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.

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.

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.

Expression of KIF3C kinesin during neural development and in vitro neuronal differentiation.

KIF3A, KIF3B and KIF3C are kinesin-related motor subunits of the KIF3 family that associate to form the kinesin-II motor complex in which KIF3C and KIF3B are alternative partners of KIF3A. We have analysed the expression of Kif3 mRNAs during prenatal murine development. Kif3c transcripts are detectable from embryonic day 12.5 and persist throughout development both in the CNS and in some peripheral ganglia. Comparison of the expression patterns of the Kif3 genes revealed that Kif3c and Kif3a mRNAs colocalize in the CNS, while only Kif3a is also present outside the CNS. In contrast, Kif3b is detectable in several non-neural tissues. We have also performed immunocytochemical analyses of the developing rat brain and have found the presence of the KIF3C protein in selected brain regions and in several fibre systems. Using neuroblastoma cells as an in vitro model for neuronal differentiation, we found that retinoic acid stimulated the expression of the three Kif3 and the kinesin-associated protein genes, although with different time courses. The selective expression of Kif3c in the nervous system during embryonic development and its up-regulation during neuroblastoma differentiation suggest a role for this motor during maturation of neuronal cells.

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.

Barhl1, a gene belonging to a new subfamily of mammalian homeobox genes, is expressed in migrating neurons of the CNS.

The BarH1 and BarH2 ( Bar ) Drosophila genes are homeobox-containing genes, which are required for the fate determination of external sensory organs in the fly. By means of a bioinformatic approach, we have identified in mouse and human two homeobox genes highly related to the Bar Drosophila genes, Barhl1 and Barhl2. While Barhl1 represents a novel gene, Barhl2 turned out to correspond to the mBH1 cDNA recently described in rat. We isolated and sequenced the full-length mouse Barhl1 and mapped both the human BARHL1 and BARHL2 genes to chromosomes 9q34 and 1p22, respectively. Detailed analysis of the murine Barhl1 expression pattern by in situ hybridization revealed that this transcript is exclusively expressed in restricted domains of the developing CNS, which suggests that this gene, similar to its Drosophila counterparts BarH1 and BarH2, may play a crucial role in cell fate determination of neural structures. In particular, Barhl1 showed specific domains of expression in the diencephalon and in the rhombencephalon where it was found to be expressed in migrating cells giving rise to the cerebellar external granular layer and to specific populations of dorsal sensory interneurons of the spinal cord. Thus, Barhl1 function may be required for the generation of these specific subtypes of neuronal progenitors. Furthermore, the mapping assignment and the expression pattern make BARHL1 an attractive positional candidate gene for a form of Joubert syndrome, a rare developmental anomaly of the cerebellum in humans.

A mouse model for valproate teratogenicity: parental effects, homeotic transformations, and altered HOX expression.

Valproate (VPA) is one of several effective anti-epileptic and mood-stabilizing drugs, many of which are also potent teratogens in humans and several other mammalian species. Variable teratogenicity among inbred strains of laboratory mice suggests that genetic factors influence susceptibility. While studying the genetic basis for VPA teratogenicity in mice, we discovered that parental factors influence fetal susceptibility to induced malformations. Detailed examination of these malformations revealed that many were homeotic transformations. To test whether VPA, like retinoic acid (RA), alters HOX expression, pluripotent human embryonal carcinoma cells were treated with VPA or RA and Hox expression assessed. Altered expression of specific Hox genes may thus account for the homeotic transformations and other malformations found in VPA-treated fetuses.

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.

A homeobox gene, vax2, controls the patterning of the eye dorsoventral axis.

We have identified a transcription factor specifically expressed in the developing vertebrate eye. We named this gene vax2 because of the high degree of sequence similarity to the recently described vax1. Both in the human and mouse genomes, vax2 is localized in the vicinity of the emx1 gene. This mapping assignment, together with the previously reported colocalization of Vax1 and Emx2 in mouse, indicates that the vax and the emx genes may be organized in clusters. vax2 has a remarkable expression domain confined to the ventral portion of the prospective neural retina in mouse, human, and Xenopus. The overexpression of either the frog Xvax2 or the human VAX2 in Xenopus embryos leads to an aberrant eye phenotype and, in particular, determines a ventralizing effect on the developing eye. The expression domain of the transcription factor Xpax2, normally confined to the ventral developing retina, extends to the dorsal region of the retina after overexpression of vax2. On the other hand, the expression of Xvent2, a molecular marker of the dorsal retina, is strongly reduced. Furthermore, vax2 overexpression induces a striking expansion of the optic stalk, a structure deriving from the ventralmost region of the eye vesicle. Altogether, these data indicate that vax2 plays a crucial role in eye development and, in particular, in the specification of the ventral optic vesicle.

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.

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

Identification and characterization of CDS2, a mammalian homolog of the Drosophila CDP-diacylglycerol synthase gene.

The general strategies of phototransduction in vertebrates and invertebrates share many similarities, but differ significantly in their underlying molecular machinery. The CDS gene encodes the CDP-diacylglycerol synthase (CDS) enzyme and is required for phototransduction in Drosophila. Using a bioinformatic approach, we have identified two novel transcripts (CDS1 and CDS2) highly homologous to the Drosophila CDS gene. We isolated and sequenced the CDS2 full-length cDNA and mapped the two genes to human chromosomes 20p13 (CDS2) and 4q21.1 (CDS1). Sequence analysis revealed that both genes are highly homologous to the Drosophila protein (64.4 and 58. 6% identity at the protein level between CDS and CDS2 and between CDS and CDS1, respectively). The mouse homologs for both genes were isolated and used in RNA in situ hybridization studies on adult and embryonic mouse tissue sections. These studies showed that Cds2 is highly expressed in the differentiating neuroblasts of the neural retina and in the central nervous system during embryonic development, while it was not detected in adult retina. Cds1, on the other hand, shows a high level of expression in the photoreceptor layer of adult retina, which strongly suggests a role for Cds1 in phototransduction. Knowledge of the expression pattern of these genes in mammals may shed light on the evolution of vision mechanisms and help in the evaluation of candidate genes for human retinopathies.

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.

Characterization of Cxorf5 (71-7A), a novel human cDNA mapping to Xp22 and encoding a protein containing coiled-coil alpha-helical domains.

The human X chromosome is known to contain several disease genes yet to be cloned. In the course of a project aimed at the construction of a transcription map of the Xp22 region, we fully characterized a novel cDNA, Cxorf5 (HGMW-approved symbol, alias 71-7A), previously mapped to this region but for which no sequence information was available. We isolated and sequenced the full-length transcript, which encodes a predicted protein of unknown function containing a large number of coiled-coild domains, typically presented in a variety of different molecules, from fibrous proteins to transcription factors. We showed that the Cxorf5 cDNA is ubiquitously expressed, undergoes alternative splicing, and escapes X inactivation. Furthermore, we precisely mapped two additional Cxorf5-related loci on the Y chromosome and on chromosome 5. By virtue of its mapping assignment to the Xp22 region, Cxorf5 represents a candidate gene for at least four human diseases, namely spondyloepiphiseal dysplasia late, oral-facial-digital syndrome type 1, craniofrontonasal syndrome, and a nonsyndromic sensorineural deafness.

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.

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.