear translocation has been documented for members of the fibroblast growth factor (FGF) family in addition to their roles as extra-cellular signalling molecules. Fusing different parts of the chicken FGF-2 open reading frame to pyruvate kinase shows that direct nuclear accumulation is mediated by the amino-termini of the two leucine initiated FGF-2 isoforms (Leu-isoforms; 21.5 and 20.0 kDa). An evolutionarily conserved glycine-arginine (GR)-motif is present in the 21.5 kDa Leu-isoform and a shorter GR-repeat in the 20.0 kDa Leu-isoform, whereas no such repeats are present in the 18.5 kDa FGF-2 isoform (Met-isoform). Expression in NIH3T3 fibroblasts shows that the 21.5 kDa Leu-isoform is predominantly nuclear, whereas the Met-isoform is predominantly cytoplasmic. Most importantly, insertion of the GR-motif into the Met-isoform results in a protein with characteristics similar to the Leu-isoforms, as shown by nuclear accumulation of the chimeric MGR-protein. Furthermore, only NIH3T3 fibroblasts expressing the Met-isoform proliferate under serum starvation conditions, whereas cells expressing either the MGR- or Leu-isoforms stay growth arrested. These studies show that the GR-signal mediates nuclear translocation of endogenous Leu-isoforms and blocks their mitogenic activity.

Nucleo-cytoplasmic translocation and secretion of fibroblast growth factor-2 during avian gastrulation.

The expression and distribution of the fibroblast growth factor-2 (FGF-2 or bFGF) proteins during early avian embryogenesis has been analysed in detail. Three FGF-2 protein isoforms of 18.5, 20.0 and 21.5 kDa are expressed during gastrulation of chicken embryos. Using whole mount immunohistochemistry, these proteins were found to be predominantly nuclear in prestreak blastodiscs during mesoderm induction. Distribution of positive cells in the epiblast was mosaic, whereas all cells of the forming hypoblast expressed the FGF-2 proteins. During primitive streak formation, the proteins started to translocate to the cytoplasm in epiblast cells but remained nuclear in the hypoblast. The FGF-2 proteins became predominantly cytoplasmic in all cells during the subsequent developmental stages. Their highest levels were detected in endodermal cells underlying Hensen's node and the newly formed notochord, the dorsal apex of all epiblast cells and, most interestingly, in the extra-cellular basal lamina separating the epiblast from newly formed mesoderm. Heparin and suramin treatment of these advanced embryos (stage 4) revealed a dose-dependent inhibition on the regression of Hensen's node and formation of mesodermal derivatives such as somites. The results are discussed with respect to current models on FGF-mediated functions during vertebrate mesoderm induction and regionalization.

The CpG island in the 5' region of the G6PD gene of man and mouse.

The nucleotide (nt) sequence of the entire CpG island in the 5' region of the human glucose-6-phosphate dehydrogenase-encoding gene (G6PD) and of the corresponding region in mouse was determined. In comparison to the human gene, the 5' region of the mouse G6PD gene has highly reduced G + C and CpG dinucleotide content, but maintains the functional features of a CpG island, as it is differentially methylated on the active vs. the inactive X chromosome. In addition to the expected conservation of exons, nt sequence comparison showed that several boxes are highly conserved between the two species in the 5' flanking DNA and in the first intron. Moreover, the conservation of the position of most CpG dinucleotides in the promoter region and in one of the upstream boxes, at about -900, gives support to the hypothesis that, in each island, specific CpGs play a major role in the regulation of gene expression.

Analysis of intrinsic pH, soluble solids and buffer effect of non-alcoholic drinks.

The aim of the present study was to determine in vitro the erosive capacity of different common drinks employing intrinsic pH, soluble solids and buffer effect at pH 5.5 and pH 7.0 as end-points. Fifty- two drinks of mass consumption were selected and grouped according to type into 3 groups: 1: Juices (n = 23); 2: soy beverages (n = 18); 3: carbonated drinks (n = 11). The mean and standard deviation were calculated for each drink and for each group of drinks. The statistical analysis of the data was performed by Analysis of Variance (ANOVA) and Tukey's test for multiple comparisons. Statistical significance was set at p < or = 0.05. The data showed that the 3 groups exhibited initial acid pH values. Natural juices had the greatest buffer effect. The soluble solids, expressed as Brix Grades, showed statistically significant differences between the regular and light forms of carbonated and soy beverages. The light and regular forms of the drinks included in the study failed to show statistically significant differences at pH 5.5 and pH 7.0.

Effect of self-brushing with acidulated phosphate fluoride (pH 5.6) on dental caries in children.

