Expression pattern of the Kallmann syndrome gene in the olfactory system suggests a role in neuronal targeting.

Kallmann syndrome is a genetic disorder characterized by a defect in olfactory system development, which appears to be due to an abnormality in the migration of olfactory axons and gonadotropin releasing hormone (Gn-RH) producing neurons. The X-linked Kallmann syndrome gene shares significant similarities with molecules involved in neural development. We have now isolated the evolutionarily conserved chicken homologue of the Kallmann gene. In the developing and adult chicken, high levels of expression were found in the mitral cells of the olfactory bulb (the target of olfactory axons) and in the Purkinje cells of the cerebellar cortex, both areas affected in patients with Kallmann syndrome. We propose a model in which the Kallmann syndrome gene product is a signal molecule required for neuronal targeting throughout life.

Different chromosomal localization of the Clcn4 gene in Mus spretus and C57BL/6J mice.

We report the unprecedented finding of a gene with a different map position in two mouse strains. The Clcn4 gene was found to map to the X chromosome in the wild Mediterrean mouse, Mus spretus but to chromosome 7 in the inbred strain of laboratory mouse C57BL/6J. These data indicate that a recent evolutionary rearrangement occurred on the mouse sex chromosomes, very close to the pseudoautosomal region. Our data provide molecular evidence for a major divergence near the pseudoautosomal region, consistent with the hypothesis that hybrid sterility in these species results from abnormal pairing of sex chromosomes during male meiosis.

Opitz G/BBB syndrome, a defect of midline development, is due to mutations in a new RING finger gene on Xp22.

Opitz syndrome (OS) is an inherited disorder characterized by midline defects including hypertelorism, hypospadias, lip-palate-laryngotracheal clefts and imperforate anus. We have identified a new gene on Xp22, MID1 (Midline 1), which is disrupted in an OS patient carrying an X-chromosome inversion and is also mutated in several OS families. MID1 encodes a member of the B-box family of proteins, which contain protein-protein interaction domains, including a RING finger, and are implicated in fundamental processes such as body axis patterning and control of cell proliferation. The association of MID1 with OS suggests an important role for this gene in midline development.

JAGGED2: a putative Notch ligand expressed in the apical ectodermal ridge and in sites of epithelial-mesenchymal interactions.

The Drosophila Notch gene and its ligands, Delta and Serrate, are involved in cell fate determination in a variety of developing tissues. Recently, several Notch, Delta and Serrate homologues have been identified in vertebrates. We report here the cloning of the human and murine JAGGED2 (JAG2), a Serrate-like gene, and the analysis of its expression pattern during embryogenesis. Jag2 was found to be expressed as early as E9 in the surface ectoderm of the branchial arches and in the apical ectodermal ridge (AER) of the developing limb. At E12.5, Jag2 expression is upregulated in differentiated neurons of the central and peripheral nervous system and in the inner neuroblastic layer of the developing retina. Outside the nervous system, Jag2 is expressed in the developing vibrissae follicles, tooth buds, thymus, submandibular gland and stomach. Our findings suggest the involvement of Jagged2 in the development of the mammalian limb, branchial arches, central and peripheral nervous systems and several tissues whose development depends upon epithelial-mesenchymal interactions.

X-linked Kallmann syndrome. A neuronal targeting defect in the olfactory system?

Kallmann syndrome is a human genetic disorder characterized by the association of hypogonadism with the inability to smell, and is due to defects in the olfactory system development (i.e. incomplete migration of olfactory axons and of gonadotropin-releasing hormone producing neurons from the olfactory epithelium to the forebrain; aplasia or hypoplasia of olfactory bulbs and tracts). The human X-linked Kallmann syndrome gene and its chicken homologue have been cloned. Their protein products contain fibronectin type III repeats and a 'four-disulfide-core' domain also found in molecules that are involved in neural development. Consistent with the human phenotype, the chicken Kallmann gene is expressed in the developing olfactory bulb. At present the molecular and cellular mechanism of action of the Kallmann syndrome gene product is unknown. Based on expression studies and the characteristics domains of the predicted protein, it is hypothesized that the protein may be involved in targeting olfactory axons to the bulb. Alternatively, the Kallmann protein could be an extracellular matrix component required for the proper formation of the multilayered structure of the olfactory bulb.

Detection of beta-nerve growth factor mRNA in the human fetal brain.

Nerve growth factor (NGF) is a trophic molecule recently demonstrated to interact with different structures in the central nervous system. The expression of the beta-NGF mRNA from human fetal cortices at the 15-16th week of gestational age has been demonstrated and quantitated by polymerase chain reaction amplification of the specific cDNA. beta-NGF mRNA expression in the human brain coincides with the period of active differentiation and synaptogenesis, suggesting that the trophic agent plays a role in the cerebral development.

