Epithelial V-like antigen (EVA), a novel member of the immunoglobulin superfamily, expressed in embryonic epithelia with a potential role as homotypic adhesion molecule in thymus histogenesis.

Thymus development depends on a complex series of interactions between thymocytes and the stromal component of the organ. To identify regulated genes during this codependent developmental relationship, we have applied an RNA fingerprinting technique to the analysis of thymus expansion and maturation induced in recombinase-deficient mice injected with anti-CD3 antibodies. This approach led us to the identification of a gene encoding a new member of the immunoglobulin superfamily, named epithelial V-like antigen (EVA), which is expressed in thymus epithelium and strongly downregulated by thymocyte developmental progression. This gene is expressed in the thymus and in several epithelial structures early in embryogenesis. EVA is highly homologous to the myelin protein zero and, in thymus-derived epithelial cell lines, is poorly soluble in nonionic detergents, strongly suggesting an association to the cytoskeleton. Its capacity to mediate cell adhesion through a homophilic interaction and its selective regulation by T cell maturation might imply the participation of EVA in the earliest phases of thymus organogenesis.

Changes in gene expression during the growth arrest of HepG2 hepatoma cells induced by reducing agents or TGFbeta1.

The growth of hepatoma cells can be inhibited by treatment with TGFbeta1 or with exogenous reducing agents. To gain information on the molecular mechanisms underlying growth arrest, we visualized and compared gene expression profiles of proliferating versus non proliferating HepG2 cells by computer-assisted gene fishing, an improved technique of RNA fingerprinting that allows the selective amplification of coding regions within transcripts. While many transcripts are selectively regulated by either treatment, a set of bands appear to be coordinately regulated by 2ME and TGFbeta1, suggesting their possible involvement in the mechanisms of growth arrest. Display tags corresponding to 18 differentially expressed genes were cloned and, in most cases, identified as known genes or, more frequently, as their homospecific/cross-specific homologues. A novel member of the kinesin superfamily was identified amongst the genes induced by both 2ME and TGFbeta1. This gene, KIF3C, is upregulated in several cell lines undergoing growth arrest. Taken together, our findings show that computer-assisted gene fishing is a powerful tool for the identification and cloning of genes involved in the control of cell proliferation and indicate that extracellular reducing agents can regulate cell growth through modulation of gene expression.

GKLF in thymus epithelium as a developmentally regulated element of thymocyte-stroma cross-talk.

Gut-enriched Krüppel-like factor (GKLF) is a transcriptional regulator expressed in differentiated epithelia. We identified GKLF transcript as a regulated element in thymic epithelium of recombinase-deficient mice during thymus development induced by anti-CD3 antibody injection. This treatment recapitulates the organogenetic process depending on productive rearrangement of T cell receptor (TCR) beta gene with thymocytes expansion and acquisition of the CD4+8+ double positive phenotype. In wildtype mice, GKLF is expressed very early in embryogenesis and becomes intensely up-regulated in thymus epithelium at day 18 of gestation when TCR beta expressing cells have selectively expanded and express both CD4 and CD8. The results presented here suggest that thymocytes may regulate GKLF transcriptionally in the cortical epithelium at the developmental check-point controlled by TCR beta gene rearrangement. Furthermore, GKLF expression in hematopoietic stroma might suggest the thus far uncharacterised participation of this factor in hematopoiesis.

Mab21, the mouse homolog of a C. elegans cell-fate specification gene, participates in cerebellar, midbrain and eye development.

A multitude of regulatory genes are involved in phylogenetically conserved developmental cascades required for the patterning, cell-type specification, and differentiation of specific central nervous system (CNS) structures. Here, we describe the distribution of a mouse transcript encoding a homolog of the C. elegans mab-21 gene. In the nematode tail, mab-21 is required for the short-range patterning and cell-fate determination events mediated by egl-5 and mab-18, two homeobox genes homologous to Abd-B and Pax6, respectively. In mouse midgestation embryogenesis, Mab21 is expressed at its highest levels in the rhombencephalon, cerebellum, midbrain, and prospective neural retina. Our data and the genetic interactions previously documented in the nematode suggest that Mab21 may represent a novel, important regulator of mammalian cerebellum and eye development.

Factors involved in the migration of neuroendocrine hypothalamic neurons.