The aim of this work was to study the effect of a preventive program on the dental plaque and on the caries incidence in school children. The program comprised weekly supervised self brushing with acidulated phosphate fluoride (APF) gel (pH 5.6; concentration: 4520 ppm of ion F-). The program involved 240 children of 1st., 3rd and 5th grade of a primary school in the City of Buenos Aires (Argentina). The population was divided in 2 groups: A (experimental) and B (control). Ninety children from both groups (45 experimental and 45 control) were submitted to baseline clinical examination (DMFT and plaque index) and microbiological analysis (total streptococci, St mutans and St. mutans and St. mutans percentage). Group A was then submitted to a preventive program which included self brushing with APF gel (4520 ppm of ion F-). The 90-children sample was monitored after 1 and 2 years of program. Results were statistically processed and they revealed the following: a--DMFT was significantly greater in the control group than in the experimental group after 1 and 2 years of program; b--an 81.43% reduction in caries increment rate at the end of the 2-years program in the experimental group as compared to the control group; c--a rise in the number of colonies of total streptococci and of St. Mutans; d--a reduction in the % of St. mutans in the total streptococci flora in the plaque of children in the experimental group; e--the presence of St. mutans colonies featuring a rough surface; f--the effectiveness of the program in the modifying the profile of the diagnosed dental pathology.

Enhanced neuronal Met signalling levels in ALS mice delay disease onset.

Signalling by receptor tyrosine kinases (RTKs) coordinates basic cellular processes during development and in adulthood. Whereas aberrant RTK signalling can lead to cancer, reactivation of RTKs is often found following stress or cell damage. This has led to the common belief that RTKs can counteract degenerative processes and so strategies to exploit them for therapy have been extensively explored. An understanding of how RTK stimuli act at cellular levels is needed, however, to evaluate their mechanism of therapeutic action. In this study, we genetically explored the biological and functional significance of enhanced signalling by the Met RTK in neurons, in the context of a neurodegenerative disease. Conditional met-transgenic mice, namely Rosa26(LacZ-stop-Met), have been engineered to trigger increased Met signalling in a temporal and tissue-specific regulated manner. Enhancing Met levels in neurons does not affect either motor neuron (MN) development or maintenance. In contrast, increased neuronal Met in amyotrophic lateral sclerosis (ALS) mice prolongs life span, retards MN loss, and ameliorates motor performance, by selectively delaying disease onset. Thus, our studies highlight the properties of RTKs to counteract toxic signals in a disease characterized by dysfunction of multiple cell types by acting in MNs. Moreover, they emphasize the relevance of genetically assessing the effectiveness of agents targeting neurons during ALS evolution.

Cell-type-specific nuclear translocation of fibroblast growth factor-2 isoforms during chicken kidney and limb morphogenesis.

Analysis of the fibroblast growth factor-2 (FGF-2 or bFGF) proteins during chicken embryonic pattern formation and organogenesis revealed that three isoforms (18.5, 20.0, and 21.5 kDa) were synthesized by alternative translation initiation from one coding region. A highly specific antiserum was raised and used for studying the temporal and spatial distribution of the FGF-2 isoforms during chicken embryogenesis. The distribution of FGF-2 proteins during limb pattern formation has been unraveled. Their presence in both ectodermal and mesenchymal cells is consistent with an involvement in regulating the balance of growth and differentiation. High levels of FGF-2 proteins were furthermore detected in all epithelial cells of the developing kidney from the pronephric stage onward. The proteins were in general predominantly cytoplasmic, but a specific subpopulation of limb mesenchymal cells and kidney epithelial cells (podocytes) showed a striking nuclear localization. Nuclear translocation of the FGF-2 proteins occurred in differentiating podocytes of meso- and metanephric glomeruli and was maintained in adult kidneys. These results, in contrast to previous in vitro studies, revealed that nuclear accumulation of FGF-2 proteins is restricted to few specific cells during embryogenesis.

FGF signalling is required for differentiation-induced cytoskeletal reorganisation and formation of actin-based processes by podocytes.

To examine the potential role of fibroblast growth factor (FGF) signalling during cell differentiation, we used conditionally immortalised podocyte cells isolated from kidneys of Fgf2 mutant and wild-type mice. Wild-type mouse podocyte cells upregulate FGF2 expression when differentiating in culture, as do maturing podocytes in vivo. Differentiating wild-type mouse podocyte cells undergo an epithelial to mesenchymal-like transition, reorganise their actin cytoskeleton and extend actin-based cellular processes; all of these activities are similar to the activity of podocytes in vivo. Molecular analysis of Fgf2 mutant mouse podocyte cells reveals a general disruption of FGF signalling as expression of Fgf7 and Fgf10 are also downregulated. These FGF mutant mouse podocyte cells in culture fail to activate mesenchymal markers and their post-mitotic differentiation is blocked. Furthermore, mutant mouse podocyte cells in culture fail to reorganise their actin cytoskeleton and form actin-based cellular processes. These studies show that FGF signalling is required by cultured podocytes to undergo the epithelial to mesenchymal-like changes necessary for terminal differentiation. Together with other studies, these results point to a general role for FGF signalling in regulating cell differentiation and formation of actin-based cellular processes during morphogenesis.

