Deletion of a conserved regulatory element required for Hmx1

RESEARCH ARTICLE
Disease Models & Mechanisms 5, 812-822 (2012) doi:10.1242/dmm.009910
Deletion of a conserved regulatory element required for
Hmx1 expression in craniofacial mesenchyme in the
dumbo rat: a newly identified cause of congenital ear
malformation
Lely A. Quina1,*, Takashi Kuramoto2,*, Daniela V. Luquetti3,4, Timothy C. Cox3,4,5, Tadao Serikawa2 and Eric E. Turner1,3,6,‡
Disease Models & Mechanisms DMM
SUMMARY
Hmx1 is a homeodomain transcription factor expressed in the developing eye, peripheral ganglia, and branchial arches of avian and mammalian
embryos. Recent studies have identified a loss-of-function allele at the HMX1 locus as the causative mutation in the oculo-auricular syndrome (OAS)
in humans, characterized by ear and eye malformations. The mouse dumbo (dmbo) mutation, with similar effects on ear and eye development, also
results from a loss-of-function mutation in the Hmx1 gene. A recessive dmbo mutation causing ear malformation in rats has been mapped to the
chromosomal region containing the Hmx1 gene, but the nature of the causative allele is unknown. Here we show that dumbo rats and mice exhibit
similar neonatal ear and eye phenotypes. In midgestation embryos, dumbo rats show a specific loss of Hmx1 expression in neural-crest-derived
craniofacial mesenchyme (CM), whereas Hmx1 is expressed normally in retinal progenitors, sensory ganglia and in CM, which is derived from mesoderm.
High-throughput resequencing of 1 Mb of rat chromosome 14 from dmbo/dmbo rats, encompassing the Hmx1 locus, reveals numerous divergences
from the rat genomic reference sequence, but no coding changes in Hmx1. Fine genetic mapping narrows the dmbo critical region to an interval
of ~410 kb immediately downstream of the Hmx1 transcription unit. Further sequence analysis of this region reveals a 5777-bp deletion located
~80 kb downstream in dmbo/dmbo rats that is not apparent in 137 other rat strains. The dmbo deletion region contains a highly conserved domain
of ~500 bp, which is a candidate distal enhancer and which exhibits a similar relationship to Hmx genes in all vertebrate species for which data are
available. We conclude that the rat dumbo phenotype is likely to result from loss of function of an ultraconserved enhancer specifically regulating
Hmx1 expression in neural-crest-derived CM. Dysregulation of Hmx1 expression is thus a candidate mechanism for congenital ear malformation,
most cases of which remain unexplained.
INTRODUCTION
Hmx1 is a homeodomain transcription factor expressed in the
developing eye, peripheral ganglia and branchial arches of avian
and mammalian embryos (Wang et al., 2000; Yoshiura et al., 1998).
Relatively little is known about the function of Hmx1, but recently
an Hmx1 allele has been identified as the causative genetic defect
1
Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle,
WA 98101, USA
2
Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University,
Kyoto 606-8501, Japan
3
Center on Human Development and Disability, University of Washington, Seattle,
WA 98101, USA
4
Department of Pediatrics (Craniofacial Medicine), University of Washington, and
Center for Tissue & Cell Sciences, Seattle Children’s Research Institute, Seattle,
WA 98101, USA
5
Department of Anatomy and Developmental Biology, Monash University, Clayton,
Victoria 3800, Australia
6
Department of Psychiatry and Behavioral Sciences, University of Washington,
Seattle, WA, USA
*These authors contributed equally to this work
‡
Author for correspondence (eric.turner@seattlechildrens.org)
Received 26 March 2012; Accepted 30 May 2012
© 2012. Published by The Company of Biologists Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution
Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), which
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that the original work is properly cited and all further distributions of the work or adaptation are
subject to the same Creative Commons License terms.
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in a human disorder, oculo-auricular syndrome (OAS) (Schorderet
et al., 2008). Patients with OAS exhibit malformations of the outer
ear (pinna) and defects of the eye, including microphthalmia,
cataract, coloboma and retinal dystrophy (Schorderet et al., 2008;
Vaclavik et al., 2011).
Animal models of OAS include the dumbo (dmbo) and misplaced
ears (mpe) mutations in mice (Munroe et al., 2009). Mouse dmbo,
mouse mpe and the known human Hmx1 allele causing OAS are
all coding mutations that affect the Hmx1 DNA-binding
homeodomain, and thus are predicted to result in loss of function.
In addition to malformed ears, dumbo mice exhibit eye
malformations, although less severe than those observed in the OAS
patients identified to date. In both mouse and man, hearing is
spared. In rats, the dumbo (dmbo) trait identified in animals kept
by amateur ‘fancy rat’ breeders has also been mapped to a 5 Mb
region encompassing the Hmx1 locus (Kuramoto et al., 2010), but
the nature of the causative mutation is unknown.
Recent work in mice has also revealed a role for Hmx1 in the
development of sensory neurons. Hmx1 is widely expressed in the
sensory peripheral nervous system, including a subset of neurons
in the trigeminal ganglion, geniculate ganglion, superior ganglion
of the IX-X ganglion complex and dorsal root ganglia (Wang et al.,
2000; Yoshiura et al., 1998). In the caudal cranial ganglia, Hmx1 is
confined to somatosensory neurons and is not expressed in the
distal viscerosensory component of these ganglia. Despite the wide
expression of Hmx1 in the sensory system, only the geniculate
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Rat dumbo Hmx1 mutation
TRANSLATIONAL IMPACT
Clinical issue
The oculo-auricular syndrome (OAS) is a rare human craniofacial disorder that
involves multiple anomalies of the eyes (retinal dystrophia, microphthalmia,
chorioretinal colobomas and cataract) and ears (lobe malformation and
simplification of ear morphology). OAS in humans has been linked to a
deletion in the gene HMX1, a transcription factor expressed in the developing
eye, peripheral nervous system and branchial arches of birds and mammals.
However, the function of this gene and the nature of the disrupted
embryological processes that result in these anomalies are not known.
Moreover, the genetic and non-genetic causes of much more prevalent
congenital anomalies of the ear, with and without associated eye
malformations, remain largely unknown. Eye and ear anomalies have profound
effects on the lives of the affected individuals. The study of animal models that
advance our understanding of these conditions, and enable specific diagnosis
and ultimately treatment of patients, are an invaluable resource.
Disease Models & Mechanisms DMM
Results
In prior work, the dumbo phenotype, named for its characteristic ear
malformation, has been linked to the Hmx1 gene in rats and mice. Our results
show that neonatal dumbo rats have a similar phenotype to that observed in
dumbo mice, with characteristic ventral displacement of the ear and a
moderate degree of microphthalmia. Dumbo rats show a specific loss of Hmx1
expression in neural-crest-derived craniofacial mesenchyme, particularly the
maxillary component and most of the mandibular component of the first
branchial arch, and the distal part of the second branchial arch. Expression of
Hmx1 in the early developing retina and peripheral nervous system is intact.
