Deletion of a conserved regulatory element required for ...dumbo ear phenotype occur in the E10.5-E14.5 developmental interval in mice. To better understand the expression of Hmx1

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INTRODUCTIONHmx1 is a homeodomain transcription factor expressed in thedeveloping eye, peripheral ganglia and branchial arches of avianand mammalian embryos (Wang et al., 2000; Yoshiura et al., 1998).Relatively little is known about the function of Hmx1, but recentlyan Hmx1 allele has been identified as the causative genetic defect

in a human disorder, oculo-auricular syndrome (OAS) (Schorderetet al., 2008). Patients with OAS exhibit malformations of the outerear (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 misplacedears (mpe) mutations in mice (Munroe et al., 2009). Mouse dmbo,mouse mpe and the known human Hmx1 allele causing OAS areall coding mutations that affect the Hmx1 DNA-bindinghomeodomain, and thus are predicted to result in loss of function.In addition to malformed ears, dumbo mice exhibit eyemalformations, although less severe than those observed in the OASpatients identified to date. In both mouse and man, hearing isspared. In rats, the dumbo (dmbo) trait identified in animals keptby amateur ‘fancy rat’ breeders has also been mapped to a 5 Mbregion encompassing the Hmx1 locus (Kuramoto et al., 2010), butthe nature of the causative mutation is unknown.

Recent work in mice has also revealed a role for Hmx1 in thedevelopment of sensory neurons. Hmx1 is widely expressed in thesensory peripheral nervous system, including a subset of neuronsin the trigeminal ganglion, geniculate ganglion, superior ganglionof the IX-X ganglion complex and dorsal root ganglia (Wang et al.,2000; Yoshiura et al., 1998). In the caudal cranial ganglia, Hmx1 isconfined to somatosensory neurons and is not expressed in thedistal viscerosensory component of these ganglia. Despite the wideexpression of Hmx1 in the sensory system, only the geniculate

Disease Models & Mechanisms 5, 812-822 (2012) doi:10.1242/dmm.009910

1Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle,WA 98101, USA2Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University,Kyoto 606-8501, Japan3Center on Human Development and Disability, University of Washington, Seattle,WA 98101, USA4Department of Pediatrics (Craniofacial Medicine), University of Washington, andCenter for Tissue & Cell Sciences, Seattle Children’s Research Institute, Seattle, WA 98101, USA5Department of Anatomy and Developmental Biology, Monash University, Clayton,Victoria 3800, Australia6Department of Psychiatry and Behavioral Sciences, University of Washington,Seattle, WA, USA*These authors contributed equally to this work‡Author for correspondence ([email protected])

Received 26 March 2012; Accepted 30 May 2012

© 2012. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), whichpermits unrestricted non-commercial use, distribution and reproduction in any medium providedthat the original work is properly cited and all further distributions of the work or adaptation aresubject to the same Creative Commons License terms.

SUMMARY

Hmx1 is a homeodomain transcription factor expressed in the developing eye, peripheral ganglia, and branchial arches of avian and mammalianembryos. 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, alsoresults from a loss-of-function mutation in the Hmx1 gene. A recessive dmbo mutation causing ear malformation in rats has been mapped to thechromosomal region containing the Hmx1 gene, but the nature of the causative allele is unknown. Here we show that dumbo rats and mice exhibitsimilar neonatal ear and eye phenotypes. In midgestation embryos, dumbo rats show a specific loss of Hmx1 expression in neural-crest-derivedcraniofacial 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 divergencesfrom the rat genomic reference sequence, but no coding changes in Hmx1. Fine genetic mapping narrows the dmbo critical region to an intervalof ~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 domainof ~500 bp, which is a candidate distal enhancer and which exhibits a similar relationship to Hmx genes in all vertebrate species for which data areavailable. We conclude that the rat dumbo phenotype is likely to result from loss of function of an ultraconserved enhancer specifically regulatingHmx1 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.

Deletion of a conserved regulatory element required forHmx1 expression in craniofacial mesenchyme in thedumbo rat: a newly identified cause of congenital earmalformationLely A. Quina1,*, Takashi Kuramoto2,*, Daniela V. Luquetti3,4, Timothy C. Cox3,4,5, Tadao Serikawa2 and Eric E. Turner1,3,6,‡

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Rat dumbo Hmx1 mutation RESEARCH ARTICLE

somatosensory neurons appear to require it for neurogenesis(Quina et al., 2012), whereas early development of the trigeminaland dorsal root ganglia appear normal in dumbo mice.

In the present study, we show that dumbo rats and mice exhibitsimilar ear malformations and extent of microphthalmia. Hmx1protein expression in dumbo rat embryos shows no change in theearly developing eye and sensory ganglia compared with controls,but dumbo embryos exhibit specific loss of Hmx1 expression inthe mesenchymal components of branchial arches 1 and 2 (BA1and BA2), which give rise to the pinna. Fate-mapping of Hmx1-expressing craniofacial mesenchyme (CM) cells in transgenic miceshows that this region of decreased Hmx1 expression in the dumborat corresponds to CM that is derived from neural crest, but notfrom cranial mesoderm. To better understand the genetic basis of

this specific loss of Hmx1 expression, we resequenced 1 Mb of ratchromosome 14 encompassing the Hmx1 locus, revealingnumerous divergences from the rat genomic reference sequence,but no coding changes in Hmx1. Single nucleotide polymorphismswithin this span allowed the dmbo critical region to be narrowedto the interval of ~410 kb immediately downstream of the Hmx1transcription unit. Within this region, we identified a 5777-bpdeletion that encompasses a ~500-bp region conserved across allvertebrate species. Together, these results suggest that the rat dmbomutation is a cis-acting regulatory allele of an ultraconservedenhancer that is necessary for Hmx1 expression in neural-crest-derived CM. Dysregulation of Hmx1 expression is thus a potentialcausative mechanism in many patients with congenital earmalformations, a common abnormality for which the etiology islargely unknown.