Expression of the G6PD locus on the human X chromosome is associated with demethylation of three CpG islands within 100 kb of DNA.

We have previously reported that expression of the G6PD locus is correlated with the methylation status of two islands of CpG dinucleotides which are 3' to the locus and in the 5' region of two adjacent genes of unknown function, P3 and GdX. We have now examined the methylation of a third CpG island in the promoter region of the G6PD gene itself in DNA from males, females and reactivants that express G6PD on the inactive X chromosome. Our results show that expression of the G6PD gene is associated with concordant demethylation of all three CpG islands in this 100-kb region of DNA.

Molecular characterization of a gene of the 'EGF family' expressed in undifferentiated human NTERA2 teratocarcinoma cells.

A novel human gene, encoding a 188 amino acid polypeptide that contains a region similar to that of the epidermal growth factor, has been isolated. The gene, expressed in undifferentiated human and mouse teratocarcinoma cells, is shut off after inducing the cells to differentiate by treatment with retinoic acid. Introduction of the cDNA under the control of a viral LTR induces transformation of NIH3T3 cells.

The murine cripto gene: expression during mesoderm induction and early heart morphogenesis.

The murine cripto gene encodes a 171-aminoacid epidermal growth factor-related protein, with 93% similarity to its human counterpart in the 'EGF-like' domain. The murine cripto mRNA contains two B1 repeats in its 3' non-coding region and a 163-nucleotide homology to the human mRNA. The mouse cripto gene is expressed at low level in specific organs of the adult animal such as spleen, heart, lung and brain. In situ hybridization analysis during murine embryogenesis (day 6.2 to day 10.5) reveals a very restricted expression pattern. cripto transcripts are first detected in a few epiblastic cells at day 6.5. During gastrulation, the transcripts are expressed in the forming mesoderm and later during development cripto gene expression is restricted to the truncus arteriosus of the developing heart. This expression pattern suggests a role for cripto gene in the determination of the epiblastic cells that subsequently give rise to the mesoderm.

Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice.

Fibroblast growth factor-2 (FGF-2) has been implicated in various signaling processes which control embryonic growth and differentiation, adult physiology and pathology. To analyze the in vivo functions of this signaling molecule, the FGF-2 gene was inactivated by homologous recombination in mouse embryonic stem cells. FGF-2-deficient mice are viable, but display cerebral cortex defects at birth. Bromodeoxyuridine pulse labeling of embryos showed that proliferation of neuronal progenitors is normal, whereas a fraction of them fail to colonize their target layers in the cerebral cortex. A corresponding reduction in parvalbumin-positive neurons is observed in adult cortical layers. Neuronal defects are not limited to the cerebral cortex, as ectopic parvalbumin-positive neurons are present in the hippocampal commissure and neuronal deficiencies are observed in the cervical spinal cord. Physiological studies showed that FGF-2-deficient adult mice are hypotensive. They respond normally to angiotensin II-induced hypertension, whereas neural regulation of blood pressure by the baroreceptor reflex is impaired. The present genetic study establishes that FGF-2 participates in controlling fates, migration and differentiation of neuronal cells, whereas it is not essential for their proliferation. The observed autonomic dysfunction in FGF-2-deficient adult mice uncovers more general roles in neural development and function.

Regional localization of the human EGF-like growth factor CRIPTO gene (TDGF-1) to chromosome 3p21.

The CRIPTO gene encodes a novel human growth factor structurally related to epidermal growth factor. We localized the CRIPTO gene to chromosome 3p21 by fluorescence in situ hybridization with a cosmid clone containing 40 kb of the CRIPTO genomic region (TDGF-1). To suppress hybridization to CRIPTO-related sequences, present in multiple copies in the human genome, hybridization was carried out in the presence of unlabeled CRIPTO cDNA in excess over the probe. Our finding confirms the provisional mapping of the CRIPTO gene to chromosome 3, and assigns it precisely to a chromosomal region involved in several rearrangements occurring in malignancy.

Differential effects of retinoic acid and a retinoid antagonist on the spatial distribution of the homeoprotein Hoxb-7 in vertebrate embryos.