Unlike the known OAS HMX1 allele in humans and the dumbo mutation in
mice (both of which prevent translation of the Hmx1 homeodomain),
sequence analysis of the chromosomal region that encompasses the rat Hmx1
locus reveals no coding changes in the Hmx1 open reading frame. Instead,
dumbo rats exhibit a novel 5777 bp deletion 79 kb downstream from Hmx1,
which includes an ‘ultraconserved’ region of ~500 bp that is likely to be crucial
for Hmx1 expression in the branchial arches.
Implications and future directions
This detailed genetic analysis of the dumbo rat suggests that the causative
mutation is deletion of a conserved enhancer specifically affecting Hmx1
expression in neural-crest-derived mesenchyme, and future studies will focus
on unraveling the nature of this regulatory allele. In future studies of
individuals with ear malformations, exon sequencing alone will not be
sufficient to exclude the Hmx1 locus; regulatory alleles of Hmx1 must also be
considered. These results also suggest that Hmx1 lies in the middle of a poorly
understood pathway for development of the pinna. Thus, genes both
upstream and downstream of Hmx1, as well as the Hmx1 locus itself, are
candidates for the site of the causative allele in studies of individuals with
syndromic or isolated eye and ear anomalies.
somatosensory neurons appear to require it for neurogenesis
(Quina et al., 2012), whereas early development of the trigeminal
and dorsal root ganglia appear normal in dumbo mice.
In the present study, we show that dumbo rats and mice exhibit
similar ear malformations and extent of microphthalmia. Hmx1
protein expression in dumbo rat embryos shows no change in the
early developing eye and sensory ganglia compared with controls,
but dumbo embryos exhibit specific loss of Hmx1 expression in
the mesenchymal components of branchial arches 1 and 2 (BA1
and BA2), which give rise to the pinna. Fate-mapping of Hmx1expressing craniofacial mesenchyme (CM) cells in transgenic mice
shows that this region of decreased Hmx1 expression in the dumbo
rat corresponds to CM that is derived from neural crest, but not
from cranial mesoderm. To better understand the genetic basis of
Disease Models & Mechanisms
RESEARCH ARTICLE
this specific loss of Hmx1 expression, we resequenced 1 Mb of rat
chromosome 14 encompassing the Hmx1 locus, revealing
numerous divergences from the rat genomic reference sequence,
but no coding changes in Hmx1. Single nucleotide polymorphisms
within this span allowed the dmbo critical region to be narrowed
to the interval of ~410 kb immediately downstream of the Hmx1
transcription unit. Within this region, we identified a 5777-bp
deletion that encompasses a ~500-bp region conserved across all
vertebrate species. Together, these results suggest that the rat dmbo
mutation is a cis-acting regulatory allele of an ultraconserved
enhancer that is necessary for Hmx1 expression in neural-crestderived CM. Dysregulation of Hmx1 expression is thus a potential
causative mechanism in many patients with congenital ear
malformations, a common abnormality for which the etiology is
largely unknown.
RESULTS
Dumbo rats exhibit congenital malformations of the pinna similar
to dumbo mice, and modest reduction in ocular size at birth
In order to compare the phenotypes of dumbo mice and rats, we
first examined the morphology of the ear and eye in newborn
dumbo animals and controls. The characteristic ventral
displacement of the ear in dumbo rats and mice, which gives the
mutation its name, has been previously described in adults
(Kuramoto et al., 2010; Munroe et al., 2009). To define an endpoint
for developmental studies, we examined the morphology of the
external ear in neonatal dumbo rats and mice at postnatal day 2
(P2) and P1, respectively. At P2, dumbo rats exhibited marked
ventral displacement of the pinna compared with heterozygous
controls (Fig. 1A,B). P1 Hmx1dm/dm mice also exhibited ventral
displacement and, in addition, the top of the pinna was rotated
posteriorly by 33° relative to controls (Fig. 1C,D).
To determine the extent of congenital microphthalmia in dumbo
rats and mice we measured the neonatal eye. The average diameter
of the newborn dumbo rat eye was modestly decreased, to 86%
that of heterozygous controls (Fig. 1E). However, the eye was of
normal size at E14 (data not shown), indicating that the ocular size
difference must be generated during the period of extensive eye
growth in late gestation. Prior characterization of the Hmx1dm/dm
mouse identified a microphthalmia phenotype in adults but did
not quantify the reduction in eye size (Munroe et al., 2009). In the
present study, comparison of P1 dumbo mice (Hmx1dm/dm) to
littermate controls (Hmx1dm/+ and Hmx1+/+) revealed that the mean
diameter and length of the orbit in Hmx1dm/dm mice was reduced
to 91% that of controls, corresponding to approximately 75% of
normal ocular volume (Fig. 1F-H). We then undertook detailed
developmental studies to examine the cellular expression of Hmx1
in the developing CM and eye in order to better understand these
deformities.
Expression of Hmx1 in the mouse craniofacial mesenchyme
In developing mice, Hmx1 mRNA can be detected in BA2 at E10.5,
and Hmx1 protein in the mesenchyme of proximal BA1 at E11.5
(Quina et al., 2012; Wang et al., 2000; Yoshiura et al., 1998).
Malformation of the pinna is evident by E14.5 in Hmx1dm/dm
embryos (data not shown). Thus the crucial events leading to the
dumbo ear phenotype occur in the E10.5-E14.5 developmental
interval in mice. To better understand the expression of Hmx1 at
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RESEARCH ARTICLE
Rat dumbo Hmx1 mutation
Disease Models & Mechanisms DMM
Region 3 consisted of an extensive region of posterior mesenchyme,
caudal to the head vein and branchial arches, ending at the top of
the limb bud (Fig. 2A-H). By contrast, extensive regions of CM,
particularly the maxillary component and most of the mandibular
component of BA1 and the proximal part of BA2, do not express
Hmx1. Based on past studies of the embryological origin of the
external ear, only Region 2 CM in the distal part of BA1 and BA2
is likely to contribute directly to the pinna.
Fig. 1. Ear and eye phenotypes in the newborn rat and mouse. Dumbo rat
and mouse pups and controls were compared at P2 and P1, respectively.
(A)Rat controls (dmbo/+) and (B) rat dumbo (dmbo/dmbo) pups. (C)External
features of controls (Hmxdm/+ or Hmx1+/+, which are indistinguishable) and (D)
dumbo mice (Hmx1dm/dm), showing characteristic rotation and ventral
displacement of the ear (dotted lines). Reduced ocular size is evident through
the closed eye (arrows). (E)Measurement of eye diameter in P2 rat embryos
(control, mean 2.5 mm, n6; dumbo, mean 2.15 mm, n4; P0.03, unpaired ttest). (F,G)P1 mouse eye, shown in lateral and frontal views for both
genotypes. D, diameter; L, length. (H)Measurement of reduced eye diameter
and length in the P1 eye of Hmx1dm/dm mice, n5 of each genotype. Diameter:
control, mean1.89 mm; Dumbo, mean1.72 mm; P0.0002 (unpaired t-test).