RESULTSDumbo rats exhibit congenital malformations of the pinna similarto dumbo mice, and modest reduction in ocular size at birthIn order to compare the phenotypes of dumbo mice and rats, wefirst examined the morphology of the ear and eye in newborndumbo animals and controls. The characteristic ventraldisplacement of the ear in dumbo rats and mice, which gives themutation its name, has been previously described in adults(Kuramoto et al., 2010; Munroe et al., 2009). To define an endpointfor developmental studies, we examined the morphology of theexternal ear in neonatal dumbo rats and mice at postnatal day 2(P2) and P1, respectively. At P2, dumbo rats exhibited markedventral displacement of the pinna compared with heterozygouscontrols (Fig. 1A,B). P1 Hmx1dm/dm mice also exhibited ventraldisplacement and, in addition, the top of the pinna was rotatedposteriorly by 33° relative to controls (Fig. 1C,D).

To determine the extent of congenital microphthalmia in dumborats and mice we measured the neonatal eye. The average diameterof the newborn dumbo rat eye was modestly decreased, to 86%that of heterozygous controls (Fig. 1E). However, the eye was ofnormal size at E14 (data not shown), indicating that the ocular sizedifference must be generated during the period of extensive eyegrowth in late gestation. Prior characterization of the Hmx1dm/dm

mouse identified a microphthalmia phenotype in adults but didnot quantify the reduction in eye size (Munroe et al., 2009). In thepresent study, comparison of P1 dumbo mice (Hmx1dm/dm) tolittermate controls (Hmx1dm/+ and Hmx1+/+) revealed that the meandiameter and length of the orbit in Hmx1dm/dm mice was reducedto 91% that of controls, corresponding to approximately 75% ofnormal ocular volume (Fig. 1F-H). We then undertook detaileddevelopmental studies to examine the cellular expression of Hmx1in the developing CM and eye in order to better understand thesedeformities.

Expression of Hmx1 in the mouse craniofacial mesenchymeIn 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 thedumbo ear phenotype occur in the E10.5-E14.5 developmentalinterval in mice. To better understand the expression of Hmx1 at

TRANSLATIONAL IMPACT

Clinical issueThe oculo-auricular syndrome (OAS) is a rare human craniofacial disorder thatinvolves multiple anomalies of the eyes (retinal dystrophia, microphthalmia,chorioretinal colobomas and cataract) and ears (lobe malformation andsimplification of ear morphology). OAS in humans has been linked to adeletion in the gene HMX1, a transcription factor expressed in the developingeye, peripheral nervous system and branchial arches of birds and mammals.However, the function of this gene and the nature of the disruptedembryological processes that result in these anomalies are not known.Moreover, the genetic and non-genetic causes of much more prevalentcongenital anomalies of the ear, with and without associated eyemalformations, remain largely unknown. Eye and ear anomalies have profoundeffects on the lives of the affected individuals. The study of animal models thatadvance our understanding of these conditions, and enable specific diagnosisand ultimately treatment of patients, are an invaluable resource.

ResultsIn prior work, the dumbo phenotype, named for its characteristic earmalformation, has been linked to the Hmx1 gene in rats and mice. Our resultsshow that neonatal dumbo rats have a similar phenotype to that observed indumbo mice, with characteristic ventral displacement of the ear and amoderate degree of microphthalmia. Dumbo rats show a specific loss of Hmx1expression in neural-crest-derived craniofacial mesenchyme, particularly themaxillary component and most of the mandibular component of the firstbranchial arch, and the distal part of the second branchial arch. Expression ofHmx1 in the early developing retina and peripheral nervous system is intact.Unlike the known OAS HMX1 allele in humans and the dumbo mutation inmice (both of which prevent translation of the Hmx1 homeodomain),sequence analysis of the chromosomal region that encompasses the rat Hmx1locus 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 crucialfor Hmx1 expression in the branchial arches.

Implications and future directionsThis detailed genetic analysis of the dumbo rat suggests that the causativemutation is deletion of a conserved enhancer specifically affecting Hmx1expression in neural-crest-derived mesenchyme, and future studies will focuson unraveling the nature of this regulatory allele. In future studies ofindividuals with ear malformations, exon sequencing alone will not besufficient to exclude the Hmx1 locus; regulatory alleles of Hmx1 must also beconsidered. These results also suggest that Hmx1 lies in the middle of a poorlyunderstood pathway for development of the pinna. Thus, genes bothupstream and downstream of Hmx1, as well as the Hmx1 locus itself, arecandidates for the site of the causative allele in studies of individuals withsyndromic or isolated eye and ear anomalies.

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the onset of pinna development, we first examined in detail theexpression of Hmx1 protein in the CM of E11.5 Hmx+/+ mouseembryos (Fig. 2). Hmx1dm/dm mouse embryos were not examinedbecause we have previously demonstrated that Hmx1 protein isnot detectable in these embryos at these stages (Quina et al., 2012).

In the E11.5 mouse embryo, Hmx1 was expressed in threeregions of the CM. Region 1 consisted of the part of proximal BA1overlying the trigeminal ganglion (Fig. 2A-D). Region 2 comprisedthe caudal half of distal BA2 (Fig. 2D-F), and a small region of thecaudal or distal tip of the mandibular component of BA1 (Fig. 2F,G).

Region 3 consisted of an extensive region of posterior mesenchyme,caudal to the head vein and branchial arches, ending at the top ofthe limb bud (Fig. 2A-H). By contrast, extensive regions of CM,particularly the maxillary component and most of the mandibularcomponent of BA1 and the proximal part of BA2, do not expressHmx1. Based on past studies of the embryological origin of theexternal ear, only Region 2 CM in the distal part of BA1 and BA2is likely to contribute directly to the pinna.