An antibody raised against the recombinant Xenopus laevis Hoxb-7 protein (López and Carrasco [1992] Mech. Dev. 36:153-164) recognizes the 30 kDa translation product of the Hoxb-7 gene in X. laevis and the cognate nuclear protein in chicken embryos. The X. laevis Hoxb-7 protein was expressed maternally and zygotically. Treatment of X. laevis and chicken embryos with either all-trans retinoic acid (RA) or the retinoid antagonist Ro 41-5253 (Ro; Apfel et al. [1992] Proc. Natl. Acad. Sci. U.S.A. 89:7129-7133) during early development induced malformations of the neural tube and complementary changes in the expression domain of the homeoprotein Hoxb-7. Treatment of X. laevis embryos with retinoic acid during gastrulation induced an anterior shift of the Hoxb-7 expression domain and was correlated with an enlargement of rhombomere r7. In addition to a reduction in rhombomere numbers and of forebrain size, various malformations involving all three germ layers were observed. Treatment of X. laevis embryos with the antagonist Ro before or during gastrulation caused a progressive reduction of the Hoxb-7 domain and also dose-dependent malformations of all three germ layers. RA or Ro treatment of chicken embryos from the beginning of gastrulation caused changes of the Hoxb-7 expression domain very similar to those observed in X. laevis. In particular, either a dose-dependent loss of the Hoxb-7 protein in the neural tube or an ectopic expression in the forebrain region was observed. The results of this study indicate that endogenous retinoids regulate the spatial expression of homeobox-containing genes in vertebrates.

Characterization of the mouse Tdgf1 gene and Tdgf pseudogenes.

Cripto protein is a member of the "EGF family" of growth factors present in colon tumors and in human and mouse undifferentiated teratocarcinoma cells. During gastrulation in the mouse, cripto-encoding transcripts are expressed in the forming mesoderm and later in the truncus arteriosus of the developing heart. As a necessary step prior to investigating the in vivo role of cripto through gene disruption, we have isolated all the genomic cripto-related sequences in the mouse. One gene (Tdgf1) and two pseudogenes (Tdgf2 and Tdgf3) have been isolated and characterized. The mouse Tdgf1 (coding for cripto), like the human gene, is divided into six exons. Comparison of the human and mouse genomic sequences reveals that mouse exons 1 and 3 are shorter than the corresponding human exons. The pseudogene Tdgf2 corresponds to about 1 kb of the mRNA and contains five base substitutions in the coding region that represent both silent and replacement substitutions. The pseudogene Tdgf3 corresponds only to the coding portion of Tdgf. Many mutations have been introduced in this pseudogene, suggesting its early origin. Alignments of the Tdgf3, human and mouse mRNA sequences, shows that this pseudogene has retained the 33 nucleotides of the human exon 3 that are missed in the Tdgf1 gene. Taken together, these data suggest that Tdgf3 is derived from an ancestral gene and that the human and mouse genes are probably evolving separately.

Isolation and characterization of the CRIPTO autosomal gene and its X-linked related sequence.

We have previously reported on the identification of a cDNA clone encoding a novel human growth factor, named "CRIPTO," that is abundantly expressed in undifferentiated human NTERA-2 clone D1 (NT2/D1) and mouse (F9) teratocarcinoma cells. We now report the organization and nucleotide sequence of two related genomic sequences. One (CR-1) corresponds to the structural gene encoding the human CRIPTO protein expressed in the undifferentiated human teratocarcinoma cells, and the other (CR-3) corresponds to a complete copy of the mRNA containing seven base substitutions in the coding region representing both silent and replacement substitutions. The 440 bp 5' to the CAP site of CR-1 are preserved in CR-3. CR-1 maps to chromosome 3, and CR-3 maps to Xq21-q22. Southern blot analysis reveals that multiple CRIPTO-related DNA sequences are present in the human as well as in the mouse genome.

Formin defines a large family of morphoregulatory genes and functions in establishment of the polarising region.

Formin was originally isolated as the gene affected by the murine limb deformity (ld) mutations, which disrupt the epithelial-mesenchymal interactions regulating patterning of the vertebrate limb autopod. More recently, a rapidly growing number of genes with similarity to formin have been isolated from many different species including fungi and plants. Genetic and biochemical analysis shows that formin family members function in cellular processes regulating either cytokinesis and/or cell polarisation. Another common feature among formin family members is their requirement in morphogenetic processes such as budding and conjugation of yeast, establishment of Drosophila oocyte polarity and vertebrate limb pattern formation. Vertebrate formins are predominantly nuclear proteins which control polarising activity in limb buds through establishment of the SHH/FGF-4 feedback loop. Formin acts in the limb bud mesenchyme to induce apical ectodermal ridge (AER) differentiation and FGF-4 expression in the posterior AER compartment. Finally, disruption of the epithelial-mesenchymal interactions controlling induction of metanephric kidneys in ld mutant embryos indicates that formin might function more generally in transduction of morphogenetic signals during embryonic pattern formation.