Length: control, mean1.60 mm; Dumbo, mean1.45 mm; P0.009. Error bars
show ± s.d.
the onset of pinna development, we first examined in detail the
expression of Hmx1 protein in the CM of E11.5 Hmx+/+ mouse
embryos (Fig. 2). Hmx1dm/dm mouse embryos were not examined
because we have previously demonstrated that Hmx1 protein is
not detectable in these embryos at these stages (Quina et al., 2012).
In the E11.5 mouse embryo, Hmx1 was expressed in three
regions of the CM. Region 1 consisted of the part of proximal BA1
overlying the trigeminal ganglion (Fig. 2A-D). Region 2 comprised
the caudal half of distal BA2 (Fig. 2D-F), and a small region of the
caudal or distal tip of the mandibular component of BA1 (Fig. 2F,G).
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Expression of Hmx1 in the dumbo rat embryo
In order to understand the expression of Hmx1 in the dumbo rat
embryo, and the basis of the rat dumbo phenotype, we examined
Hmx1 expression in E13 rat embryos (equivalent to the E11.5 mouse
described above) and in E14 rat embryos (equivalent to an E12.5
mouse). Dumbo rats and embryos were generated by interbreeding
KFRS4/Kyo parental rats. Age-matched controls were generated
from crosses of the PVG/seac strain (see Methods).
Examination of BA1 in E13 dumbo rat embryos and controls
revealed loss of Hmx1 expression in the proximal part of BA1,
overlying the trigeminal ganglion, defined in controls as Region 1
(Fig. 3B-I). The thickness of the mesenchyme overlying the trigeminal
ganglion, measured as the distance between the trigeminal ganglion
itself and the surface ectoderm, was not altered, indicating that there
was loss of Hmx1 expression in this domain rather than loss of the
CM cells as such. Expression of Hmx1 was also diminished in the
region of branchial arches 3 and 4, overlying the superior ganglion
(Fig. 3F-G). Remarkably, expression of Hmx1 was not affected in the
sensory neurons residing at the same axial levels, including the
mandibular lobe of the trigeminal ganglion (Fig. 3H,I), the
somatosensory neurons of the geniculate ganglion or the Hmx1expressing neurons in the statoacoustic ganglion (Fig. 3J,K). The
Hmx1 signal in the retina and the characteristic nasotemporal
gradient of retinal expression were also unchanged in dumbo
embryos compared with controls (Fig. 4B,C).
Similar loss of Hmx1 expression was seen in the ventral branchial
arches of E13 dumbo rat embryos, designated as Region 2. Hmx1
was expressed in the caudal part of distal BA2 in control embryos,
whereas dumbo embryos revealed a complete loss of Hmx1
expression in this region (Fig. 4D-G). By contrast, expression was
unchanged in the posterior mesenchyme designated as Region 3
(Fig. 4D,E). The cervical dorsal root ganglia (DRG) of dumbo
embryos also maintained normal Hmx1 expression (Fig. 4H,I), as
did the cranial sensory ganglia.
In E14 rat embryos (equivalent to an E12.5 mouse), Hmx1 was
prominently expressed in the lateral facial mesenchyme, including
a region of expression posterior to the eye, and dorsal to the
branchial arches, which was not evident at E13 (Fig. 5). Doublelabeling experiments with Hmx1 and Ap2a demonstrated that
Hmx1 is expressed in the mesenchyme of this region, but not in
the surface ectoderm (supplementary material Fig. S1). To better
define the Hmx1 expression differences in dumbo embryos, serial
sections were examined throughout this dorsal domain, and the
extent of Hmx1 expression was measured using semi-quantitative
immunofluorescence (Fig. 5A-C). The overall size of the area of
expression was reduced moderately (44%) in the most dorsal
region of expression, at the level of the eye and lateral to the
hypothalamus (Fig. 5B,C, level 0), and severely (74-92%) in the
ventral region of expression, lateral to the mandibular lobe of the
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Disease Models & Mechanisms DMM
Rat dumbo Hmx1 mutation
Fig. 2. Hmx1 expression in developing mouse craniofacial mesenchyme.
(A-H)Serial sections of a wild-type E11.5 mouse embryo showing the
expression domains of Hmx1 in the CM. Successively more ventral planes of
section show distribution of Hmx1-expressing cells in the CM. Arrows in F and
G indicate the region of expression in ventral BA1. (I)Planes of section in A-H.
Shaded areas indicate regions of Hmx1 expression: 1, part of proximal BA1
overlying the trigeminal ganglion; 2, the caudal half of distal BA2 and a small
region of the caudal/distal tip of the mandibular component of BA1; 3, an
extensive region of posterior mesenchyme, caudal to the head vein and
branchial arches, ending at the top of the limb bud. BA1, branchial arch 1; BA2,
branchial arch 2; BA3, branchial arch 3; C1, brachial (pharyngeal) cleft (groove)
1; C2, brachial cleft 2; Cor, heart; Di, diencephalon; DRG, dorsal root ganglion;
EAM, external auditory meatus; FL, forelimb; Hb, hindbrain; GG, geniculate
ganglion; Inf X, inferior ganglion of the IX-X ganglion complex
(nodose/petrosal ganglion); Mn, mandibular process (of BA1); Mx, maxillary
process (of BA1); mnTG, mandibular lobe, trigeminal ganglion; mxTG, maxillary
lobe, trigeminal ganglion; OV, otic vesicle; P, pons; RP, Rathke’s pouch; SAG,
statoacoustic ganglion; SC, spinal cord; SG, superior ganglion; V, head vein.
Scale bar: 200m.
trigeminal ganglion and pons (Fig. 5B,C, levels 7-9; P1.7⫻10–4 for
all ten planes of section). In the intermediate sections, where there
was partial loss of Hmx1 expression, the decreased expression
occurred mainly rostral to the midpoint of the trigeminal ganglion
(Fig. 5C, arrows).