Expression of Hmx1 in the dumbo rat embryoIn order to understand the expression of Hmx1 in the dumbo ratembryo, and the basis of the rat dumbo phenotype, we examinedHmx1 expression in E13 rat embryos (equivalent to the E11.5 mousedescribed above) and in E14 rat embryos (equivalent to an E12.5mouse). Dumbo rats and embryos were generated by interbreedingKFRS4/Kyo parental rats. Age-matched controls were generatedfrom crosses of the PVG/seac strain (see Methods).

Examination of BA1 in E13 dumbo rat embryos and controlsrevealed 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 trigeminalganglion, measured as the distance between the trigeminal ganglionitself and the surface ectoderm, was not altered, indicating that therewas loss of Hmx1 expression in this domain rather than loss of theCM cells as such. Expression of Hmx1 was also diminished in theregion of branchial arches 3 and 4, overlying the superior ganglion(Fig. 3F-G). Remarkably, expression of Hmx1 was not affected in thesensory neurons residing at the same axial levels, including themandibular lobe of the trigeminal ganglion (Fig. 3H,I), thesomatosensory neurons of the geniculate ganglion or the Hmx1-expressing neurons in the statoacoustic ganglion (Fig. 3J,K). TheHmx1 signal in the retina and the characteristic nasotemporalgradient of retinal expression were also unchanged in dumboembryos compared with controls (Fig. 4B,C).

Similar loss of Hmx1 expression was seen in the ventral branchialarches of E13 dumbo rat embryos, designated as Region 2. Hmx1was expressed in the caudal part of distal BA2 in control embryos,whereas dumbo embryos revealed a complete loss of Hmx1expression in this region (Fig. 4D-G). By contrast, expression wasunchanged in the posterior mesenchyme designated as Region 3(Fig. 4D,E). The cervical dorsal root ganglia (DRG) of dumboembryos also maintained normal Hmx1 expression (Fig. 4H,I), asdid the cranial sensory ganglia.

In E14 rat embryos (equivalent to an E12.5 mouse), Hmx1 wasprominently expressed in the lateral facial mesenchyme, includinga region of expression posterior to the eye, and dorsal to thebranchial arches, which was not evident at E13 (Fig. 5). Double-labeling experiments with Hmx1 and Ap2a demonstrated thatHmx1 is expressed in the mesenchyme of this region, but not inthe surface ectoderm (supplementary material Fig. S1). To betterdefine the Hmx1 expression differences in dumbo embryos, serialsections were examined throughout this dorsal domain, and theextent of Hmx1 expression was measured using semi-quantitativeimmunofluorescence (Fig. 5A-C). The overall size of the area ofexpression was reduced moderately (44%) in the most dorsalregion of expression, at the level of the eye and lateral to thehypothalamus (Fig. 5B,C, level 0), and severely (74-92%) in theventral region of expression, lateral to the mandibular lobe of the

Fig. 1. Ear and eye phenotypes in the newborn rat and mouse. Dumbo ratand mouse pups and controls were compared at P2 and P1, respectively.(A)Rat controls (dmbo/+) and (B) rat dumbo (dmbo/dmbo) pups. (C)Externalfeatures of controls (Hmxdm/+ or Hmx1+/+, which are indistinguishable) and (D)dumbo mice (Hmx1dm/dm), showing characteristic rotation and ventraldisplacement of the ear (dotted lines). Reduced ocular size is evident throughthe 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 t-test). (F,G)P1 mouse eye, shown in lateral and frontal views for bothgenotypes. D, diameter; L, length. (H)Measurement of reduced eye diameterand 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 barsshow ± s.d.

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trigeminal ganglion and pons (Fig. 5B,C, levels 7-9; P1.7�10–4 forall ten planes of section). In the intermediate sections, where therewas partial loss of Hmx1 expression, the decreased expressionoccurred mainly rostral to the midpoint of the trigeminal ganglion(Fig. 5C, arrows).

By contrast, no difference was observed in Hmx1 expression inthe mandibular lobe of the trigeminal ganglion (Fig. 5D,E). In theretina, the overall expression of Hmx1 in the E14 retinalneuroepithelium was reduced compared with E13, but no differencewas noted between dumbo embryos and controls. Onset of Hmx1expression was noted in early differentiating retinal ganglion cells(neurons), which first appear at this stage, some of which alsoexpressed Brn3a (Fig. 5F,G). The normal expression of Hmx1 inthe eye at this stage is consistent with our observation that ocular

size was also normal at E14. We conclude that the mildmicrophthalmia observed in neonatal dumbo rats is due todiminished ocular growth in late gestation.

Loss of Hmx1 expression in the Dumbo rat embryo occursspecifically in neural-crest-derived mesenchymeDetailed examination of E13 and E14 rat embryos shows that theloss of Hmx1 expression in dumbo embryos is not uniform, butinstead shows marked regional specificity, with profound loss inCM in the branchial arches and relative sparing of expression inregions of Hmx1 expression in the parietal region adjacent to thehindbrain.

The craniofacial mesenchyme, which gives rise to the bones andcartilage of the head, is known to originate from both the cranialneural crest and mesoderm, depending on location (Le Douarin etal., 1993). Our observation that Hmx1 expression in dumbo ratembryos is lost only in specific regions of CM led us to hypothesizethat the effect of the rat dmbo mutation might differ according tothe embryological origin of the affected CM cells. In mice, tissuederived from neural crest in the cranium and branchial arches canbe identified by embryological fate mapping, using a Wnt1-Cre

Fig. 2. Hmx1 expression in developing mouse craniofacial mesenchyme.(A-H)Serial sections of a wild-type E11.5 mouse embryo showing theexpression domains of Hmx1 in the CM. Successively more ventral planes ofsection show distribution of Hmx1-expressing cells in the CM. Arrows in F andG 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 BA1overlying the trigeminal ganglion; 2, the caudal half of distal BA2 and a smallregion of the caudal/distal tip of the mandibular component of BA1; 3, anextensive region of posterior mesenchyme, caudal to the head vein andbranchial 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, geniculateganglion; Inf X, inferior ganglion of the IX-X ganglion complex(nodose/petrosal ganglion); Mn, mandibular process (of BA1); Mx, maxillaryprocess (of BA1); mnTG, mandibular lobe, trigeminal ganglion; mxTG, maxillarylobe, 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: 200m.

