The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains

Abstract

Mouse Tabby (Ta) and X chromosome-linked human EDA share the features of hypoplastic hair, teeth, and eccrine sweat glands. We have cloned the Ta gene and find it to be homologous to the EDA gene. The gene is altered in two Ta alleles with a point mutation or a deletion. The gene is expressed in developing teeth and epidermis; no expression is seen in corresponding tissues from Ta mice. Ta and EDA genes both encode alternatively spliced forms; novel exons now extend the 3' end of the EDA gene. All transcripts recovered have the same 5' exon. The longest Ta cDNA encodes a 391-residue transmembrane protein, ectodysplasin-A, containing 19 Gly-Xaa-Yaa repeats. The isoforms of ectodysplasin-A may correlate with differential roles during embryonic development.

Figure 1

(A) The location of the Tabby (Ta) locus on the mouse X chromosome genetic map (17) and EDA on a syntenic physical map of the human X chromosome (18). (B) Partial genomic organization of the Ta gene and corresponding transcript isoforms (Ta A, Ta B, and Ta C). Transcript size and protein length for each transcript are shown in brackets. Translated regions for exons (boxes) are drawn to scale and numbered. Note that no exon 2 corresponding to human EDA exon 2 has been detected in mouse. Numbers below each box represent amino acids encoded by the respective exon. Arrowheads show the locations of the exon–intron junctions in mouse genomic DNA. Split codons (see text) are shown by extensions within a box. Alternative exons (1a in Ta C and 3a in Ta B) are indicated. Potential splice site sequences are shown at 3′ ends of exon 1 and exon 3 in Ta C and Ta B transcripts. Sequence at the exon 4–intron junction (–TCTGgtgagt—) is not shown. Polyadenylylation site AATAAA or AGTAAA and poly(A) tail are indicated. (C) Nucleotide sequence (first 1,375 bp of 5,004-bp sequence, GenBank accession no. AF016628), predicted amino acid sequence of Ta A, and predicted peptide sequence of Ta B and Ta C. The predicted start of translation at +1 nucleotide with the in-frame stop codon (TAG) at nucleotides 1,174–1,176 yields an ORF of 1,176 bp that encodes a predicted protein of 391 amino acids in Ta A. A putative transmembrane domain is underlined, and the Gly-Xaa-Yaa collagenous repeats are in underlined italics. The locations of identified introns are indicated by arrowheads. Cysteine residues are indicated in underlined boldface. Amino acids in lowercase type are translated by in-frame adjacent intron sequences in alternatively spliced cDNAs Ta B and Ta C. (D) Amino acid sequence comparison of the mouse Ta B protein with its human EDA homologue. Identities are shown as dots.

Figure 2

Analysis of rearrangement in Ta gene in two Tabby alleles. (A) PCR-based detection of exons deleted in Tabby alleles. Products for exons 1, 3 ,and 5 were amplified using oligonucleotide primer pairs and visualized on EtBr-stained agarose gels. Deletion is evident for exon 1 in one Ta allele (lane 2). Lanes: 1 and 3, control mouse DNAs, respectively, for Ta (lane 2) and Tabby 〈6J〉 (lane 4); 5–7, other mouse DNAs; 8, water; 9, human DNA. (B) Exon 1 sequence analysis of mutated Tabby 〈6J〉 allele and respective mouse control. Point mutation 550delT is indicated by an arrow. Sequence reads in the sense direction from bottom to top (Upper). (Lower) Nucleotide sequence and translated amino acids of normal control (wild type) and mutant Ta 〈6J〉 allele. Amino acids shown in italics are due to a frame-shift mutation and results in premature stop codon (*).

Figure 3

Northern blot analysis of the expression of Ta gene. A 589-bp PCR product specific for exon 1 region was hybridized to RNA samples derived from staged mouse embryos (Left) and mouse adult tissues (Right).

Figure 4

Expression of Ta mRNA in embryonic tooth and skin. In situ hybridization was performed with a ³³P-labeled antisense RNA probe. (a and b) A wild-type late cap stage E15 tooth. Ta mRNA is expressed in the outer enamel epithelium (arrow) and the dental lamina. No expression in the inner enamel epithelium (arrowhead) and the mesenchyme. (c and d) In corresponding Tabby teeth there is no expression (arrow indicate outer enamel epithelium). (e and f) Neck skin from a wild-type E14 embryo. Tabby expression is concentrated in the epidermis (arrow). (g and h) In Tabby neck skin there is no expression. (a, c, e, and g) Digital bright-field images overlaid with expression in red. (b, d, f, and h) Digital dark-field images. e, epithelium; m, mesenchyme.