Neuroendocrine control of physiological functions needs a complex developmental organisation of the hypothalamic parvicellular neurons, which synthesise and release hypophysiotropic hormones. Among the hypothalamic neuroendocrine cells, Gonadotropin-releasing hormone (GnRH) neurons represent a unique class; they are generated in the olfactory placode and, during embryonic life, migrate to the septo/hypothalamic region along terminal and vomeronasal nerves. At this level GnRH neurons undergo terminal differentiation and start to release GnRH to modulate the secretion of pituitary gonadotropins. All these steps are under the strict control of several developmental cues and their defect might represent a cause of clinical disorders. A number of factors have been proposed to be involved in the migration of GnRH neurons, but their role is still unclear. By using gene knockout techniques it has been found that mice carrying a targeted deletion of Ebf2 gene, a component of Olf/Ebf bHLH transcription factors, show a defective migration of GnRH neurons, providing the first evidence of a mouse model of such defect. Since the investigation of GnRH neurons is hindered by their peculiar anatomical distribution, other studies has been forwarded by the availability of immortalized GnRH-expressing neurons (GN11 cells) that retain a strong chemomigratory response "in vitro". Among the factors analysed, we found that hepatocyte growth factor/scatter factor (HGF/SF) and vascular endothelial growth factor (VEGF) induce specific chemotaxis of GN 11 neurons, suggesting that migratory signals can arise from nasal mesenchyme and from the concomitant vasculogenesis. Finally, anosmin-1 (the product of the gene responsible of the X-linked form of Kallmann's disease) was found to induce a significant chemotactic response of GN11 cells, confirming a permissive/instructive role of KAL1 gene product in the migratory behaviour of GnRH neurons. In conclusion, the migration of the GnRH neurons appears to be a complex process, which involves the interplay of multiple molecular cues. These studies may provide new insights on the etiopathogenesis of the large proportion of reproductive dysfunctions that affect humans and could provide novel insights on common biochemical events controlling neuronal development and migration.

Ezrin gene, coding for a membrane-cytoskeleton linker protein, is regionally expressed in the developing mouse neuroepithelium.

Ezrin is a member of the Ezrin, Radixin, Moesin (ERM) proteins family that are proposed to act as linkers between the cytoskeleton and plasma membrane. Ezrin regulates cell-cell and cell-matrix interactions playing a role in the regulation of cellular adhesion, movement and morphology in epithelia. Alterations in the expression of Ezrin and other members of ERM family have also been observed in brain tumours. Here we report the expression pattern of Ezrin during mouse neural development, from early stages to postnatal stages. In young and middle gestation embryos, Ezrin is expressed in the roof plate of the neural tube, in the presumptive domain of the choroidal plexus, and in some precise domains of ventricular epithelium. These domains are distributed in basal and alar neuroepithelial regions, some of them in relation to the expression of cadherins. At later gestation and postnatal stages, Ezrin expression is maintained on the mature choroidal plexus and is weakly detected in the proliferative regions of the mature brain.

Neurodevelopment. Parasympathetic ganglia derive from Schwann cell precursors.

Neural crest cells migrate extensively and give rise to most of the peripheral nervous system, including sympathetic, parasympathetic, enteric, and dorsal root ganglia. We studied how parasympathetic ganglia form close to visceral organs and what their precursors are. We find that many cranial nerve-associated crest cells coexpress the pan-autonomic determinant Paired-like homeodomain 2b (Phox2b) together with markers of Schwann cell precursors. Some give rise to Schwann cells after down-regulation of PHOX2b. Others form parasympathetic ganglia after being guided to the site of ganglion formation by the nerves that carry preganglionic fibers, a parsimonious way of wiring the pathway. Thus, cranial Schwann cell precursors are the source of parasympathetic neurons during normal development.

Pattern formation during development of the embryonic cerebellum.

The patterning of the embryonic cerebellum is vital to establish the elaborate zone and stripe architecture of the adult. This review considers early stages in cerebellar Purkinje cell patterning, from the organization of the ventricular zone to the development of Purkinje cell clusters-the precursors of the adult stripes.

Local insulin-like growth factor I expression is essential for Purkinje neuron survival at birth.

