Title

Isolation, characterization, mapping and expression analysis of the human neuralized gene

Date of Completion

January 1998

Keywords

Biology, Neuroscience|Biology, Genetics|Health Sciences, Human Development

Degree

Ph.D.

Abstract

Split hand/split foot malformation (SHSF) is a human developmental defect characterized by the abscence of central digits of the hand and/or foot, accompanied by deep median clefts resulting in a claw-like appearance of the autopod. We have previously mapped four split hand/split foot families to human chromosome 10q24-25, a major autosomal dominant locus for this disease, designated as SHFM3.^ Searching for candidate genes within the SHFM3 critical region, we identified a human EST with significant homology to the D. melanogaster neuralized gene. Given the fact that the fly neuralized gene was expressed in the wing margin of the wing imaginal discs, we thought that it would be an attractive candidate for SHFM3. Subsequently, we proceeded to screen a human cerebellum cDNA library using the EST as a probe. Three positive clones were isolated and sequenced. The sequences were assembled into a cDNA that contained a 1724 bp open reading frame. The coding sequence exhibited significant homology with the D. melanogaster Neuralized gene and was thus considered to be the human homologue of Neuralized. The cDNA was predicted to encode for a 574 amino acid protein with a putative zinc finger motif at its carboxy terminus.^ We identified a genomic Pac clone that contained the human Neuralized gene and we mapped it to chromosome 10q24-25, within the SHFM3 critical region. The gene is composed of five exons extending over 7 kb of genomic DNA. Northern analysis revealed a single 4.4 kb transcript.^ In situ hybridization analysis in sections from a 6 week old human embryo showed high Neuralized expression in cartilage primordia of all major bones of the limbs, cranium and thorax. Strong uniform expression was observed throughout the skin ectoderm. Other sites of expression included muscles, meninges and the choroid plexus. In situs through an E16 mouse were in agreement with the above expression pattern and also revealed Neuralized transcripts in the cephalic mesenchyme, heart, lung, and liver. These results implicate vertebrate Neuralized in skeletogenesis and epithelial differentiation. ^