ARTICLE

Function of Rx, But Not Pax6, Is Essential for theFormation of Retinal Progenitor Cells in MiceLi Zhang,1 Peter H. Mathers,2 and Milan Jamrich1,3,4*1Program in Developmental Biology, Baylor College of Medicine, Houston, Texas2Departments of Otolaryngology, Biochemistry and Ophthalmology, West Virginia University School of Medicine,Morgantown, West Virginia3Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas4Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas

Received 3 August 2000; Accepted 28 September 2000

Summary: Rx plays a critical role in eye formation. Tar-geted elimination of Rx results in embryos that do notdevelop eyes. In this study, we have investigated the ex-pression of Otx2, Six3, and Pax6 in Rx deficient embryos.We find that these genes show normal activation in theanterior neural plate in Rx2/2 embryos, but they are notupregulated in the area of the neural plate that would formthe primordium of the optic vesicle. In contrast, in homozy-gous Small eye embryos that lack Pax6 function, Rx showsnormal activation in the anterior neural plate and normalupregulation in the optic vesicle/retinal progenitor cells.This suggests that neither Rx expression nor the formationof retinal progenitor cells is dependent on a functionalcopy of the Pax6 gene, but that Pax6 expression and theformation of the progenitor cells of the optic cup is depen-dent on a functional copy of the Rx gene. genesis 28:135–142, 2000. © 2000 Wiley-Liss, Inc.

Key words: Rx; Otx2; Pax6; Six3; Small eye; eye primor-dium; retinal progenitor cells

INTRODUCTION

Vertebrate eye formation requires the regionalizationand specification of the anterior neural plate, formationof the optic vesicles, inductive contact between theoptic vesicle and presumptive lens ectoderm, and finally,the cellular differentiation of the lens and retina. Severalhomeobox genes such as Otx2, Six3, and Pax6 areinvolved in these processes (reviewed in Jean et al.,1998; Oliver and Gruss, 1997).

We have previously shown that Rx homeobox genesare essential for eye development (Mathers et al., 1997).They belong to a subfamily of paired-like homeoboxgenes (Galliot et al., 1999). Different species have vari-able numbers of Rx genes. In mouse, only one Rx genehas been isolated (Furukawa et al., 1997; Mathers et al.,1997). This gene is activated between embryonic day 7.5and 8.0 in the presumptive anterior neural plate. At laterstages, its expression becomes progressively restrictedto the optic cups and ventral hypothalamus.

Functional studies revealed that Rx genes are essentialfor eye development. Overexpression of Xrx in Xenopusembryos leads to the formation of ectopic retinal tissue,hyperproliferation of the retina, and duplication of theanterior neural tube (Mathers et al., 1997). Elimination ofRx activity in mouse by a targeted knock out strategyeliminates eye formation (Mathers et al., 1997). Duringnormal mouse development, the first morphological signof eye development is the formation of the optic sulciand optic pits, bilateral indentations in the cephalicneural plate, at E8.5 (Theiler, 1989). At E9.0, these evagi-nations sequentially give rise to the optic vesicles andoptic cups, which come into contact with the prospec-tive lens ectoderm at E9.5, and induce lens formation.The optic cup eventually becomes the retina. Rx2/2embryos do not develop optic sulci, optic cups, or anymorphologically visible optic structures. This indicatesthat Rx is involved in the initial stages of eye formation.

There are several possible reasons why optic struc-tures might not be visible in this mutant. First, Rx mightbe necessary for the specification of progenitor cells ofthe optic vesicle and in a Rx deficient background,progenitor cells of the optic vesicle do not form. Wedefine retinal progenitor cells as cells in the optic vesicleor its primordium that display gene expression charac-teristic of a developing optic cup. Second, Rx might benecessary for the proliferation of progenitor cells of theoptic vesicle. In this case, optic vesicle progenitor cellsmight be specified but they fail to proliferate and form amorphologically visible optic field. Finally, Rx mightcontrol the morphogenetic movements that lead to for-mation of a visible optic structure. In this case, progen-itor cells form in sufficient numbers, but their specifica-

* Correspondence to: Milan Jamrich, Associate Professor, Department ofCell Biology, N620 Baylor College of Medicine, One Baylor Plaza, Houston,Texas 77030.

E-mail: jamrich@bcm.tmc.eduContract grant sponsor: National Eye Institute; Contract grant

number: 1 RO1 EY12163-01A1.

© 2000 Wiley-Liss, Inc. genesis 28:135–142 (2000)

tion is uncoupled from the morphogenesis of the opticfield. This is a realistic possibility because it has beenshown that the conditional medaka mutation eyelessuncouples eye-specific gene expression and morphogen-esis of the eye (Winkler et al., 2000).

To distinguish between these possibilities, we haveinvestigated the expression of Pax6, Six3, and Otx2 inRx deficient embryos. Expression of these homeoboxcontaining genes is characteristic of the early stages ofeye formation, and mutations in these genes lead toabnormal eye development.

The transcription factor Pax6 plays a critical role invertebrate and invertebrate eye formation. Mutations inPax6 result in eye malformations known as aniridia,Peter’s anomaly and congenital cataracts in humans (Gla-ser et al., 1994; Hanson et al., 1994, 1999; Jordan et al.,1992; Ton et al., 1991). In mice, Pax6 mutations resultin Small eye syndrome (Hill et al., 1991). The Drosoph-ila homologue of Pax6, eyeless, is necessary for correcteye development (Quiring et al., 1994). Misexpressionof eyeless, or murine Pax6, leads to the formation ofectopic eyes in Drosophila (Halder et al., 1995). For thisreason, Pax6 has been proposed to be the “master gene”of eye development (Gehring, 1996).