Fig. 3. Loss of Hmx1 expression in proximal BA1 in the E13 Dumbo rat. E13rat embryos, developmentally equivalent to E11.5 mice, were examined usingimmunofluorescence for Hmx1 and for Brn3a, which identifies somatosensoryneurons. (A)Plane of section in subsequent views. (B-E)Control (B,D) anddumbo (C,E) embryos showing loss of expression of Hmx1 in the dorsalmostpart of BA1 in the mutant. The CM overlying the TG is present (dashed line inE), but fails to express Hmx1. Expression of Hmx1 is unaffected in the mnTGand SAG. (F-K)Control (F,H,J) and dumbo (G,I,K) embryos showing loss ofHmx1 expression in BA1 in dumbo embryos. Some loss of Hmx1 expression isalso 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, trigeminalganglion; 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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driver and a LoxP-mediated reporter (Danielian et al., 1998). Inmid-gestation embryos, a clear boundary can be shown betweenneural-crest-derived frontonasal structures and mesoderm-derivedparietal structures (Jiang et al., 2002). Because no such approachis available for rats, we chose to determine the embryological originof the Hmx1-expressing CM in a transgenic mouse model.

To assess the embryological origin of the Hmx1-expressing cellsin the CM, we crossed mice carrying Wnt1-Cre with a reporterstrain, Ai6, which conditionally expresses the fluorescent reporterZS-green from a modified Rosa26 locus (Madisen et al., 2010). InE12.5 Wnt1-Cre/Ai6 mouse embryos, expression of the inducedmarker gene revealed an occult boundary between the neural-crest-derived CM in frontonasal and branchial arch regions and themesoderm-derived CM in the parietal region (Fig. 6), which wasconsistent with prior studies (Jiang et al., 2002; Yoshida et al., 2008).

Hmx1-expressing CM cells were found in both the neural crest andmesoderm compartments, but the embryological origin of theHmx1 cells differed markedly according to axial level. In the mostdorsal and rostral domain of expression, at the level of thediencephalon (Fig. 6B), Hmx1-expressing CM cells were derivedentirely from mesoderm. At the level of the trigeminal andgeniculate ganglia and the future pons, an increasing proportionof the Hmx1-expressing CM cells resided in the neural-crest-

Fig. 4. Loss of Hmx1 expression in BA2 in the E13 Dumbo rat. (A)Plane ofsection in subsequent views. (B,C)Hmx1 is expressed in a nasotemporalgradient in the retina which is unchanged in dumbo embryos. (D-G)Expression of Hmx1 in the distal branchial arches; top of figures isanterior. Hmx1 is not expressed in distal BA1 at this stage. Hmx1 expression indistal BA2 is lost in the dumbo embryo (G). Expression of Hmx1 in posteriormesenchyme is not affected (D,E arrowheads). Some neurons in the inferiorpart of the tenth ganglion are weakly positive for Brn3a. (H-I�)Expression ofHmx1 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, brachialpouch 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) and100m (D).

Fig. 5. Altered Hmx1 expression in the E14 dumbo rat. Hmx1 expressionwas examined by immunofluorescence in the lateral cranial mesenchyme ofE14 embryos, at which stage the branchial arches are no longer anatomicallydistinct structures. (A)Planes of section for levels analyzed in subsequentviews. (B,C)Semiquantitative analysis of Hmx1 expression in BA1 at E14. Thearea and intensity of Hmx1 expression was measured at 160-m intervalsencompassing the cranial mesenchyme lateral to the trigeminal ganglion andpons. The overall area of Hmx1 immunofluorescence staining weresignificantly diminished in the dumbo embryo (paired t-test, n10, P0.00017).In the more dorsal sections, the loss of expression occurred mainly rostral to aboundary at about the midpoint of the trigeminal ganglion (arrows). The maineffect on Hmx1 expression was to reduce the area of expression, but theintensity of the immunofluorescence signal was also somewhat diminished inthe expressing area (paired t-test, n10, P0.003). (D,E)Expression of Hmx1 inmandibular lobe of trigeminal ganglion. (F,G)Expression of Hmx1 indifferentiating 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, trigeminalganglion; NE, neuroepithelium; Ret, retina; V, head vein. Scale bars: 100m(C,D) and 50m (F).

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derived compartment in progressively more ventral and caudalsections (Fig. 6C-E). Caudal to the otic region, in CM overlying thesuperior ganglion and future medulla, Hmx1 was again expressedpredominantly in the mesoderm-derived CM (Fig. 6F).

Although it is not possible to directly fate-map the neural crestand parietal mesoderm in rat embryos, the loss of Hmx1 expressionin the dumbo rat appears to be largely confined to the neural crestcompartment, with persistence of Hmx1 expression in themesoderm-derived CM (compare Fig. 5, L5 with Fig. 6D and Fig.5, L7 with Fig. 6E). However, the Hmx1 expression is not affectedin the trigeminal or dorsal root ganglia, which are also largelyneural-crest-derived. Thus, it appears that the dumbo rat phenotyperesults from loss of Hmx1 expression specifically within the neural-crest-derived CM, suggesting a tissue-specific defect in theregulation of Hmx1 expression.