IGF1, an anabolic and neuroprotective factor, promotes neuronal survival by blocking apoptosis. It is released into the bloodstream by the liver, or synthesized locally by muscles and neural cells, acting in an autocrine or paracrine fashion. Intriguingly, genetic studies conducted in invertebrate and murine models also suggest that an excess of IGF1 signaling may trigger neurodegeneration. This emphasizes the importance of gaining a better understanding of the mechanisms controlling IGF1 regulation and gene transcription. In the cerebellum, Igf1 expression is activated just before birth in a subset of Purkinje cells (PCs). Mice carrying a null mutation for HLH transcription factor EBF2 feature PC apoptosis at birth. We show that Igf1 is sharply downregulated in Ebf2 null PCs starting before the onset of PC death. In vitro, EBF2 binds a conserved distal Igf1 promoter region. The pro-survival PI3K signaling pathway is strongly inhibited in mutant cerebella. Finally, Ebf2 null organotypic cultures respond to IGF1 treatment by inhibiting PC apoptosis. Consistently, wild type slices treated with an IGF1 competitor feature a sharp increase in PC death. Our findings reveal that IGF1 is required for PC survival in the neonatal cerebellum, and identify a new mechanism regulating its local production in the CNS.

Purkinje cell subtype specification in the cerebellar cortex: early B-cell factor 2 acts to repress the zebrin II-positive Purkinje cell phenotype.

The mammalian cerebellar cortex is highly compartmentalized. First, it is subdivided into four transverse expression domains: the anterior zone (AZ), the central zone (CZ), the posterior zone (PZ), and the nodular zone (NZ). Within each zone, the cortex is further subdivided into a symmetrical array of parasagittal stripes. The most extensively studied compartmentation antigen is zebrin II/aldolase c, which is expressed by a subset of Purkinje cells forming parasagittal stripes. Stripe phenotypes are specified early in cerebellar development, in part through the action of early B-cell factor 2 (Ebf2), a member of the atypical helix-loop-helix transcription factor family Collier/Olf1/EBF. In the murine cerebellum, Ebf2 expression is restricted to the zebrin II-immunonegative (zebrin II-) Purkinje cell population. We have identified multiple cerebellar defects in the Ebf2 null mouse involving a combination of selective Purkinje cell death and ectopic expression of multiple genes normally restricted to the zebrin II- subset. The nature of the cerebellar defect in the Ebf2 null is different in each transverse zone. In contrast to the ectopic expression of genes characteristic of the zebrin II+ Purkinje cell phenotype, phospholipase Cbeta4 expression, restricted to zebrin II- Purkinje cells in control mice, is well maintained, and the normal number of stripes is present. Taken together, these data suggest that Ebf2 regulates the expression of genes associated with the zebrin II+ Purkinje cell phenotype and that the zebrin II- Purkinje cell subtype is specified independently.

cDNA sequence, map, and expression of the murine homolog of GTBP, a DNA mismatch repair gene.

DNA mismatch repair genes (MMR) have been actively studied in humans for their role in cancer susceptibility. Recently, GTBP, a new member of the MMR gene family, has been cloned and found to encode a 160-kDa protein that forms a heterodimer with hMSH2. Our group has isolated and characterized the mouse homolog of GTBP. The transcript, termed Gtmbp (G/T mismatch-binding protein), encodes a protein highly similar to its human counterpart. The gene is markedly conserved across species and maps to the distal portion of mouse chromosome 17, in a region homologous to human 2p. One Gtmbp-related sequence maps to proximal mouse chromosome 4. Studies of the expression of Gtmbp suggest that its regulated transcription is tightly linked to the level of DNA replication, consistent with the protein's DNA proofreading function.

EDF-1, a novel gene product down-regulated in human endothelial cell differentiation.

Endothelial cell differentiation is a crucial step in angiogenesis. Here we report the identification of EDF-1, a novel gene product that is down-regulated when endothelial cells are induced to differentiate in vitro. The cDNA encoding EDF-1 was isolated by RNA fingerprinting from human endothelial cells exposed to human immunodeficiency virus type 1 Tat, a viral protein known to be angiogenic. The deduced amino acid sequence of EDF-1 encodes a basic intracellular protein of 148 amino acids that is homologous to MBF1 (multiprotein-bridging factor 1) of the silkworm Bombyx mori and to H7, which is implicated in the early developmental events of Dictyostelium discoideum. Interestingly, human immunodeficiency virus type 1 Tat, which affects endothelial functions, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and culture on fibrin gels, which promote endothelial differentiation in vitro, all down-regulate EDF-1 expression both at the RNA and protein levels. In addition, the inhibition of EDF-1 translation by an antisense anti-EDF-1 construct results in the inhibition of endothelial cell growth and in the transition from a nonpolar cobblestone phenotype to a polar fibroblast-like phenotype. These data suggest that EDF-1 may play a role in the regulation of human endothelial cell differentiation.