By contrast, no difference was observed in Hmx1 expression in
the mandibular lobe of the trigeminal ganglion (Fig. 5D,E). In the
retina, the overall expression of Hmx1 in the E14 retinal
neuroepithelium was reduced compared with E13, but no difference
was noted between dumbo embryos and controls. Onset of Hmx1
expression was noted in early differentiating retinal ganglion cells
(neurons), which first appear at this stage, some of which also
expressed Brn3a (Fig. 5F,G). The normal expression of Hmx1 in
the eye at this stage is consistent with our observation that ocular
Disease Models & Mechanisms
RESEARCH ARTICLE
Fig. 3. Loss of Hmx1 expression in proximal BA1 in the E13 Dumbo rat. E13
rat embryos, developmentally equivalent to E11.5 mice, were examined using
immunofluorescence for Hmx1 and for Brn3a, which identifies somatosensory
neurons. (A)Plane of section in subsequent views. (B-E)Control (B,D) and
dumbo (C,E) embryos showing loss of expression of Hmx1 in the dorsalmost
part of BA1 in the mutant. The CM overlying the TG is present (dashed line in
E), but fails to express Hmx1. Expression of Hmx1 is unaffected in the mnTG
and SAG. (F-K)Control (F,H,J) and dumbo (G,I,K) embryos showing loss of
Hmx1 expression in BA1 in dumbo embryos. Some loss of Hmx1 expression is
also noted in posterior mesenchyme overlying the head vein in the mutant
(arrowheads, F). CM, craniofacial mesenchyme; Di, diencephalon; GG,
geniculate ganglion; GGS, geniculate ganglion, somatosensory component;
GGV, geniculate ganglion, viscerosensory component; Hb, hindbrain; mnTG,
mandibular lobe, trigeminal ganglion; mxTG, maxillary lobe, trigeminal
ganglion; OV, otic vesicle; SAG, statoacoustic ganglion; SG, superior ganglion
(of IX-X ganglion complex); TG, trigeminal ganglion, V, head vein. Scale bar:
200m.
size was also normal at E14. We conclude that the mild
microphthalmia observed in neonatal dumbo rats is due to
diminished ocular growth in late gestation.
Loss of Hmx1 expression in the Dumbo rat embryo occurs
specifically in neural-crest-derived mesenchyme
Detailed examination of E13 and E14 rat embryos shows that the
loss of Hmx1 expression in dumbo embryos is not uniform, but
instead shows marked regional specificity, with profound loss in
CM in the branchial arches and relative sparing of expression in
regions of Hmx1 expression in the parietal region adjacent to the
hindbrain.
The craniofacial mesenchyme, which gives rise to the bones and
cartilage of the head, is known to originate from both the cranial
neural crest and mesoderm, depending on location (Le Douarin et
al., 1993). Our observation that Hmx1 expression in dumbo rat
embryos is lost only in specific regions of CM led us to hypothesize
that the effect of the rat dmbo mutation might differ according to
the embryological origin of the affected CM cells. In mice, tissue
derived from neural crest in the cranium and branchial arches can
be identified by embryological fate mapping, using a Wnt1-Cre
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Disease Models & Mechanisms DMM
RESEARCH ARTICLE
Fig. 4. Loss of Hmx1 expression in BA2 in the E13 Dumbo rat. (A)Plane of
section in subsequent views. (B,C)Hmx1 is expressed in a nasotemporal
gradient in the retina which is unchanged in dumbo embryos.
(D-G)Expression of Hmx1 in the distal branchial arches; top of figures is
anterior. Hmx1 is not expressed in distal BA1 at this stage. Hmx1 expression in
distal BA2 is lost in the dumbo embryo (G). Expression of Hmx1 in posterior
mesenchyme is not affected (D,E arrowheads). Some neurons in the inferior
part of the tenth ganglion are weakly positive for Brn3a. (H-I⬘)Expression of
Hmx1 in the DRG is unchanged in dumbo embryos. Top of figure is dorsal.
BA2, branchial arch 2; BC1, brachial cleft 1; BC2, brachial cleft 2; BP1, brachial
pouch 1; BP1, brachial pouch 2; DRG, dorsal root ganglion; L, lens; mxBA1,
maxillary component of BA1; mnBA1, mandibular component of BA1; n, nasal
(aspect of retina); Nd, inferior tenth (nodose) cranial ganglion; t, temporal
(aspect of retina); SC, spinal cord; V, head vein. Scale bars: 200m (B) and
100m (D).
driver and a LoxP-mediated reporter (Danielian et al., 1998). In
mid-gestation embryos, a clear boundary can be shown between
neural-crest-derived frontonasal structures and mesoderm-derived
parietal structures (Jiang et al., 2002). Because no such approach
is available for rats, we chose to determine the embryological origin
of the Hmx1-expressing CM in a transgenic mouse model.
To assess the embryological origin of the Hmx1-expressing cells
in the CM, we crossed mice carrying Wnt1-Cre with a reporter
strain, Ai6, which conditionally expresses the fluorescent reporter
ZS-green from a modified Rosa26 locus (Madisen et al., 2010). In
E12.5 Wnt1-Cre/Ai6 mouse embryos, expression of the induced
marker gene revealed an occult boundary between the neural-crestderived CM in frontonasal and branchial arch regions and the
mesoderm-derived CM in the parietal region (Fig. 6), which was
consistent with prior studies (Jiang et al., 2002; Yoshida et al., 2008).
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Rat dumbo Hmx1 mutation
Fig. 5. Altered Hmx1 expression in the E14 dumbo rat. Hmx1 expression
was examined by immunofluorescence in the lateral cranial mesenchyme of
E14 embryos, at which stage the branchial arches are no longer anatomically
distinct structures. (A)Planes of section for levels analyzed in subsequent
views. (B,C)Semiquantitative analysis of Hmx1 expression in BA1 at E14. The
area and intensity of Hmx1 expression was measured at 160-m intervals
encompassing the cranial mesenchyme lateral to the trigeminal ganglion and
pons. The overall area of Hmx1 immunofluorescence staining were
significantly diminished in the dumbo embryo (paired t-test, n10, P0.00017).
In the more dorsal sections, the loss of expression occurred mainly rostral to a
boundary at about the midpoint of the trigeminal ganglion (arrows). The main
effect on Hmx1 expression was to reduce the area of expression, but the
intensity of the immunofluorescence signal was also somewhat diminished in
the expressing area (paired t-test, n10, P0.003). (D,E)Expression of Hmx1 in
mandibular lobe of trigeminal ganglion. (F,G)Expression of Hmx1 in
differentiating retinal ganglion cells, some of which also express Brn3a. CM,
craniofacial mesenchyme; GCL, ganglion cell layer; Hb, hindbrain; mnTG,
mandibular lobe, trigeminal ganglion; mxTG, maxillary lobe, trigeminal
ganglion; NE, neuroepithelium; Ret, retina; V, head vein. Scale bars: 100m
(C,D) and 50m (F).
