Three novel Pax6 alleles in the mouse leading to the same small-eye phenotype caused by different consequences at target promoters.
OBJECTIVE
To characterize three new mouse small-eye mutants detected during ethylnitrosourea mutagenesis programs.


METHODS
Three new mouse small-eye mutants were morphologically characterized, particularly by in situ hybridization. The mutations were mapped, and the candidate gene was sequenced. The relative amount of Pax6-specific mRNA was determined by real-time PCR. Reporter gene analysis used Crygf and Six3 promoter fragments in front of a luciferase gene and HEK293 cells as recipients.


RESULTS
The new mutations--ADD4802, Aey11, and Aey18--were mapped to chromosome 2; causative mutations have been characterized in Pax6 (Aey11: C-->T substitution in exon 8, creating a stop codon just in front of the homeobox; ADD4802: G-->A substitution at the beginning of intron 8 changes splicing and leads to an altered open reading frame and then to a premature stop codon; Aey18: G-->A exchange in the last base of intron 5a leads also to a splice defect, skipping exons 5a and 6). Real-time PCR indicated nonsense-mediated decay in Pax6Aey11 and Pax6Aey18 mutants but not in Pax6ADD4802. This result is supported by the functional analysis of corresponding expression constructs in cell culture, where the Aey11 and Aey18 alleles did not show a stimulation of the Six3 promotor or an inhibition of the Crygf promoter (as wild-type constructs do). However, the Pax6ADD4802 allele stimulated both promoters.


CONCLUSIONS
Together with functional analysis in a reporter gene assay and immunohistochemistry using Pax6 antibodies, it is suggested that the Pax6Aey11 and Pax6Aey18 alleles act through a loss of function, whereas ADD4802 represents a gain-of-function allele.
Graw
Jochen
J
Institute of Developmental Genetics, GSF-National Research Center for Environment and Health, Neuherberg, Germany. graw@gsf.de
Löster
Jana
J
Puk
Oliver
O
Münster
Doris
D
Haubst
Nicole
N
Soewarto
Dian
D
Fuchs
Helmut
H
Meyer
Birgit
B
Nürnberg
Peter
P
Pretsch
Walter
W
Selby
Paul
P
Favor
Jack
J
Wolf
Eckhard
E
Hrabé de Angelis
Martin
M
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005038:Ethylnitrosourea / Q000633:toxicity; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000235:genetics*; D017403:In Situ Hybridization; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008850:Microphthalmos / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics*; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000097607:Homeobox Protein SIX3
D005038:Ethylnitrosourea; D005136:Eye Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012333:RNA, Messenger; D012097:Repressor Proteins; D000097607:Homeobox Protein SIX3; C000605732:Pax6 protein, mouse