Expression of the Xvax2 gene demarcates presumptive ventral telencephalon and specific visual structures in Xenopus laevis.

vax2 is a recently isolated homeobox gene, that plays an important role in controlling the dorso-ventral patterning of the retina. In this paper we present a thorough description of the Xvax2 expression pattern all along Xenopus embryogenesis, and compare this pattern in detail to that shown by Xvax1b and Xpax2, two genes also involved in ventral eye development. At early neurula stages, while Xpax2 starts to be expressed within the eye field, both Xvax2 and Xvax1b are exclusively activated in the presumptive ventral telencephalon. Since midneurula stages, Xvax2 and Xvax1b are also transcribed in the medial aspect of the eye field. At tailbud and tadpole stages, Xvax2, Xvax1b and Xpax2 expression overlaps in the optic stalk and nerve and in the optic disk, while Xvax2 and Xvax1b also display specific activation domains in the ventral retina as well as in the ventral telencephalon and diencephalon. Finally, during metamorphosis a high level of both Xvax2 and Xvax1b transcription is maintained in the optic chiasm. In addition, Xvax1b is transcribed in the ventral hypothalamus and in the hypophysis, whereas a strong Xvax2 expression is retained in the ventral portion of the mature retina.

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.

Identification and characterization of AFG3L2, a novel paraplegin-related gene.

We recently identified a gene responsible for an autosomal recessive form of hereditary spastic paraplegia (HSP). This gene encodes paraplegin, a mitochondrial protein highly homologous to the yeast mitochondrial ATPases Afg3p and Rcalp, which have both proteolytic and chaperone-like activities at the inner mitochondrial membrane. By screening the Expressed Sequence Tag database, we identified and characterized a novel human cDNA, ATPase family gene 3-like 2 (AFG3L2, Human Gene Nomenclature Committee-approved symbol), which is closely related to paraplegin. This cDNA encodes a 797-amino-acid predicted protein highly similar to paraplegin as well as to yeast Afg3p and Rca1p. Immunofluorescence studies revealed that AFG3L2 and paraplegin share a similar expression pattern and the same subcellular localization, the mitochondrial compartment. We subsequently mapped AFG3L2 to chromosome 18p11 by radiation hybrid analysis. AFG3L2 may represent a candidate gene for other forms of HSPs and possibly for other neurodegenerative disorders.

The embryonic expression pattern of 40 murine cDNAs homologous to Drosophila mutant genes (Dres): a comparative and topographic approach to predict gene function.

Nature often utilizes the same metabolic 'core groups' of interacting genes or 'pathways' in completely different organs, tissues and cellular compartments. Deciphering the physiological role of a particular gene in a living organism is therefore critical to understanding not only how a gene/protein works, but also where (in which tissue/organ) and when (at what developmental stage) it functions. We have performed systematic RNA in situ hybridization on a subset of murine genes homologous to Drosophila mutant genes, called Drosophila -related expressed sequences (Dres). This approach combines functional information derived from cross-species sequence comparisons and biochemical, physiological and pathological studies performed in the fly with knowledge of the spatial and temporal distribution of gene expression. Forty murine Dres were tested by RNA in situ hybridization on sagittal, coronal and transverse sections at three developmental stages, E10.5, E12.5 and E17.5. For some of them, whole mount in situ hybridization was performed at earlier stages. These data are valuable for establishing how the function of these genes and the genetic programs underlying the development of a particular tissue or organ have evolved during evolution. For example, six Dres genes showed restricted expression domains within the murine retina, suggesting a different role for each of these genes in eye development and functioning. Furthermore, the information derived from this combined approach will be instrumental in predicting the phenotypic consequences of gene dysfunction in both mouse mutants and human genetic diseases.

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.

A mammalian homologue of the Drosophila retinal degeneration B gene: implications for the evolution of phototransduction mechanisms.

Comparative analysis of homologous genes in distantly related species provides important insights into the evolution of complex physiological processes. The Drosophila retinal degeneration B (rdgB) gene encodes a protein involved in phototransduction in the fly. We have isolated a human gene, DRES9, and its murine homologue (Dres9), which show a high degree of similarity to the Drosophila rdgB gene. RNA in situ hybridization studies performed on mouse-embryo tissue sections at various developmental stages revealed that Dres9 is expressed at very high levels in the neural retina and in the central nervous system (CNS), similar to its Drosophila counterpart. The high level of sequence conservation and similarities in the expression patterns of rdgB and DRES9 during development in Drosophila and mammals indicate that Dres9 is the orthologue of RdgB, and strongly suggest a possible functional conservation of these proteins during evolution. DRES9 encodes a phosphatidylinositol-transfer protein, suggesting that phosphatidylinositol may have a role as an intracellular messenger in vertebrate phototransduction. The identification of this gene and the study of its expression pattern in mammals will help shed new light on the evolution of vision mechanisms and suggest DRES9 as a candidate gene for human retinopathies.

Identification and mapping of human cDNAs homologous to Drosophila mutant genes through EST database searching.

Cross-species comparison is an effective tool used to identify genes and study their function in both normal and pathological conditions. We have applied the power of Drosophila genetics to the vast resource of human cDNAs represented in the expressed sequence tag (EST) database (dbEST) to identify novel human genes of high biological interest. Sixty-six human cDNAs showing significant homology to genes causing Drosophila mutant phenotypes were identified by screening dbEST using the "text string' option, and their map position was determined using both fluorescence in situ hybridization (FISH) and radiation hybrid mapping. Comparison between these genes and their putative partners in Drosophila may provide important insights into their function in mammals. Furthermore, integration of these genes into the transcription map of the human genome contributes to the positional candidate approach for disease gene identification.