Human fetal brain beta-nerve growth factor cDNA: molecular cloning of 5' and 3' untranslated regions.

The mRNA of beta-nerve growth factor (beta-NGF) has been demonstrated to be present in the human brain, both in adult and fetal stages of development. However, its complete nucleotide sequence is unknown since a full-length cDNA has yet to be isolated. We report here the cloning and complete analysis of the human fetal brain beta-NGF transcript. cDNA synthesis was performed from total brain RNA and overlapping regions of beta-NGF cDNA were enzymatically amplified and sequenced. The central portion of the cDNA was amplified using primers designed on the known genomic DNA sequence. The 5' and 3' unknown regions were amplified by the anchored polymerase chain reaction (PCR) and by complementary DNA Ends-PCR respectively. The latter method is an original variation of Inverted PCR. The sequenced transcript is very similar to the most common form of beta-NGF mRNA present in the mouse central nervous system. In addition, both the 5' and 3' untranslated regions show a high degree of homology to the corresponding murine sequences.

Maintained cellular function of adrenal medullary cells in parkinsonian dysautonomia.

Adrenal gland involvement in Parkinson's disease was reported by different authors. Further studies became relevant after adrenal was proposed as dopaminergic donor for neurotransplantation. Chromaffin cells were grown in culture and the effects of nerve growth factor (NGF) tested: no differences were observed between parkinsonian and control cells. The expression of the beta-NGF mRNA in the parkinsonian adrenal was analyzed: a specific cDNA was synthesized and a 168 bp portion amplified using PCR. The products were identified and the identity of the fragment was confirmed by sequencing. Quantitative PCR demonstrated a beta-NGF mRNA concentration exceeding 5 fg/micrograms of total adrenal RNA. These findings demonstrate the retained functional capacity of the parkinsonian adrenal to respond to NGF and express the beta-NGF mRNA.

Reelin: a novel extracellular matrix protein involved in brain lamination.

Normal development of the nervous system is achieved through an elaborate program of guided neuronal migration and axonal growth. In the last few years, a flood of research has dissected the molecular bases of these phenomena, and several cell-surface and extracellular matrix molecules, which are implicated in neuronal and axonal targeting processes, have been recognized. Taking this knowledge a step further, a recent paper by Tom Curran's group reports the molecular cloning of the gene deleted in the autosomal recessive mouse mutation reeler, affecting cortical neuronal migration. This gene encodes reelin, a novel extracellular matrix protein.

The Kallmann syndrome gene product expressed in COS cells is cleaved on the cell surface to yield a diffusible component.

Kallmann syndrome is characterized by hypogonadotropic hypogonadism and anosmia and caused by a defect of migration and targeting of gonadotropin-releasing hormone-secreting neurons and olfactory axons during embryonic development. We previously cloned the gene responsible for the X-linked form of the disease encoding a 680 amino acid protein, KAL, which displays the unusual combination of a protease inhibitor domain with fibronectin type III repeats. Previous expression studies by northern blot and RNA in situ hybridization in human and chick indicated that the gene is expressed at very low levels in the olfactory bulb during development. Therefore, low abundance of the protein has hampered a detailed biochemical characterization. By overexpressing both the human and chick KAL cDNAs in eukaryotic cells, we now provide evidence that KAL is a glycosylated peripheral membrane protein with an apparent molecular weight of approximately 100 kDa. We show that this 100 kDa protein is proteolytically processed on the cell membrane to yield a 45 kDa diffusible component, which is detectable with an antisera against the C-terminal part of the protein and binds tightly to cell surfaces. These data provide a first step toward understanding KAL function in neuronal interactions and neurite extension in the olfactory bulb and suggest that KAL might be a diffusible chemoattractant molecule for olfactory axons.

Characterization of a human and murine gene (CLCN3) sharing similarities to voltage-gated chloride channels and to a yeast integral membrane protein.

We describe the isolation and characterization of a human gene (CLCN3) and its murine homologue (Clcn3) sharing significant sequence and structural similarities with all previously identified members of the voltage-gated chloride channel (ClC) family. This gene is expressed primarily in tissues derived from neuroectoderm. Within the brain, Clcn3 expression is particularly evident in the hippocampus, olfactory cortex, and olfactory bulb. CLCN3 encodes a 760-amino-acid protein that differs by only 2 amino acid residues from the protein encoded by Clcn3. CLCN3 protein also shows a high similarity with GEF1, an integral membrane protein of the yeast Saccharomyces cerevisiae known to be involved in respiration and iron-limited cell growth, and with the predicted protein product of a DNA sequence from the mold Septoria nodorum. This high degree of sequence conservation in very distantly related species such as human and yeast indicates that this gene has retained a fundamental function throughout evolution.