Hmx1-expressing CM cells were found in both the neural crest and
mesoderm compartments, but the embryological origin of the
Hmx1 cells differed markedly according to axial level. In the most
dorsal and rostral domain of expression, at the level of the
diencephalon (Fig. 6B), Hmx1-expressing CM cells were derived
entirely from mesoderm. At the level of the trigeminal and
geniculate ganglia and the future pons, an increasing proportion
of the Hmx1-expressing CM cells resided in the neural-crestdmm.biologists.org
Disease Models & Mechanisms DMM
Rat dumbo Hmx1 mutation
Fig. 6. Hmx1 is expressed in CM of neural crest and non-crest origin. A
Wnt1-Cre transgenic line was interbred with the reporter strain Ai6, expressing
the fluorescent protein ZEG from a modified Rosa26 locus. Mouse embryos
were analyzed at E12.5. (A)Whole-mount E12.5 embryo showing planes of
section for levels analyzed in subsequent views. The ganglia and projections
of the sensory peripheral nervous system are marked by expression of a LacZ
transgene targeted to the Brn3a locus. (B-F)Progressively more ventral and
caudal sections showing expression of Hmx1 in crest-derived and mesodermderived CM. The endogenous fluorescence of the ZEG reporter appears in
green. Dashed lines demarcate the border of the cranial neural-crest-derived
tissue. BA1-4, branchial arch 1-4; Di, diencephalon; GG, geniculate ganglion;
Hb, hindbrain; inf IX/X, inferior part of IX-X ganglion complex (nodose and
petrosal ganglia); mnTG, mandibular lobe, trigeminal ganglion; mxTG,
maxillary lobe, trigeminal ganglion; Ret, retina; SAG, statoacoustic ganglion;
SG, superior ganglion (of the IX-X ganglion complex).
derived compartment in progressively more ventral and caudal
sections (Fig. 6C-E). Caudal to the otic region, in CM overlying the
superior ganglion and future medulla, Hmx1 was again expressed
predominantly in the mesoderm-derived CM (Fig. 6F).
Although it is not possible to directly fate-map the neural crest
and parietal mesoderm in rat embryos, the loss of Hmx1 expression
in the dumbo rat appears to be largely confined to the neural crest
compartment, with persistence of Hmx1 expression in the
mesoderm-derived CM (compare Fig. 5, L5 with Fig. 6D and Fig.
5, L7 with Fig. 6E). However, the Hmx1 expression is not affected
in the trigeminal or dorsal root ganglia, which are also largely
neural-crest-derived. Thus, it appears that the dumbo rat phenotype
results from loss of Hmx1 expression specifically within the neuralcrest-derived CM, suggesting a tissue-specific defect in the
regulation of Hmx1 expression.
Sequence of the rat dumbo critical region reveals deletion of a
unique distal conserved region.
Tissue-specific loss of Hmx1 expression in neural-crest-derived CM
could in principle result from the loss of a trans-acting regulatory
factor governing expression in this domain, or from the disruption
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RESEARCH ARTICLE
of a cis-acting neural-crest- or CM-specific regulatory element at
the Hmx1 locus. However, prior mapping of the rat dmbo allele to
a critical region of ~5 Mb encompassing the Hmx1 locus suggested
a cis-acting mechanism (Kuramoto et al., 2010).
In an effort to determine the nature of the dmbo mutation, we
performed resequencing of a 1.0-Mb region of the dmbo/dmbo rat
strain KFRS4, spanning the interval from 80.5 to 81.5 Mb of
chromosome 14, which includes the core of the dmbo critical region
(see Methods). Comparison of this region with the Brown Norway
(BN) reference sequence revealed 31 single nucleotide
polymorphisms (SNPs) residing in the coding sequences of nine of
the ten annotated genes in this interval (supplementary material
Table S1), as well as numerous SNPs in the intergenic regions. Eight
of the SNPs resulted in conservative amino acid changes, and 23
were synonymous. The single SNP identified within the Hmx1
coding sequence was silent (L251L).
The identification of additional SNPs distinguishing KFRS4 from
the BN reference sequence allowed further refinement of the
genetic map of the dmbo critical region. To fine-map the dmbo locus,
we assessed the progeny of a (BN/SsNSlc ⫻ KFRS4/Kyo)F1 ⫻
KFRS4/Kyo backcross. Transmission or non-transmission of the
dmbo allele was determined by the appearance of the pinna at 3
weeks of age. A total of 614 backcross progeny were examined, of
which 322 expressed the dumbo ear phenotype and 292 had normal
ears. Recombination between the chromosomal markers D14Rat10
and D14Rat57 was observed in 31 of the backcross progeny, and
these animals were used for fine mapping (Fig. 7). By genotyping
of recombinant progeny with Ablim2_SNP(H42Y) and
Hmx1_SNP(L251L), the minimal dmbo critical region was refined
to a 411.5-kb segment between 80.58 and 81.0 Mb of Chr14 (Fig.
7A). The haplotypes of animals used to define the critical region
appear in supplementary material Fig. S2.
Only two mis-sense mutations were localized to this refined dmbo
critical region (supplementary material Table S1), Cpz_S23P and
Acox3_N287H. However, these SNPs are not specific to the
KFRS4/Kyo strain. Cpz_S23P has been identified as a homozygous
SNP (rs64860291) between the BN reference strain and the Sprague
Dawley (SD) strain, which does not have dumbo ears.
Acox3_N287H has been identified by our group as a homozygous
mutation in the NER rat strain (data not shown), an inbred line
derived from Crj:Wistar rats, which does not exhibit dumbo ears.
Although resequencing provided ~99% coverage of the dmbo
critical region, we wished to determine whether small
discontinuities detected in the sequence data represented genomic
rearrangements. To search for deletions or insertions, we used long
PCR to amplify segments of DNA from dumbo and control rats
spanning the 120-kb intergenic region from the 3⬘ end of the Hmx1
gene to the 3⬘ end of the Cpz gene. One primer set spanning the
region 80-90 kb downstream of the Hmx1 gene yielded a ~6 kb
shorter product in dumbo samples than in controls, and a mixed
product in heterozygotes (supplementary material Table S2).
Sequencing of the PCR product from this region revealed a 5777bp deletion in KFRS4/Kyo genomic sequence relative to the
reference strain (Fig. 7B). The deletion was not detected in a panel
of 137 other rat strains, strongly suggesting that it is the causative
allele for the rat dumbo phenotype. To further verify the
relationship between this deletion and the dumbo phenotype, we
amplified the breakpoint region in DNA samples obtained from
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Rat dumbo Hmx1 mutation
Fig. 7. Structure of the rat dmbo critical region. (A)Genetic linkage map
(left) and physical map (right) of the region of rat chromosome 14
encompassing the dmbo allele. The linkage map was derived from
examination of 614 progeny of a (BN/SsNSlc ⫻ KFRS4/Kyo)F1 ⫻ KFRS4/Kyo
backcross. Recombination events between the markers Ablim2_SNP(H42Y)
and Hmx1_SNP(L251L) place the dmbo allele critical region within a 410-kb
interval. Resequencing data revealed a novel 5777-bp deletion between the
Cpz and Hmx1 genes (red). (B)Structure of the KFRS4/Kyo deletion breakpoint.
four dumbo rats with diverse genetic backgrounds, obtained from
an enthusiast breeder in California. Each of these animals exhibited
the same 5777-bp deletion 3⬘ to the Hmx1 transcribed region
observed in KFRS4/Kyo (supplementary material Fig. S3), and no
coding changes in the Hmx1 exonic sequence.