Sequence of the rat dumbo critical region reveals deletion of aunique distal conserved region.Tissue-specific loss of Hmx1 expression in neural-crest-derived CMcould in principle result from the loss of a trans-acting regulatoryfactor governing expression in this domain, or from the disruption

of a cis-acting neural-crest- or CM-specific regulatory element atthe Hmx1 locus. However, prior mapping of the rat dmbo allele toa critical region of ~5 Mb encompassing the Hmx1 locus suggesteda cis-acting mechanism (Kuramoto et al., 2010).

In an effort to determine the nature of the dmbo mutation, weperformed resequencing of a 1.0-Mb region of the dmbo/dmbo ratstrain KFRS4, spanning the interval from 80.5 to 81.5 Mb ofchromosome 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 nucleotidepolymorphisms (SNPs) residing in the coding sequences of nine ofthe ten annotated genes in this interval (supplementary materialTable S1), as well as numerous SNPs in the intergenic regions. Eightof the SNPs resulted in conservative amino acid changes, and 23were synonymous. The single SNP identified within the Hmx1coding sequence was silent (L251L).

The identification of additional SNPs distinguishing KFRS4 fromthe BN reference sequence allowed further refinement of thegenetic 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 thedmbo allele was determined by the appearance of the pinna at 3weeks of age. A total of 614 backcross progeny were examined, ofwhich 322 expressed the dumbo ear phenotype and 292 had normalears. Recombination between the chromosomal markersD14Rat10and D14Rat57 was observed in 31 of the backcross progeny, andthese animals were used for fine mapping (Fig. 7). By genotypingof recombinant progeny with Ablim2_SNP(H42Y) andHmx1_SNP(L251L), the minimal dmbo critical region was refinedto 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 regionappear in supplementary material Fig. S2.

Only two mis-sense mutations were localized to this refined dmbocritical region (supplementary material Table S1), Cpz_S23P andAcox3_N287H. However, these SNPs are not specific to theKFRS4/Kyo strain. Cpz_S23P has been identified as a homozygousSNP (rs64860291) between the BN reference strain and the SpragueDawley (SD) strain, which does not have dumbo ears.Acox3_N287H has been identified by our group as a homozygousmutation in the NER rat strain (data not shown), an inbred linederived from Crj:Wistar rats, which does not exhibit dumbo ears.

Although resequencing provided ~99% coverage of the dmbocritical region, we wished to determine whether smalldiscontinuities detected in the sequence data represented genomicrearrangements. To search for deletions or insertions, we used longPCR to amplify segments of DNA from dumbo and control ratsspanning the 120-kb intergenic region from the 3� end of the Hmx1gene to the 3� end of the Cpz gene. One primer set spanning theregion 80-90 kb downstream of the Hmx1 gene yielded a ~6 kbshorter product in dumbo samples than in controls, and a mixedproduct in heterozygotes (supplementary material Table S2).Sequencing of the PCR product from this region revealed a 5777-bp deletion in KFRS4/Kyo genomic sequence relative to thereference strain (Fig. 7B). The deletion was not detected in a panelof 137 other rat strains, strongly suggesting that it is the causativeallele for the rat dumbo phenotype. To further verify therelationship between this deletion and the dumbo phenotype, weamplified the breakpoint region in DNA samples obtained from

Fig. 6. Hmx1 is expressed in CM of neural crest and non-crest origin. AWnt1-Cre transgenic line was interbred with the reporter strain Ai6, expressingthe fluorescent protein ZEG from a modified Rosa26 locus. Mouse embryoswere analyzed at E12.5. (A)Whole-mount E12.5 embryo showing planes ofsection for levels analyzed in subsequent views. The ganglia and projectionsof the sensory peripheral nervous system are marked by expression of a LacZtransgene targeted to the Brn3a locus. (B-F)Progressively more ventral andcaudal sections showing expression of Hmx1 in crest-derived and mesoderm-derived CM. The endogenous fluorescence of the ZEG reporter appears ingreen. Dashed lines demarcate the border of the cranial neural-crest-derivedtissue. BA1-4, branchial arch 1-4; Di, diencephalon; GG, geniculate ganglion;Hb, hindbrain; inf IX/X, inferior part of IX-X ganglion complex (nodose andpetrosal 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).

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four dumbo rats with diverse genetic backgrounds, obtained froman enthusiast breeder in California. Each of these animals exhibitedthe same 5777-bp deletion 3� to the Hmx1 transcribed regionobserved in KFRS4/Kyo (supplementary material Fig. S3), and nocoding changes in the Hmx1 exonic sequence.

Because the 5.8-kb dmbo deletion lies in a presumed intergenicregion, we used BLAST and VISTA genomic search tools todetermine whether the deletion region contained sequenceelements conserved across species. A BLAST search revealed a highdegree of similarity between the rat dmbo deletion region andsequences in the Hmx1-Cpz intergenic region in the mouse (Chr:5)and human (Chr:4) genomes. The BLAST search did not revealsignificant homology to identified transcripts or expressed sequencetags in any species, confirming the intergenic nature of the deletionregion and suggesting instead a regulatory function for theconserved sequence.

Because of the limited annotation of the rat genome, we usedthe homologous region of the mouse genome to generate VISTAalignments (Dubchak et al., 2009) of conserved sequences in thedmbo deletion region across multiple species. VISTA analysisrevealed a highly conserved region of ~500 bp within the deletionregion that is present in all mammalian species (Fig. 8A). MultiZalignment revealed that a core sequence is conserved acrossvertebrate classes, including in the chicken, frog and zebrafishgenomes (Fig. 8B). This Hmx distal conserved region (DCR) occursconsistently in a syntenic chromosomal region including the Hmxlocus or loci and the Cpz locus in all species for which completedata are available. In chicken and zebrafish, two closely relatedHmx-class genes are present in tandem in this region. Althoughthe genomic distance between the Hmx transcribed region and the

DCR varies from 45 kb (zebrafish) to 326 kb (opossum), nointervening identified transcripts occur between the Hmx loci andthe DCR in any species. We conclude that the Hmx DCR is a strongcandidate for an ancient distal enhancer that regulates Hmxexpression in the branchial arch CM.

