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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
Center for Integrative Brain Research, Seattle Childrens 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 Childrens 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
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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.
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.
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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, n6; dumbo, mean 2.15 mm, n4; P0.03, 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, n5 of each genotype. Diameter:
control, mean1.89 mm; Dumbo, mean1.72 mm; P0.0002 (unpaired t-test).
Length: control, mean1.60 mm; Dumbo, mean1.45 mm; P0.009. Error bars
show s.d.
trigeminal ganglion and pons (Fig. 5B,C, levels 7-9; P1.7104 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
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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:
200m.
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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: 200m (B) and
100m (D).
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, n10, P0.00017).
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, n10, P0.003). (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: 100m
(C,D) and 50m (F).
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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.
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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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