Because the 5.8-kb dmbo deletion lies in a presumed intergenic
region, we used BLAST and VISTA genomic search tools to
determine whether the deletion region contained sequence
elements conserved across species. A BLAST search revealed a high
degree of similarity between the rat dmbo deletion region and
sequences in the Hmx1-Cpz intergenic region in the mouse (Chr:5)
and human (Chr:4) genomes. The BLAST search did not reveal
significant homology to identified transcripts or expressed sequence
tags in any species, confirming the intergenic nature of the deletion
region and suggesting instead a regulatory function for the
conserved sequence.
Because of the limited annotation of the rat genome, we used
the homologous region of the mouse genome to generate VISTA
alignments (Dubchak et al., 2009) of conserved sequences in the
dmbo deletion region across multiple species. VISTA analysis
revealed a highly conserved region of ~500 bp within the deletion
region that is present in all mammalian species (Fig. 8A). MultiZ
alignment revealed that a core sequence is conserved across
vertebrate classes, including in the chicken, frog and zebrafish
genomes (Fig. 8B). This Hmx distal conserved region (DCR) occurs
consistently in a syntenic chromosomal region including the Hmx
locus or loci and the Cpz locus in all species for which complete
data are available. In chicken and zebrafish, two closely related
Hmx-class genes are present in tandem in this region. Although
the genomic distance between the Hmx transcribed region and the
818
Fig. 8. Comparative genomics of the Hmx1 distal conserved region.
(A)VISTA alignment of the dmbo deletion region. The reference sequence for
the alignment is chr5:35,807,810-35,813,977 (version NCBI37/mm9) of the
mouse genome. A highly conserved region of ~500 bp is detected across all
mammalian species, and a core sequence is conserved across vertebrate
classes. (B)Map of the relationship of the DCR to Hmx and Cpz genes for all
vertebrate species for which contiguous genomic sequence is available. Red
arrows indicate the 3⬘ end of the actual or predicted Hmx1 and Cpz
transcripts, and the direction of transcription. The distance from Hmx1 to the
DCR in the human genome is unknown because of a gap in genomic
coverage. The chick genome contains two Hmx1 homologues, Hmx1 and
SOHo1, transcribed on the same strand; similarly, the zebrafish genome
contains two Hmx1 homologues, Hmx1 and Hmx4. For each species, the
genome build version and genomic coordinates of the core conserved
sequence (~300 bp) are as follows: Mouse, mm9, chr5:35,808,825-35,809,130;
Rat, rn4, chr14:80,916,323-80,916,629; Human, hg18, chr4:8,753,059-8,753,365;
Chimp, panTro2, chr4_random:6,195,662-6,195,968; Dog, canFam2,
chr3:63,192,444-63,192,750; Cow, bosTau3, chr6:108,192,356-108,192,675;
Opossum, monDom4, chr5:228,001,978-228,002,289; Chick, galGal3,
chr4:84,267,370-84,267,675; Lizard, anoCar1, scaffold_326:1,558,1351,558,406; Frog, xenTro2, scaffold_583:238,403-238,706; Zebrafish, danRer7,
chr1:41,246,062-41,246,105.
DCR varies from 45 kb (zebrafish) to 326 kb (opossum), no
intervening identified transcripts occur between the Hmx loci and
the DCR in any species. We conclude that the Hmx DCR is a strong
candidate for an ancient distal enhancer that regulates Hmx
expression in the branchial arch CM.
DISCUSSION
The dumbo mouse and rat strains were named for their most
obvious physical characteristic, displaced and malformed ears, prior
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Disease Models & Mechanisms DMM
Rat dumbo Hmx1 mutation
to any knowledge of the genetic mechanism underlying these
malformations. The dumbo mouse phenotype was identified in a
mutagenesis screen (Wilson et al., 2005), whereas the dumbo rat
mutation arose spontaneously among ‘fancy’ rats kept by enthusiast
breeders, probably in the western United States, and has become
a popular pet strain on the basis of its novel appearance. Given the
large number of genes that are likely to play a role in the
morphogenesis of the pinna, it is remarkable that both the mouse
and rat dumbo strains converged on the same gene locus. The
appearance of the mouse and rat dumbo ears are quite similar, but
at the cellular level the phenotypes of the mouse and rat mutants
reveal other striking differences. In recent work, we have shown
that Hmx1dm/dm mice exhibit marked loss of geniculate ganglion
neurons and the associated posterior auricular nerve (Quina et al.,
2012). The cranial nerve defects in dumbo mice were identified
using transgenic tract-tracing, and this method cannot not be used
in the rat, but the geniculate ganglion itself appears normal in
dumbo rat embryos. Furthermore, Hmx1 expression is normal in
dumbo rats during the critical developmental period in which the
geniculate ganglion defects appear in mice. In general, we infer that
the sensory ganglia develop normally in dumbo rats, because
sensory expression of Hmx1 does not appear to be affected.
Even without the sequence of the dumbo rat Hmx1 locus, it is
clear that the fundamental genetic mechanism of the dmbo
mutation is distinctive in the mouse and rat. The mouse Hmx1dm
allele is a nonsense mutation preceding the Hmx1 homeodomain
(Munroe et al., 2009), and Hmx1 protein is undetectable in
Hmx1dm/dm mice, probably because the truncated protein is rapidly
degraded (Quina et al., 2012). Similarly, the causative allele in
human oculoauricular syndrome is predicted to result in loss of
protein function (Schorderet et al., 2008). By contrast, in dumbo
rat embryos, Hmx1 immunoreactivity appears normal in the retina,
sensory ganglia and the part of the CM that is not derived from
neural crest, findings that are not compatible with a nonsense
mutation. Sequence data confirm that the dumbo rat Hmx1 open
reading frame and splice junctions are intact. Instead, the KFRS4
strain of dumbo rats exhibits a 5777-bp deletion approximately 79
kb distal to the end of the final Hmx1 exon that is not found in any
of 137 other rat strains tested. This, combined with the
identification of a highly conserved region in the dumbo deletion
region, strongly suggests that the functional rat dmbo allele consists
of the loss of an enhancer that regulates expression in neural-crestderived CM.