Neuroretinal rim area in normal eyes: a study on a randomized group of 30 subjects.

This study was undertaken in order to evaluate the range of the neuroretinal rim area in a normal sample and the repeatability of manual photogrammetric measurements. Thirty randomly chosen eyes from 30 subjects without ocular disease were examined: the mean disc area (2.20 +/- SD 0.58 mm2), the mean cup area (0.36 +/- 0.29 mm2) and the mean rim area (1.83 +/- 0.37 mm2) were evaluated. All the measurements were performed by two independent observers and were corrected to the actual size by measuring refraction and axial length of each eye. A linear correlation among disc areas (r = 0.71), cup areas (r = 0.93) and rim areas (r = 0.74) between the two observers was found. Furthermore, a highly significant correlation between disc area and rim area was observed (r = 0.90) together with a correlation between the disc and cup area (r = 0.82). A correlation between disc area and axial length was finally found (r = 0.59). The neuroretinal rim area has been expressed as a percentage of the total disc area. Percent values (+/- SD) were clustered around a mean of 84.83 +/- 8.8%. Despite their high degree of repeatability, our data are slightly different from those previously reported by other authors.

Xebf3 is a regulator of neuronal differentiation during primary neurogenesis in Xenopus.

During primary neurogenesis in Xenopus, a cascade of helix--loop--helix (HLH) transcription factors regulates neuronal determination and differentiation. While XNeuroD functions at a late step in this cascade to regulate neuronal differentiation, the factors that carry out terminal differentiation are still unknown. We have isolated a new Xenopus member of the Ebf/Olf-1 family of HLH transcription factors, Xebf3, and provide evidence that, during primary neurogenesis, it regulates neuronal differentiation downstream of XNeuroD. In developing Xenopus embryos, Xebf3 is turned on in the three stripes of primary neurons at stage 15.5, after XNeuroD. In vitro, XEBF3 binds the EBF/OLF-1 binding site and functions as a transcriptional activator. When overexpressed, Xebf3 is able to induce ectopic neurons at neural plate stages and directly convert ectodermal cells into neurons in animal cap explants. Expression of Xebf3 can be activated by XNeuroD both in whole embryos and in animal caps, indicating that this new HLH factor might be regulated by XNeuroD. Furthermore, in animal caps, XNeuroD can activate Xebf3 in the absence of protein synthesis, suggesting that, in vitro, this regulation is direct. Similar to XNeuroD, but unlike Xebf2/Xcoe2, Xebf3 expression and function are insensitive to Delta/Notch-mediated lateral inhibition. In summary, we conclude that Xebf3 functions downstream of XNeuroD and is a regulator of neuronal differentiation in Xenopus.

A computer-driven approach to PCR-based differential screening, alternative to differential display.

MOTIVATION: Polymerase chain reaction (PCR)-based RNA fingerprinting is a powerful tool for the isolation of differentially expressed genes in studies of neoplasia, differentiation or development. Arbitrarily primed RNA fingerprinting is capable of targeting coding regions of genes, as opposed to differential display techniques, which target 3' non-coding cDNA. In order to be of general use and to permit a systematic survey of differential gene expression, RNA fingerprinting has to be standardized and a number of highly efficient and selective arbitrary primers must be identified. RESULTS: We have applied a rational approach to generate a representative panel of high-efficiency oligonucleotides for RNA fingerprinting studies, which display marked affinity for coding portions of known genes and, as shown by preliminary results, of novel ones. The choice of oligonucleotides was driven by computer simulations of RNA fingerprinting reverse transcriptase (RT)-PCR experiments, performed on two custom-generated, non-redundant nucleotide databases, each containing the complete collection of deposited human or murine cDNAs. The simulation approach and experimental protocol proposed here permit the efficient isolation of coding cDNA fragments from differentially expressed genes. AVAILABILITY: Freely available on request from the authors. CONTACT: fesce.riccardo@hsr.it

The human skeletal muscle glycogenin gene: cDNA, tissue expression and chromosomal localization.

