ABSTRACT
Recent genetic analysis of the Drosophila dachshund (dac) gene has established that dac encodes a novel nuclear protein that is involved in both eye and leg development. In the Drosophila eye, dac expression appears to be controlled by the product of the eyeless/Pax6 gene. In order to analyze the Pax6 pathway in vertebrates we have isolated and characterized the cDNA and genomic clones corresponding to the human and mouse homologues of Drosophila dac. A full-length human cDNA encoding dachshund (DACH) encodes the 706 amino acids protein with predicted molecular weight of 73 kDa. A 109 amino acid domain located at the N-terminus of the DACH showed significant sequence and secondary structure homologies to the ski/sno oncogene products. Northern blot analysis found human DACH predominantly in adult kidney, heart, and placenta, with less expression detected in the brain, lung, skeletal muscle and pancreas. A panel of human cell lines was studied and most notably a large proportion of neuroblastomas expressed DACH mRNA. Mouse Dach encodes a protein of 751 amino acids with predicted molecular weight of 78 kDa that is 95% identical to the human DACH. RNase protection analysis showed the highest Dach mRNA expression in the adult mouse kidney and lung, whereas lower expression was detected in the brain and testis. RT/PCR analysis readily detected Dach mRNA in the adult mouse cornea and retina. Dach mRNA expression in the mouse E11.5 embryo was observed primarily in the fore and hind limbs, as well as in the somites.
Subject(s)
Drosophila Proteins , Embryonic and Fetal Development , Gene Expression Regulation , Nuclear Proteins/genetics , Transcription, Genetic , Adult , Amino Acid Sequence , Animals , Cloning, Molecular , Drosophila/genetics , Female , Gene Expression Regulation, Developmental , Humans , Mice , Molecular Sequence Data , Nuclear Proteins/biosynthesis , Nuclear Proteins/chemistry , Organ Specificity , Pregnancy , RNA, Messenger/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Transcription factors Pax-4 and Pax-6 are known to be key regulators of pancreatic cell differentiation and development. We report on the cloning of a mouse Pax-4 gene, which contains 10 exons, spanning a 4. 7-kbp region. The gene-targeting experiments revealed that Pax-4 and Pax-6 cannot substitute for each other in tissue with overlapping expression of both genes. We identified DNA-binding specificities of Pax-4 paired domain and paired-type homeodomain. Despite the different Pax-4 amino acid residues in positions responsible for Pax-6 paired-domain specificity, the DNA-binding specificities of Pax-4 and Pax-6 are similar. The Pax-4 homeodomain was shown to preferentially dimerize on DNA sequences consisting of an inverted TAAT motif, separated by 4-nucleotide spacing. The Pax-4 transactivation domain was localized within its C-terminal region, which transactivated GAL-based reporter 2.5-fold less than the C-terminal region of Pax-6. We believe that Pax-4 can act as a Pax-6 "repressor," due to the competition for binding sites and lower transactivation potential of Pax-4.
Subject(s)
DNA-Binding Proteins/physiology , Homeodomain Proteins/physiology , Transcription Factors/physiology , Transcriptional Activation/physiology , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Cloning, Molecular , Dose-Response Relationship, Drug , Exons , Eye Proteins , Humans , Introns , Kidney/metabolism , Luciferases/metabolism , Mice , Molecular Sequence Data , PAX6 Transcription Factor , Paired Box Transcription Factors , Repressor Proteins , Sequence Homology, Amino AcidABSTRACT
On the basis of developmental gene expression, the vertebrate central nervous system comprises: a forebrain plus anterior midbrain, a midbrain-hindbrain boundary region (MHB) having organizer properties, and a rhombospinal domain. The vertebrate MHB is characterized by position, by organizer properties and by being the early site of action of Wnt1 and engrailed genes, and of genes of the Pax2/5/8 subfamily. Wada and others (Wada, H., Saiga, H., Satoh, N. and Holland, P. W. H. (1998) Development 125, 1113-1122) suggested that ascidian tunicates have a vertebrate-like MHB on the basis of ascidian Pax258 expression there. In another invertebrate chordate, amphioxus, comparable gene expression evidence for a vertebrate-like MHB is lacking. We, therefore, isolated and characterized AmphiPax2/5/8, the sole member of this subfamily in amphioxus. AmphiPax2/5/8 is initially expressed well back in the rhombospinal domain and not where a MHB would be expected. In contrast, most of the other expression domains of AmphiPax2/5/8 correspond to expression domains of vertebrate Pax2, Pax5 and Pax8 in structures that are probably homologous - support cells of the eye, nephridium, thyroid-like structures and pharyngeal gill slits; although AmphiPax2/5/8 is not transcribed in any structures that could be interpreted as homologues of vertebrate otic placodes or otic vesicles. In sum, the developmental expression of AmphiPax2/5/8 indicates that the amphioxus central nervous system lacks a MHB resembling the vertebrate isthmic region. Additional gene expression data for the developing ascidian and amphioxus nervous systems would help determine whether a MHB is a basal chordate character secondarily lost in amphioxus. The alternative is that the MHB is a vertebrate innovation.