DISCUSSIONThe dumbo mouse and rat strains were named for their mostobvious physical characteristic, displaced and malformed ears, prior

Fig. 7. Structure of the rat dmbo critical region. (A)Genetic linkage map(left) and physical map (right) of the region of rat chromosome 14encompassing the dmbo allele. The linkage map was derived fromexamination of 614 progeny of a (BN/SsNSlc � KFRS4/Kyo)F1 � KFRS4/Kyobackcross. Recombination events between the markers Ablim2_SNP(H42Y)and Hmx1_SNP(L251L) place the dmbo allele critical region within a 410-kbinterval. Resequencing data revealed a novel 5777-bp deletion between theCpz and Hmx1 genes (red). (B)Structure of the KFRS4/Kyo deletion breakpoint.

Fig. 8. Comparative genomics of the Hmx1 distal conserved region.(A)VISTA alignment of the dmbo deletion region. The reference sequence forthe alignment is chr5:35,807,810-35,813,977 (version NCBI37/mm9) of themouse genome. A highly conserved region of ~500 bp is detected across allmammalian species, and a core sequence is conserved across vertebrateclasses. (B)Map of the relationship of the DCR to Hmx and Cpz genes for allvertebrate species for which contiguous genomic sequence is available. Redarrows indicate the 3� end of the actual or predicted Hmx1 and Cpztranscripts, and the direction of transcription. The distance from Hmx1 to theDCR in the human genome is unknown because of a gap in genomiccoverage. The chick genome contains two Hmx1 homologues, Hmx1 andSOHo1, transcribed on the same strand; similarly, the zebrafish genomecontains two Hmx1 homologues, Hmx1 and Hmx4. For each species, thegenome build version and genomic coordinates of the core conservedsequence (~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_326:1,558,135-1,558,406; Frog, xenTro2, scaffold_583:238,403-238,706; Zebrafish, danRer7,chr1:41,246,062-41,246,105.

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to any knowledge of the genetic mechanism underlying thesemalformations. The dumbo mouse phenotype was identified in amutagenesis screen (Wilson et al., 2005), whereas the dumbo ratmutation arose spontaneously among ‘fancy’ rats kept by enthusiastbreeders, probably in the western United States, and has becomea popular pet strain on the basis of its novel appearance. Given thelarge number of genes that are likely to play a role in themorphogenesis of the pinna, it is remarkable that both the mouseand rat dumbo strains converged on the same gene locus. Theappearance of the mouse and rat dumbo ears are quite similar, butat the cellular level the phenotypes of the mouse and rat mutantsreveal other striking differences. In recent work, we have shownthat Hmx1dm/dm mice exhibit marked loss of geniculate ganglionneurons and the associated posterior auricular nerve (Quina et al.,2012). The cranial nerve defects in dumbo mice were identifiedusing transgenic tract-tracing, and this method cannot not be usedin the rat, but the geniculate ganglion itself appears normal indumbo rat embryos. Furthermore, Hmx1 expression is normal indumbo rats during the critical developmental period in which thegeniculate ganglion defects appear in mice. In general, we infer thatthe sensory ganglia develop normally in dumbo rats, becausesensory expression of Hmx1 does not appear to be affected.

Even without the sequence of the dumbo rat Hmx1 locus, it isclear that the fundamental genetic mechanism of the dmbomutation 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 inHmx1dm/dm mice, probably because the truncated protein is rapidlydegraded (Quina et al., 2012). Similarly, the causative allele inhuman oculoauricular syndrome is predicted to result in loss ofprotein function (Schorderet et al., 2008). By contrast, in dumborat embryos, Hmx1 immunoreactivity appears normal in the retina,sensory ganglia and the part of the CM that is not derived fromneural crest, findings that are not compatible with a nonsensemutation. Sequence data confirm that the dumbo rat Hmx1 openreading frame and splice junctions are intact. Instead, the KFRS4strain of dumbo rats exhibits a 5777-bp deletion approximately 79kb distal to the end of the final Hmx1 exon that is not found in anyof 137 other rat strains tested. This, combined with theidentification of a highly conserved region in the dumbo deletionregion, strongly suggests that the functional rat dmbo allele consistsof the loss of an enhancer that regulates expression in neural-crest-derived CM.

The physical distance between the Hmx1 transcription unit andthe Hmx1 DCR, from ~80 kb in mouse and rat up to 326 kb in theopossum, is remarkable. However, there are examples of verifiedenhancer function at even greater distances. One example is a highlyconserved cis-acting element that regulates Shh expression in thedeveloping limb and is the site of mutations resulting in limbabnormalities in humans and mice, despite the fact that it is located~1 Mb from the Shh transcription unit, in an intron of an upstreamgene (Lettice and Hill, 2005; Lettice et al., 2002; Sagai et al., 2005;Sagai et al., 2004). Although not identified in prior studies, theHmx1 DCR has key characteristics of the class of regulatorysequences that have been called ‘conserved non-coding elements’(Woolfe et al., 2005), and ‘ultraconserved enhancers’ (Pennacchioet al., 2006). In addition to recognizable sequence conservationbetween higher and lower vertebrates, these elements are often

located adjacent to developmental regulators, such as tissue-specific transcription factors. Although in some cases the targeteddeletion of ultraconserved regions has failed to show an obviousphenotype (Ahituv et al., 2007), in the case of the rat Hmx1 DCRthe loss-of-function phenotype is clearly highly penetrant, resultingin characteristic ear deformities in the large range of geneticbackgrounds maintained by the rat-enthusiast breeder community.