Glycogen synthesis is impaired in first degree relatives of subjects with non-insulin-dependent diabetes mellitus and genes relevant to this metabolic pathway are considered reasonable candidates in the pathogenesis of the disease. In skeletal muscle the de novo synthesis of glycogen in primed by an enzyme named glycogenin. We have cloned the glycogenin cDNA from human skeletal muscle mRNA: human glucogenin is a 333 amino acid protein exhibiting 93% identity with rabbit glycogenin. A single transcript of about 2.4 kb, prominent in skeletal muscle, was detected by Northern blot analysis. In situ hybridization unequivocally located the human glycogenin gene to chromosome 3q25.1. Furthermore, we mapped two intronless glycogenin-related sequences to human chromosomes 12 and 13.

Two murine and human homologs of mab-21, a cell fate determination gene involved in Caenorhabditis elegans neural development.

We report the cloning and genetic characterization of one human and two murine homologs of the mab-21 cell fate specification gene. mab-21 participates in the formation of sensory organs in the male nematode tail, and is essential for other developmental functions elsewhere in the Caenorhabditis elegans embryo. The expanding mab-21 gene family, which is strikingly conserved in evolution, includes two putative Drosophila members. The two mammalian genes, encoding 41 kDa nuclear basic proteins, are expressed in partially overlapping territories in the embryonic brain, eye and limbs, as well as in neural crest derivatives. Recent genetic data implicating mab-21 as a downstream target of TGF-beta signaling, together with the distribution of mab-21 transcripts in the mouse embryo, propose these novel genes as relevant factors in various aspects of vertebrate neural development.

Isolation of the human chromosomal band Xq28 within somatic cell hybrids by fragile X site breakage.

The chromosomal fragile-site mapping to Xq27.3 is associated with a frequent form of mental retardation and is prone to breakage after induced deoxyribonucleotide pool perturbation. The human hypoxanthine phosphoribosyltransferase (HPRT) and glucose-6-phosphate dehydrogenase (G6PD) genes flank the fragile X chromosome site and can be used to monitor integrity of the site in human-hamster somatic cell hybrids deficient in the rodent forms of these activities. After induction of the fragile X site, negative selection for HPRT and positive enrichment for G6PD resulted in 31 independent colonies of HPRT-,G6PD+ phenotype. Southern blot analysis demonstrated the loss of all tested markers proximal to the fragile X site with retention of all tested human Xq28 loci in a majority of the hybrids. In situ hybridization with a human-specific probe demonstrated the translocation of a small amount of human DNA to rodent chromosomes in these hybrids, suggesting chromosome breakage at the fragile X site and the subsequent translocation of Xq28. Southern blot hybridization of hybrid-cell DNA, resolved by pulsed-field gel electrophoresis, for human-specific repetitive sequences revealed abundant CpG-islands within Xq28, consistent with its known gene density. The electrophoretic banding patterns of human DNA among the hybrids were remarkably consistent, suggesting that fragile X site breakage is limited to a relatively small region in Xq27-28. These somatic cell hybrids, containing Xq27.3-qter as the sole human DNA, will aid the search for DNA associated with the fragile X site and will augment the high resolution genomic analysis of Xq28, including the identification of candidate genes for genetic-disease loci mapping to this region.

Reduced fertility and spermatogenesis defects in mice lacking chromosomal protein Hmgb2.

High mobility group 2 protein (Hmgb2) is a member of the HMGB protein family, which includes the ubiquitous Hmgb1 and the embryo-specific Hmgb3. The three proteins are more than 80% identical at the amino acid level and their biochemical properties are indistinguishable. Hmgb1 is an abundant component of all mammalian nuclei and acts as an architectural factor that bends DNA and promotes protein assembly on specific DNA targets. Cells that lack Hmgb1 can survive, although mutant mice die shortly after birth. As Hmgb2 is present in all cultured cells and is abundant in thymus, the preferred source for HMGB proteins, it was considered a ubiquitous variant of Hmgb1. We show that in adult mice Hmgb2 is restricted mainly to lymphoid organs and testes, although it is widely expressed during embryogenesis. Mice that lack Hmgb2 are viable. However, male Hmgb2(-/-) mice have reduced fertility, that correlates with Sertoli and germ cell degeneration in seminiferous tubules and immotile spermatozoa. Significantly, Hmgb2 is expressed at very high levels in primary spermatocytes, while it is barely detectable in spermatogonia and elongated spermatids. This peculiar pattern of expression and the phenotype of mutants indicate that Hmgb2 has a specialised role in germ cell differentiation.