Expression of the Kallmann syndrome gene in human fetal brain and in the manipulated chick embryo.

Kallmann syndrome is an inherited disorder characterized by an abnormality in olfactory system development. The gene for the X-linked form of this disorder (KAL) maps to Xp22.3 and encodes a protein sharing homologies with molecules involved in neuronal migration and axonal pathfinding. Here we report the expression pattern of the KAL gene in various parts of the human fetal brain. We found KAL transcripts in granule cells of the olfactory bulb and the cerebellum, in the dorsomedial thalamus and in the developing cerebral cortex. To determine whether or not signals from the olfactory nerve are required for KAL expression in the olfactory bulb, we analyzed chick embryos in which the olfactory placode was surgically removed. Those embryos lacking an olfactory nerve had a histologically abnormal bulb which nevertheless expressed the KAL gene at high levels. These findings indicate that, while the development of the proper cytoarchitecture of the olfactory bulb requires the innervation by olfactory axons, the expression of KAL is independent of such developmental processes.

Cloning of a human homologue of the Xenopus laevis APX gene from the ocular albinism type 1 critical region.

Ocular albinism type 1 (OA1) is an X-linked recessive disorder characterized by a major impairment of visual acuity, nystagmus, strabismus, photophobia and retinal hypopigmentation. From the analysis of patients carrying deletions and translocations involving the distal short arm of the X chromosome (Xp22.3) we have identified a region of approximately 110 kb in which the OA1 gene must lie. We have extensively searched for genes in this region using a variety of techniques which included exon amplification, cDNA selection and direct hybridization of cosmid inserts to cDNA libraries. Putative exons identified by exon amplification were used to screen a human retina cDNA library and several cDNA clones corresponding to an approximately 7.5 kb transcript were isolated and characterized. Transcripts of this newly identified gene were found to be abundant in retina and melanoma and could also be detected in brain, placenta, lung, kidney and pancreas. Interestingly, sequence analysis revealed that this new gene encodes a 1616 amino acid protein sharing significant similarities with the Apical Protein from Xenopus laevis (APX) which is implicated in amiloride-sensitive sodium channel activity. The gene, termed APXL (APX-Like), spans approximately 160 kb, contains 10 exons and covers over 70% of the 110 kb critical region for OA1. A truncated pseudogene sharing very high levels of homology with the rat eIF-5 gene, a eukaryotic translation initiation factor, was found to lie in the middle of intron 1. APXL was found deleted in two patients with contiguous gene syndromes including OA1 and in one patient with isolated OA1. Mapping, expression and patient analysis data led us to consider the APXL gene a strong candidate for the OA1 gene. DNA from 57 unrelated patients with OA1 was, therefore, scanned for mutations in the coding region, using both SSCP analysis and direct sequencing. No functionally significant mutation was identified, suggesting that APXL is not directly involved in OA1. Further studies are needed to clarify the physiologic role of this highly conserved gene.

A new region of conservation is defined between human and mouse X chromosomes.

Comparative mapping of the X chromosome in eutherian mammals has revealed distinct regions of conservation as well as evolutionary rearrangements between human and mouse. Recently, we and others mapped the murine homologue of CLCN4 (Chloride channel 4) to band F4 of the X chromosome in Mus spretus but to chromosome 7 in laboratory strains. We now report the mapping of the murine homologues of APXL (Apical protein Xenopus laevis-like) and OA1 (Ocular albinism type I), two genes that are located on the human X chromosome at band p22. 3 and in close proximity to CLCN4. Interestingly, Oa1 and Apxl map to bands F2-F3 in both M. spretus and the laboratory strain C57BL/6J, defining a new rearrangement between human and mouse X chromosomes.

Beta-nerve growth factor (beta-NGF) mRNA expression in the parkinsonian adrenal gland.

The adrenal gland is a well-demonstrated source for different neurotrophic factors. The presence of the beta-nerve growth factor (beta-NGF) mRNA in the adrenal tissue used for grafting in a Parkinsonian patient is reported here. Adrenal samples were obtained on the day of implantation, and a specific cDNA was synthesized after the extraction of total RNA using a synthetic oligonucleotide as a reverse transcription primer. A 168-bp portion of the cDNA was amplified using two other oligonucleotides as Taq polymerase primers in a polymerase chain reaction. Thirty-two cycles of amplification were performed. The amplification products were identified by agarose gel electrophoresis and Southern blot analysis as a single DNA band hybridizing with a third beta-NGF specific oligonucleotide. The identity of the fragment was confirmed by DNA sequencing. Quantitative analysis demonstrated a beta-NGF mRNA concentration exceeding 5 fg/micrograms of total adrenal RNA. These findings add NGF to the other neurotrophic factors produced by the gland (i.e., basic fibroblast growth factor) and demonstrate the retained functional capacity of the Parkinsonian adrenal to express the beta-NGF mRNA. All these data may assume relevant meaning for neurotransplantation research.

