An ENU-induced mutation in AP-2a leads to middle ear
and ocular defects in Doarad mice
* Alexandra Erven,
* Helmut Fuchs,
Martin Hrabe de Angelis,
Karen B. Avraham,
Karen P. Steel
Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
MRC Institute of Hearing Research, University Park, Nottingham, UK
GSF Research Center for Environment and Health, Institute of Experimental Genetics, Neuherberg, Germany
Departments of Craniofacial Biology and of Cell and Structural Biology, University of Colorado Health Sciences Center, Denver,
Colorado, 80262, USA
Received: 9 October 2003 / Accepted: 10 February 2004
One of the advantages of N-ethyl- N-nitrosourea
(ENU)-induced mutagenesis is that, after randomly
causing point mutations, a variety of alleles can be
generated in genes leading to diverse phenotypes. For
example, transcription factor AP-2a (Tcfap2a) null
homozygote mice show a large spectrum of devel-
opmental defects, among them missing middle ear
bones and tympanic ring. This is the usual occur-
rence, where mutations causing middle ear anoma-
lies usually coincide with other abnormalities.
Using ENU-induced mutagenesis, we discovered a
new dominant Tcfap2a mutant named Doarad (Dor)
that has a missense mutation in the PY motif of its
transactivation domain, leading to a misshapen
malleus, incus, and stapes without any other
observable phenotype. Dor homozygous mice die
perinatally, showing prominent abnormal facial
structures and ocular defects. In vitro assays suggest
that this mutation causes a ‘‘gain of function’’ in the
transcriptional activation of AP-2a. These mice en-
able us to address more specifically the develop-
mental role of Tcfap2a in the eye and middle ear and
are the first report of a mutation in a gene specifi-
cally causing middle ear abnormalities, leading to
conductive hearing loss.
The embryological origin of the middle ear is di-
verse, since the middle ear and tympanic membrane
arise from different developmental origins. The
middle ear ossicles are formed from the first two
branchial arches that originate from neural crest
cells (NCC) that migrate from the developing mid-
brain and hindbrain. The differentiation and survival
of these cells in the branchial arches is thought to be
mediated by the transcription factor AP-2a (locus
Tcfap2a), among others (Mallo 1998).
AP-2a is a member of a family of transcription
factors that function in a wide range of biological
roles, including developmental processes, apoptosis,
and cell cycle control (Hilger–Eversheim et al. 2000).
It has a helix–span–helix motif in its C-terminal re-
gion that allows homodimerization while the basic
helical DNA-binding motif enables binding to vari-
ous GC-rich DNA sequences. Its N-terminal region
is proline and glutamine rich and functions as a
transcriptional activation region, and hence is
named the transactivation domain.
Gene-targeted mutagenesis of the Tcfap2a gene
leads to phenotypically normal heterozygous mice,
except for 4% that show dental malocclusions
(Zhang et al. 1996), while homozygous mice die
perinatally, showing a wide range of severe devel-
opmental defects (Schorle et al. 1996; Zhang et al.
1996). The phenotype is first observed at embryonic
day 9.5, with a failure in closure of the cranial neural
folds that leads to anencephaly and missing ventral
craniofacial structures such as a mouth, snout, or
ears. Tcfap2a knockouts (Tcfap2a
) also have
achordal skeleton defects with missing maxilla,
cranial vault, middle ear bones, and tympanic ring.
They also display other skeletal defects such as
*These authors contributed equally to this study.
Present address: The Wellcome Trust Sanger Institute, Wellcome
Trust Genome Campus, Hinxton, Cambridge, UK.
Correspondence to: Karen B. Avraham, E-mail: firstname.lastname@example.org.
DOI: 10.1007/s00335-004-2334-z Volume 15, 424–432 (2004) Springer-Verlag New York, Inc. 2004