The physical distance between the Hmx1 transcription unit and
the Hmx1 DCR, from ~80 kb in mouse and rat up to 326 kb in the
opossum, is remarkable. However, there are examples of verified
enhancer function at even greater distances. One example is a highly
conserved cis-acting element that regulates Shh expression in the
developing limb and is the site of mutations resulting in limb
abnormalities in humans and mice, despite the fact that it is located
~1 Mb from the Shh transcription unit, in an intron of an upstream
gene (Lettice and Hill, 2005; Lettice et al., 2002; Sagai et al., 2005;
Sagai et al., 2004). Although not identified in prior studies, the
Hmx1 DCR has key characteristics of the class of regulatory
sequences that have been called ‘conserved non-coding elements’
(Woolfe et al., 2005), and ‘ultraconserved enhancers’ (Pennacchio
et al., 2006). In addition to recognizable sequence conservation
between higher and lower vertebrates, these elements are often
Disease Models & Mechanisms
RESEARCH ARTICLE
located adjacent to developmental regulators, such as tissuespecific transcription factors. Although in some cases the targeted
deletion of ultraconserved regions has failed to show an obvious
phenotype (Ahituv et al., 2007), in the case of the rat Hmx1 DCR
the loss-of-function phenotype is clearly highly penetrant, resulting
in characteristic ear deformities in the large range of genetic
backgrounds maintained by the rat-enthusiast breeder community.
Prior to the mapping of the rat dmbo mutation to the Hmx1
locus, dysregulation of the homeobox genes Msx1 and Dlx1 was
proposed as a causative mechanism for the rat dumbo phenotype
(Katerji et al., 2009). Loss-of-function mutations in Msx1/2 and
Dlx1/2 result in much more extensive defects in craniofacial
development than the phenotypes observed in the dumbo rat and
mouse (Ishii et al., 2005; Qiu et al., 1997; Qiu et al., 1995). Clearly,
the causative mutation does not reside in these genes, but they are
potential upstream regulators of Hmx1.
The phenotypes of the dumbo rat and mouse help to clearly
distinguish the role of Hmx1 from that of the structurally related
factors Hmx2 and Hmx3 (Wang et al., 2000; Yoshiura et al., 1998),
which are highly coexpressed, and have well-characterized and
partially redundant roles in vestibular and hypothalamic
development in mice (Wang et al., 2001; Wang et al., 2004; Wang
et al., 1998). Two Hmx1 homologues have been identified in the
chicken, GH6/Hmx1 and SOHo1. The expression of genes encoding
these factors in the branchial arches, retina and cranial and spinal
sensory ganglia suggest that they are the functional homologues
of mouse and rat Hmx1, not Hmx2/3 (Deitcher et al., 1994; Stadler
and Solursh, 1994). The DCR identified in the dumbo rat is located
downstream of the chick Hmx1 and SOHo1 transcription units,
providing further confirmation that they are the functional
homologues of mammalian Hmx1. Like mouse Hmx1, GH6 and
SOHo1 are expressed in a nasal-temporal gradient in the eye, and
misexpression of these factors alters the topographic mapping of
retinal axons onto their midbrain targets (Schulte and Cepko, 2000).
However, these findings do not explain the ocular defects observed
in OAS patients (coloboma) or dumbo rats and mice
(microphthalmia), and further studies of the role of Hmx1 in retinal
development are necessary.
The distinctive nature of the Hmx1 mutant phenotypes,
resulting either from the loss of protein function or defective
regulation, is underscored by comparison with the effects of other
genes that regulate morphogenesis of the pinna. One example is
Hoxa2, which affects ear development through specification of
segmental identity in the branchial arches, a classic homeotic
function. BA2 development is particularly influenced by the loss
of Hoxa2, which results in transformation of BA2-derived
structures into BA1 phenotypes and absence of the pinna
(Santagati et al., 2005). Although Hmx1 is a homeodomain
protein, its late expression in a restricted set of neural and
mesenchymal cell types across all cranial segments excludes a
Hox-like role in the assignment of the segmental identity.
Consistent with this, Hmx1 mutants do not show the extensive
changes in multiple structures derived from BA2, such as the inner
ear and brainstem, that are observed in Hoxa2 mutants. In chick
embryos, mis-expression of Hoxa2 in BA1 can induce ectopic
expression of the chick Hmx1-class factor SOHo1, suggesting that
Hmx1 might lie downstream of this Hox gene (Grammatopoulos
et al., 2000). However, this cannot reflect an exclusive requirement
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RESEARCH ARTICLE
for Hoxa2 because the normal boundaries of Hmx1 expression
extend beyond the Hoxa2 expression domain.
An Hmx gene is also present in the Drosophila genome [H6like-homeobox, FlyBase ID FBgn0264005 (Wang et al., 2000)]. The
regulatory function of the Drosophila Hmx protein overlaps that
of mouse Hmx2/3, because Drosophila Hmx can partially rescue
the hypothalamic and inner ear phenotypes observed in Hmx2/3
double mutant mouse embryos (Wang et al., 2004). The ‘nonhomeotic homeobox’ role of the Hmx family is underscored by the
fact that although insertional mutants of Drosophila Hmx are
known (http://flybase.org/reports/FBgn0264005.html), they
apparently do not exhibit homeotic transformations and there are
no published reports of Hmx function in flies.
Mutations affecting development of the external ear might also
result from defective neural crest development, a mechanism
exemplified by the role of Tcof1/Treacle in Treacher Collins
Syndrome (TCS), an autosomal dominant disorder that frequently
includes hypoplasia of the facial bones, cleft palate and middle ear
defects in addition to malformation of the pinna (Dixon et al., 2007).
Treacle is expressed throughout the dorsal neural tube and early
neural crest. Mice haplo-insufficient for Tcof1 replicate some of
the core features of TCS and demonstrate a crucial role for Tcof1
in the craniofacial neural crest (Dixon et al., 2006). By contrast,
Hmx1 is not expressed in the early neural crest, and our fatemapping studies have shown that Hmx1 is expressed in the CM
without respect to the crest-derived compartments in the
developing head. Thus the developmental roles of Tcof1 and Hmx1
might intersect but are not analogous.
These findings on the unique developmental role of Hmx1 have
significant implications for understanding human congenital
disorders that affect the ear and lateral face and that have a major
impact on individuals affected by these disorders. Abnormal
morphology and malpositioning of the ear (typically described as
‘low-set’ and ‘posteriorly rotated’) are among the most common
malformations seen in these patients. Ear anomalies might occur
in dysmorphic syndromes, where they represent one of a multitude
of abnormally developed structures, or in isolation (Calzolari et al.,
1999; Carey et al., 2006; Suutarla et al., 2007). It is also not surprising
that the stigma associated with ear malformation and the burden
of one or more corrective surgeries can have significant
psychosocial sequelae (Steffen et al., 2010).
Much progress has been made in the identification of genes
responsible for many dysmorphic syndromes, including TCS,
Miller syndromes (DHODH, Ng et al., 2010), CHARGE (CHD7,
Lalani et al., 2004; Vissers et al., 2004), the 22q11.2 deletion
syndrome (TBX1, Chieffo et al., 1997) and Townes-Brock syndrome
(SALL1, Kohlhase et al., 1998). However, malformation of the ear
is more commonly observed as an isolated finding (Canfield et al.,
2009; Carey et al., 2006; Forrester and Merz, 2005; Harris et al.,
1996; Mastroiacovo et al., 1995; Shaw et al., 2004; Suutarla et al.,
2007). To our knowledge, the Hmx1-linked allele responsible for
the rat dumbo phenotype is the first genetic variant to be identified
in any species with such a high degree of specificity for
malformation of the pinna. It suggests that neural-crest-specific
regulatory alleles at the Hmx1 locus could underlie isolated
malformations of the pinna in humans, as a variant of the OAS
that does not include severe eye deformities. It also provides a
causative model for common ear malformations and is potentially
820
Rat dumbo Hmx1 mutation
the key to a developmental pathway for pinna morphogenesis that
underlies these disorders.