A gene from the Xp22.3 region shares homology with voltage-gated chloride channels.

In the framework of constructing a comprehensive transcript map of the human Xp22.3 region, we identified an evolutionary conserved CpG island and cloned the corresponding gene. The predicted 760 amino acid protein encoded by this gene contains 12 hydrophobic domains and shares significant sequence and structural similarities with all the previously isolated members of a recently identified family of voltage-gated chloride channels (the 'CIC family'). This gene, termed CICN4 (Chloride Channel 4), contains at least 10 exons spanning 60 to 80 kb on the X chromosome. In contrast to most genes isolated from the human Xp22.3 region, the CICN4 gene does not share homology with the Y chromosome and it is conserved in mouse and hamster. Expression studies revealed the presence of a 7.5 kb transcript which is particularly abundant in skeletal muscle and is also detectable in brain and heart. These data suggest that we have identified a new voltage-gated chloride channel which is encoded by a gene located in the distal short arm of the X chromosome.

Evidence of evolutionary up-regulation of the single active X chromosome in mammals based on Clc4 expression levels in Mus spretus and Mus musculus.

Previous studies have shown that the chloride channel gene Clc4 is X-linked and subject to X inactivation in Mus spretus, but that the same gene is autosomal in laboratory strains of mice. This exception to the conservation of linkage of the X chromosome in one of two interfertile mouse species was exploited to compare expression of Clc4 from the X chromosome to that from the autosome. Clc4 was found to be highly expressed in brain tissues of both mouse species. Quantitative analyses of species-specific expression of Clc4 in brain tissues from mice resulting from M. spretus x laboratory strain crosses, demonstrate that each autosomal locus has half the level of Clc4 expression as compared with the single active X-linked locus. In contrast expression of another chloride channel gene, Clc3, which is autosomal in both mouse species is equal between alleles in F1 animals. There is no evidence of imprinting of the Clc4 autosomal locus. These results are consistent with Ohno's hypothesis of an evolutionary requirement for a higher expression of genes on the single active X chromosome to maintain balance with autosomal gene expression [Ohno, S. (1967) Sex Chromosomes and Sex-Linked Genes (Springer, Berlin)].

A novel human serine-threonine phosphatase related to the Drosophila retinal degeneration C (rdgC) gene is selectively expressed in sensory neurons of neural crest origin.

Through our transcriptional mapping effort in the Xp22 region, we have isolated by exon trapping a new transcript highly homologous to the Drosophila retinal degeneration C (rdgC) gene. rdgC encodes a serine/threonine phosphatase protein and is required in Drosophila to prevent light-induced retinal degeneration. This human gene is the first mammalian member of the serine-threonine phosphatase with EF hand motif gene family, and was thus named PPEF (Protein Phosphatase with EF calcium-binding domain). The expression pattern of the mouse Ppef gene was studied by RNA in situ hybridization on embryonic tissue sections. While rdgC is expressed in the visual system of the fly, as well as in the mushroom bodies of the central brain, we found that Ppef is highly expressed in sensory neurons of the dorsal root ganglia (DRG) and neural crest-derived cranial ganglia. The selective pattern of expression makes PPEF an important marker for sensory neuron differentiation and suggests a role for serine-threonine phosphatases in mammalian development.

The mouse Mid1 gene: implications for the pathogenesis of Opitz syndrome and the evolution of the mammalian pseudoautosomal region.

We have recently reported isolation of the gene responsible for X-linked Opitz G/BBB syndrome, a defect of midline development. MID1 is located on the distal short arm of the human X chromosome (Xp22. 3) and encodes a novel member of the B box family of zinc finger proteins. We have now cloned the murine homolog of MID1 and performed preliminary expression studies during development. Mid1 expression in undifferentiated cells in the central nervous, gastrointestinal and urogenital systems suggests that abnormal cell proliferation may underlie the defect in midline development characteristic of Opitz syndrome. We have also found that Mid1 is located within the mouse pseudoautosomal region (PAR) in Mus musculus , while it seems to be X-specific in Mus spretus. Therefore, Mid1 is likely to be a recent acquisition of the M. musculus PAR. Genetic and FISH analyses also demonstrated a high frequency of unequal crossovers in the murine PAR, creating spontaneous deletion/duplication events involving Mid1. These data provide evidence for the first time that genetic instability of the PAR may affect functionally important genes. In addition, we show that MID1 is the first example of a gene subject to X-inactivation in man while escaping it in mouse. These data contribute to a better understanding of the molecular content and evolution of the rodent PAR.