High-resolution mapping of tlt, a mouse mutant lacking otoconia
Huacheng C. Ying,
* Bele´n Hurle´,
* Yunxia Wang,
Barbara A. Bohne,
Mary K. Wuerffel,
David M. Ornitz
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA
Department of Otolaryngology, Washington University School of Medicine, St. Louis, Missouri 63110, USA
Received: 17 November 1998 / Accepted: 1 February 1999
Abstract. The ability to sense gravity is enhanced by an extra-
cellular structure that overlies the macular sensory epithelium.
This complex consists of high density particles, otoconia, embed-
ded within a gelatinous membrane. The tilted mouse specifically
lacks otoconia, yet has no other detectable anatomic lesions. Fur-
thermore, the penetrance of the tilted phenotype is nearly 100%.
This mouse provides a model to identify genes that are involved in
the development and function of vestibular otoconia. Using SSLP
markers, we have mapped the tilted (tlt) gene on mouse Chromo-
some (Chr) 5 between D5Mit421 and D5Mit353/D5Mit128/
D5Mit266/D5Mit267 by analysis of the progeny of an intersub-
intercross. We also mapped the fibroblast growth factor
receptor 3 (Fgfr3) gene, a potential candidate for tlt, and the Hun-
tington’s disease homolog (Hdh) gene to D5Mit268, approxi-
mately 4.3 centiMorgans (cM) from the tilted locus. This study
excludes both Fgfr3 and Hdh as candidate genes for tlt and iden-
tifies closely linked microsatellite markers that will be useful for
the positional cloning of tlt.
The ability to sense gravity and to maintain spatial orientation
relies on specialized sensory organs within the vestibular portion
of the inner ear. The three semicircular canals and adjoining cristae
respond to angular acceleration, whereas the macular epithelium,
located within the saccule and utricle, responds to the forces of
gravity and linear acceleration (Hunter-Duvar and Hinojosa 1984).
Because orientation with respect to gravity is essential for the
survival of complex organisms, the gravity receptor is one of the
phylogenetically oldest sensory systems, and the special adapta-
tions to sense gravity and linear acceleration are highly conserved.
Enhanced sensitivity to gravity depends on a specialized extracel-
lular structure overlying the macular epithelium. This structure
consists of high density particles called otoconia embedded within
a gelatinous membrane.
In mammals, otoconia are composed of proteins (otoconins)
and calcium carbonate crystals (calcite; Pote and Ross 1991). Oto-
conin-90 (OC90), a ∼90 kDa glycoprotein, accounts for 90% of the
organic matrix of otoconia (Pote and Ross 1986; Wang et al.
1998). Oc90 has recently been cloned and mapped to mouse Chr
15 (Wang et al. 1998, 1999; Verpy et al. 1999). In addition to the
OC90, 4–5 less abundant otoconins are visible by 2D-PAGE; how-
ever, their identity remains elusive (Ornitz et al. 1998; Wang et al.
Several mouse (Lim et al. 1978; Trune and Lim 1983; Rolfsen
and Erway 1984; Lane 1986; Kitamura et al. 1991; Steel 1995;
Bergstrom et al. 1998; Kozel et al. 1998) and zebrafish mutants
(Riley and Grunwald 1996; Whitfield et al. 1996) have been iden-
tified that have defects in balance and coordination, providing
potential models to study the development and function of the
vestibular system. However, many of these mutations result in
degeneration of vestibular structures and/or show variable pen-
etrance, circumstances that make it difficult to identify the specific
genes that are involved in vestibular pathology. Only the tilted,
head tilt, and tilted head mutants appear to have pathology limited
to vestibular otoconia (Lim et al. 1978; Sweet 1980; Lane 1986,
1987; Bergstrom et al. 1998; Ornitz et al. 1998).
The tilted mutation arose spontaneously in a cross of (C3H ×
with the p
tester stock (M. Lyon, personal communi-
cation) and has subsequently been bred onto a C57BL/6J back-
ground and cryopreserved at The Jackson Laboratory (Lane 1986,
1987; Ornitz et al., 1998). Homozygous tilted mice can readily be
identified by observing their inability to swim (Ornitz et al. 1998).
This phenotype has a penetrance of nearly 100%. Under normal
laboratory conditions, many tilted mice exhibit a head-tilting pos-
ture but show no other behavioral abnormalities such as circling or
head-tossing. Anatomical and histological analyses showed that
tilted mice specifically lack otoconia without defects in the sensory
epithelium, gelatinous membrane, or other vestibular structures
(Ornitz et al. 1998).
The tilted locus has been mapped to mouse Chr 5 between
hammer toe (Hm, 6.9 cM) and Kit (Kit
, 42.0 cM) (Hm–6.9–
; Lane 1986, 1987). These data placed tlt, with
very low resolution, at 22.9 cM, in a region that appears to be
syntenic with human Chr 4p16 (Grosson et al. 1994; Nasir et al.
1994). Within the region of 4p16.3 is the Huntington’s disease
gene (HD or IT15; Thompson et al. 1991) and the closely linked
fibroblast growth factor receptor 3 gene (FGFR3; Shiang et al.,
In this study we have constructed a detailed genetic linkage
map around the tilted locus based on a large intersubspecific F
intercross between C57BL/6J-tlt and Mus musculus castaneus. The
refined map places tlt at 16.9 cM from the top of mouse Chr 5 on
the MIT map, between D5Mit421 and D5Mit353/D5Mit128/
D5Mit267/D5Mit266. To further define the synteny between
mouse Chr 5 and the region around human 4p16.3 and to rule out
allelism between tlt, Fgfr3, and the mouse Huntington’s disease
homolog (Hdh, Barnes et al. 1994), these genes were also mapped
on this cross. We show that both Fgfr3 and Hdh colocalize with
D5Mit268, 4.3 cM centromeric to tlt.
Materials and methods
C57BL/6J-tlt mice were obtained from the embryo stocks
at The Jackson Laboratory (Bar Harbor, Me.) and have been maintained on
this background by sequentially mating tlt/tlt mice to wild-type C57BL/6J,
and in the F
generation intercrossing two tlt/+ siblings. Homozygous tlt/tlt
mice were identified based on their inability to swim. An F
cific cross was established to map markers proximal to the tilted locus. F
hybrid mice were generated by mating homozygous C57BL/6J-tlt mice
with wild-type Mus musculus castaneus. The F
intercross mice were de-
Correspondence to: D. Ornitz
* Authors have made equal contribution to this work.
Mammalian Genome 10, 544–548 (1999).
© Springer-Verlag New York Inc. 1999