METHODS
Animals and genotyping
Mice bearing the dmbo allele (Hmx1dm), strain B6;C3FeHmx1dmbo/Rw/JcsJKjn, were obtained from Jackson Laboratories
(Stock #008677). The Hmx1dm allele was originally the result of
ENU mutagenesis on a C57BL/6 genetic background (Wilson et
al., 2005). Mice were crossed to C57BL/6N (Charles River) for two
to five additional generations prior to the experiments described.
Experiments were performed with littermate controls. Genotyping
of the Hmx1dm and wild-type Hmx1 (Hmx1+) alleles was performed
by real-time PCR using oligonucleotide primers having 3⬘ termini
at the point mutation that characterizes the dmbo mutation, as
previously described (Quina et al., 2012).
The founder rat expressing the dumbo ear phenotype [SRR04,
(Kuramoto et al., 2010)] was obtained from a commercial breeder
in the United States and crossed with the PVG/seac strain of Black
Hooded rats to generate the strain KFRS4, which is homozygous
for the recessive alleles dumbo (dmbo) and the coat color allele
head spot (hs). In prior work, the dmbo locus was mapped to the
5.7-Mb region defined by D14Rat10 and D14Rat57 (Kuramoto et
al., 2010).
To further refine the dmbo locus, we produced 614 (BN/SsNSlc
⫻ KFRS4/Kyo)F1 ⫻ KFRS4/Kyo backcross progeny. The
BN/SsNSlc rat strain was purchased from Japan SLC (Hamamatsu,
Japan). Genotypes of the dmbo locus were determined by the
appearance of the outer ear (dumbo malformation or wild-type) at
3 weeks of age. Animals with recombination events between
D14Rat10 and D14Rat57 were used for the fine mapping of the dmbo
critical region.
Newborn dumbo rats and E13 and E14 dumbo embryos were
generated by interbreeding KFRS4/Kyo parental rats. For Hmx1
expression studies, age-matched wild-type controls were generated
from PVG/seac rats. For measurements of ocular diameter,
littermate controls were used. Mouse embryos were staged
according to a published method (Theiler, 1972) and the
corresponding rat embryo stages were identified according to
Witschi (Altman and Katz, 1962).
Resequencing of the rat dmbo critical region
Regions for targeted resequencing spanned from 80.5 to 81.5 Mb
of the rat chromosome 14 and contained the dmbo critical region.
The 1.0-Mb target region of KFRS4/Kyo DNA was enriched using
the SureSelect oligonucleotide hybridization solution-based capture
technique (Agilent). The enriched fragment library was then
subjected to PCR amplification, followed by sequencing on an
Illumina GAIIx platform. Coverage of 40⫻ or greater was obtained
for 98.86% of the target region. The reads generated were mapped
to the rat genome reference sequence (Baylor HGSC v3.4/rn4) using
Burrows-Wheeler alignment (Li and Durbin, 2009). The known
variants between KFRS4 and the rat genome reference sequence
were identified using the Ensembl BioMart search function from
the Ensembl variation 66 database. Further resequencing of the
dmbo critical region was performed using long PCR using
PrimeSTAR GXL DNA polymerase (Takara, Otsu, Japan). VISTA
alignment of the dmbo deletion region was performed using the
dmm.biologists.org
Rat dumbo Hmx1 mutation
Disease Models & Mechanisms DMM
Lawrence Berkeley National Laboratory and US Department of
Energy
Joint
Genome
Institute
VISTA
browser
http://genome.lbl.gov/vista/index.shtml (Dubchak et al., 2009).
Identification of conserved sequences across mammalian and
vertebrate species was performed using MultiZ alignment using
the UCSC genome browser http://genome.ucsc.edu/ (Kent et al.,
2002).
Immunofluorescence
Embryos for immunofluorescence were fixed in 4%
paraformaldehyde in phosphate-buffered saline, equilibrated in 15%
then 30% sucrose, embedded in OCT medium and cryo-sectioned
at 14-20 m, depending on stage. Antisera to Hmx1 was prepared
in rabbits to a glutathione-S-transferase (GST)/Hmx1 fusion
protein containing amino acids 2-188 of the Hmx1 protein,
excluding the conserved homeodomain, as previously described
(Quina et al., 2012). The Hmx1 antiserum shows no reactivity to
CM in Hmx1dm/dm mice. Polyclonal rabbit and guinea pig antisera
against Brn3a have also been previously described (Quina et al.,
2005). Mouse monoclonal anti-Ap2a antibody (3B5) was obtained
from the Developmental Studies Hybridoma Bank. Alexa-Fluorconjugated species-specific secondary antibodies were obtained
from Life Technologies (Grand Island, NY).
For semiquantitative immunofluorescence measurement of
Hmx1 expression, serial sections of dumbo and control rat embryos
were processed, immunostained and imaged in parallel, with the
same imaging parameters and no post-processing. Areas of interest
in the CM were defined manually, and the area, total fluorescence
signal and mean fluorescence signal were calculated using ImageJ.
ACKNOWLEDGEMENTS
We would like to thank Janell McMorran of Ratterfly Rattery, Bonita CA, as well as
Sondra Truffat and Madeleine Truffat for introducing E.E.T. to dumbo rats, and
Nicole Fung for initial work on the dumbo rat coding sequence. We would also like
to thank Mayuko Yokoe for her technical assistance and the National BioResource
Project – Rat for providing the KFRS4/Kyo strain (NBRP#0572).
COMPETING INTERESTS
The authors declare that they do not have any competing or financial interests.
AUTHOR CONTRIBUTIONS
L.Q., E.E.T., T.K. and T.S. conceived and designed the experiments. L.Q. and T.K.
performed the experiments. L.Q., E.E.T., T.K. and T.C.C. analyzed the data. L.Q., E.E.T.,
T.K., D.V.L. and T.C.C. wrote the paper.
FUNDING
Supported by Department of Veterans Affairs MERIT funding, and the National
Institutes of Health [grant numbers HD33442, MH065496 and NS064933, to E.E.T.].
E.E.T. is a NARSAD Investigator. Also supported by grants-in-aid for Scientific
Research from the Japan Society for the Promotion of Science [grant number
21300153 to T.K.] and a grant-in-aid for Cancer Research from the Ministry of
Health, Labour and Welfare.
SUPPLEMENTARY MATERIAL
Supplementary material for this article is available at
http://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.009910/-/DC1
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