Prior to the mapping of the rat dmbo mutation to the Hmx1locus, dysregulation of the homeobox genes Msx1 and Dlx1 wasproposed as a causative mechanism for the rat dumbo phenotype(Katerji et al., 2009). Loss-of-function mutations in Msx1/2 andDlx1/2 result in much more extensive defects in craniofacialdevelopment than the phenotypes observed in the dumbo rat andmouse (Ishii et al., 2005; Qiu et al., 1997; Qiu et al., 1995). Clearly,the causative mutation does not reside in these genes, but they arepotential upstream regulators of Hmx1.

The phenotypes of the dumbo rat and mouse help to clearlydistinguish the role of Hmx1 from that of the structurally relatedfactors Hmx2 and Hmx3 (Wang et al., 2000; Yoshiura et al., 1998),which are highly coexpressed, and have well-characterized andpartially redundant roles in vestibular and hypothalamicdevelopment in mice (Wang et al., 2001; Wang et al., 2004; Wanget al., 1998). Two Hmx1 homologues have been identified in thechicken, GH6/Hmx1 and SOHo1. The expression of genes encodingthese factors in the branchial arches, retina and cranial and spinalsensory ganglia suggest that they are the functional homologuesof mouse and rat Hmx1, not Hmx2/3 (Deitcher et al., 1994; Stadlerand Solursh, 1994). The DCR identified in the dumbo rat is locateddownstream of the chick Hmx1 and SOHo1 transcription units,providing further confirmation that they are the functionalhomologues of mammalian Hmx1. Like mouse Hmx1, GH6 andSOHo1 are expressed in a nasal-temporal gradient in the eye, andmisexpression of these factors alters the topographic mapping ofretinal axons onto their midbrain targets (Schulte and Cepko, 2000).However, these findings do not explain the ocular defects observedin OAS patients (coloboma) or dumbo rats and mice(microphthalmia), and further studies of the role of Hmx1 in retinaldevelopment are necessary.

The distinctive nature of the Hmx1 mutant phenotypes,resulting either from the loss of protein function or defectiveregulation, is underscored by comparison with the effects of othergenes that regulate morphogenesis of the pinna. One example isHoxa2, which affects ear development through specification ofsegmental identity in the branchial arches, a classic homeoticfunction. BA2 development is particularly influenced by the lossof Hoxa2, which results in transformation of BA2-derivedstructures into BA1 phenotypes and absence of the pinna(Santagati et al., 2005). Although Hmx1 is a homeodomainprotein, its late expression in a restricted set of neural andmesenchymal cell types across all cranial segments excludes aHox-like role in the assignment of the segmental identity.Consistent with this, Hmx1 mutants do not show the extensivechanges in multiple structures derived from BA2, such as the innerear and brainstem, that are observed in Hoxa2 mutants. In chickembryos, mis-expression of Hoxa2 in BA1 can induce ectopicexpression of the chick Hmx1-class factor SOHo1, suggesting thatHmx1 might lie downstream of this Hox gene (Grammatopouloset al., 2000). However, this cannot reflect an exclusive requirement

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for Hoxa2 because the normal boundaries of Hmx1 expressionextend beyond the Hoxa2 expression domain.

An Hmx gene is also present in the Drosophila genome [H6-like-homeobox, FlyBase ID FBgn0264005 (Wang et al., 2000)]. Theregulatory function of the Drosophila Hmx protein overlaps thatof mouse Hmx2/3, because Drosophila Hmx can partially rescuethe hypothalamic and inner ear phenotypes observed in Hmx2/3double mutant mouse embryos (Wang et al., 2004). The ‘non-homeotic homeobox’ role of the Hmx family is underscored by thefact that although insertional mutants of Drosophila Hmx areknown (http://flybase.org/reports/FBgn0264005.html), theyapparently do not exhibit homeotic transformations and there areno published reports of Hmx function in flies.

Mutations affecting development of the external ear might alsoresult from defective neural crest development, a mechanismexemplified by the role of Tcof1/Treacle in Treacher CollinsSyndrome (TCS), an autosomal dominant disorder that frequentlyincludes hypoplasia of the facial bones, cleft palate and middle eardefects in addition to malformation of the pinna (Dixon et al., 2007).Treacle is expressed throughout the dorsal neural tube and earlyneural crest. Mice haplo-insufficient for Tcof1 replicate some ofthe core features of TCS and demonstrate a crucial role for Tcof1in the craniofacial neural crest (Dixon et al., 2006). By contrast,Hmx1 is not expressed in the early neural crest, and our fate-mapping studies have shown that Hmx1 is expressed in the CMwithout respect to the crest-derived compartments in thedeveloping head. Thus the developmental roles of Tcof1 and Hmx1might intersect but are not analogous.

These findings on the unique developmental role of Hmx1 havesignificant implications for understanding human congenitaldisorders that affect the ear and lateral face and that have a majorimpact on individuals affected by these disorders. Abnormalmorphology and malpositioning of the ear (typically described as‘low-set’ and ‘posteriorly rotated’) are among the most commonmalformations seen in these patients. Ear anomalies might occurin dysmorphic syndromes, where they represent one of a multitudeof abnormally developed structures, or in isolation (Calzolari et al.,1999; Carey et al., 2006; Suutarla et al., 2007). It is also not surprisingthat the stigma associated with ear malformation and the burdenof one or more corrective surgeries can have significantpsychosocial sequelae (Steffen et al., 2010).

Much progress has been made in the identification of genesresponsible 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 deletionsyndrome (TBX1, Chieffo et al., 1997) and Townes-Brock syndrome(SALL1, Kohlhase et al., 1998). However, malformation of the earis 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 forthe rat dumbo phenotype is the first genetic variant to be identifiedin any species with such a high degree of specificity formalformation of the pinna. It suggests that neural-crest-specificregulatory alleles at the Hmx1 locus could underlie isolatedmalformations of the pinna in humans, as a variant of the OASthat does not include severe eye deformities. It also provides acausative model for common ear malformations and is potentially

the key to a developmental pathway for pinna morphogenesis thatunderlies these disorders.

METHODSAnimals and genotypingMice bearing the dmbo allele (Hmx1dm), strain B6;C3Fe-Hmx1dmbo/Rw/JcsJKjn, were obtained from Jackson Laboratories(Stock #008677). The Hmx1dm allele was originally the result ofENU mutagenesis on a C57BL/6 genetic background (Wilson etal., 2005). Mice were crossed to C57BL/6N (Charles River) for twoto five additional generations prior to the experiments described.Experiments were performed with littermate controls. Genotypingof the Hmx1dm and wild-type Hmx1 (Hmx1+) alleles was performedby real-time PCR using oligonucleotide primers having 3� terminiat the point mutation that characterizes the dmbo mutation, aspreviously described (Quina et al., 2012).

The founder rat expressing the dumbo ear phenotype [SRR04,(Kuramoto et al., 2010)] was obtained from a commercial breederin the United States and crossed with the PVG/seac strain of BlackHooded rats to generate the strain KFRS4, which is homozygousfor the recessive alleles dumbo (dmbo) and the coat color allelehead spot (hs). In prior work, the dmbo locus was mapped to the5.7-Mb region defined by D14Rat10 and D14Rat57 (Kuramoto etal., 2010).

To further refine the dmbo locus, we produced 614 (BN/SsNSlc� KFRS4/Kyo)F1 � KFRS4/Kyo backcross progeny. TheBN/SsNSlc rat strain was purchased from Japan SLC (Hamamatsu,Japan). Genotypes of the dmbo locus were determined by theappearance of the outer ear (dumbo malformation or wild-type) at3 weeks of age. Animals with recombination events betweenD14Rat10 and D14Rat57 were used for the fine mapping of the dmbocritical region.

Newborn dumbo rats and E13 and E14 dumbo embryos weregenerated by interbreeding KFRS4/Kyo parental rats. For Hmx1expression studies, age-matched wild-type controls were generatedfrom PVG/seac rats. For measurements of ocular diameter,littermate controls were used. Mouse embryos were stagedaccording to a published method (Theiler, 1972) and thecorresponding rat embryo stages were identified according toWitschi (Altman and Katz, 1962).

Resequencing of the rat dmbo critical regionRegions for targeted resequencing spanned from 80.5 to 81.5 Mbof the rat chromosome 14 and contained the dmbo critical region.The 1.0-Mb target region of KFRS4/Kyo DNA was enriched usingthe SureSelect oligonucleotide hybridization solution-based capturetechnique (Agilent). The enriched fragment library was thensubjected to PCR amplification, followed by sequencing on anIllumina GAIIx platform. Coverage of 40� or greater was obtainedfor 98.86% of the target region. The reads generated were mappedto the rat genome reference sequence (Baylor HGSC v3.4/rn4) usingBurrows-Wheeler alignment (Li and Durbin, 2009). The knownvariants between KFRS4 and the rat genome reference sequencewere identified using the Ensembl BioMart search function fromthe Ensembl variation 66 database. Further resequencing of thedmbo critical region was performed using long PCR usingPrimeSTAR GXL DNA polymerase (Takara, Otsu, Japan). VISTAalignment of the dmbo deletion region was performed using the

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Lawrence Berkeley National Laboratory and US Department ofEnergy Joint Genome Institute VISTA browserhttp://genome.lbl.gov/vista/index.shtml (Dubchak et al., 2009).Identification of conserved sequences across mammalian andvertebrate species was performed using MultiZ alignment usingthe UCSC genome browser http://genome.ucsc.edu/ (Kent et al.,2002).

ImmunofluorescenceEmbryos for immunofluorescence were fixed in 4%paraformaldehyde in phosphate-buffered saline, equilibrated in 15%then 30% sucrose, embedded in OCT medium and cryo-sectionedat 14-20 m, depending on stage. Antisera to Hmx1 was preparedin rabbits to a glutathione-S-transferase (GST)/Hmx1 fusionprotein 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 toCM in Hmx1dm/dm mice. Polyclonal rabbit and guinea pig antiseraagainst Brn3a have also been previously described (Quina et al.,2005). Mouse monoclonal anti-Ap2a antibody (3B5) was obtainedfrom the Developmental Studies Hybridoma Bank. Alexa-Fluor-conjugated species-specific secondary antibodies were obtainedfrom Life Technologies (Grand Island, NY).

For semiquantitative immunofluorescence measurement ofHmx1 expression, serial sections of dumbo and control rat embryoswere processed, immunostained and imaged in parallel, with thesame imaging parameters and no post-processing. Areas of interestin the CM were defined manually, and the area, total fluorescencesignal and mean fluorescence signal were calculated using ImageJ.ACKNOWLEDGEMENTSWe would like to thank Janell McMorran of Ratterfly Rattery, Bonita CA, as well asSondra Truffat and Madeleine Truffat for introducing E.E.T. to dumbo rats, andNicole Fung for initial work on the dumbo rat coding sequence. We would also liketo thank Mayuko Yokoe for her technical assistance and the National BioResourceProject – 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 CONTRIBUTIONSL.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.

FUNDINGSupported by Department of Veterans Affairs MERIT funding, and the NationalInstitutes 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 ScientificResearch from the Japan Society for the Promotion of Science [grant number21300153 to T.K.] and a grant-in-aid for Cancer Research from the Ministry ofHealth, Labour and Welfare.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.009910/-/DC1

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Deletion of a conserved regulatory element required for ...dumbo ear phenotype occur in the E10.5-E14.5 developmental interval in mice. To better understand the expression of Hmx1