Six3 is the vertebrate ortholog of the Drosophila sineoculis and Optix genes (Cheyette et al., 1994; Serikakuand O’Tousa, 1994; Toy et al., 1998). In mouse embryos,it is expressed in the anterior neural plate and later in thedeveloping eyes (Oliver et al., 1995). Otx2 is activatedduring gastrulation and is responsible for the specifica-tion of the rostral neuroectoderm. It is expressed indeveloping eyes at the same time as Rx (Pannese et al.,1995; Simeone et al., 1993).

These three genes, like Rx, are initially activated in theanterior neural plate and are subsequently upregulatedin the primordia of the optic cup (retinal progenitorcells). We have compared expression patterns of thesegenes in Rx deficient and wild-type embryos. We havefound that all three genes are initially activated in theanterior neural plate of Rx deficient embryos, but theyare not upregulated in the ventral neuroectoderm thatnormally forms the progenitor cells of the optic cup.This suggests that the function of Rx is necessary for theformation of progenitor cells of the optic cup. In con-trast, Rx expression, like that of Six3 and Otx2, is notaffected in Small eye embryos that lack Pax6 function.This demonstrates that Pax6 is not necessary for theactivation of Rx gene and for the specification of retinalprogenitor cells in mice.

RESULTS

Phenotype of Rx2/2 EmbryosMorphological analysis shows that the phenotype of

Rx2/2 embryos is not entirely uniform. Some of theembryos lack large parts of their forebrain whereas oth-ers lack only the eyes and ventral hypothalamus. Em-bryos with severe forebrain deficiencies can be distin-guished from their mildly affected siblings by embryonic

day 9, allowing us to focus on embryos with a relativelynormal forebrain development for this study. In Rx de-ficient embryos displaying the mild forebrain phenotype,the dorsal and lateral brain structures seem normal atE12 (Fig. 1B and D). However, the ventral neuroecto-derm is clearly thinner in the Rx deficient embryos, andthere is little or no intervening mesenchyme betweenthe oral ectoderm and neuroectoderm (Fig. 1B and D),compared to their wild type siblings (Fig. 1A and C). Rxis not expressed in the mesenchyme, suggesting thatsignals from Rx expressing ventral neuroectoderm arerequired for the homing, proliferation, and/or survival ofmesenchyme in this region. However, the primordiumof the pituitary, Rathke’s pouch, which is known to beinduced by signals from the ventral hypothalamus (Ta-kuma et al., 1998), is initially formed in Rx2/2 embryos(Fig. 1D), indicating that the ventral neuroectoderm is atleast partially functional. Further analysis of Rx2/2 ven-tral neuroectoderm will be required to understand thealterations in the signaling events.

Expression of Eye-Specific Genesin Rx2/2 Mutant

As previously described, there are no visible signs ofeye formation in the anterior neural plate of Rx2/2embryos. Nevertheless, it is possible that eye-specificgene expression is initiated in the absence of morpho-logical changes in the architecture of the neural plate.We therefore analyzed the expression of Otx2, Six3, andPax6, the early markers of eye development, by wholemount in situ hybridization in E9.0 and E10.5 embryos.These stages are most suitable to examine the initialactivation of these three genes in the anterior neuroec-toderm and their subsequent upregulation in retinal pro-genitor cells.

Otx2

Otx2 is normally expressed in forebrain, midbrain,eyes, and otic vesicles (Simeone et al., 1993). In thedeveloping eye, its expression temporarily overlaps withthat of Rx. At E10.5, Otx2 is expressed in the midbrain,otic vesicles, and optic vesicles in wild-type embryos(Fig. 2A). In mutant embryos, we can not detect upregu-lation of Otx2 expression in the presumptive retinal field(Fig. 2B). However, Otx2 expression in the midbrain andotic vesicle is not affected. The differences in Otx2expression can be detected as early as E9.0 (Fig. 2C andD). At this stage, there is strong expression of Otx2 inthe midbrain of mutant embryos as well as in the wildtype, but there is no increased Otx2 expression in thepresumptive retinal area of the mutant embryos as canbe seen in the wild-type embryos. These experimentsshow that the activation of Otx2 in the anterior neuro-ectoderm of Rx2/2 embryos is not dependent on Rxfunction but that Otx2 upregulation/stabilization in theeye field is Rx dependent.

136 ZHANG ET AL.

Six3

Six3 is initially expressed in the anterior neural plateand later in the olfactory placodes, optic vesicles, lensplacodes, midbrain, and ventral forebrain. In situ hybrid-ization of E10.5 wild-type and Rx mutant embryos showsthat there is expression in the olfactory placodes andmidbrain, but there is no upregulation of Six3 expres-sion in the presumptive eye field of mutant embryos as isobserved in the wild-type embryos (Fig. 3A and B). Thedifference in Six3 expression between the mutant andwild-type embryos is distinguishable at E9.0 (Fig. 3C and

D). At this stage, an increase in Six3 expression in theoptic area is clearly visible in the wild-type embryosalthough there is no such upregulation apparent in themutant Rx2/2 embryos. At the same time, normal Six3expression outside the eye field in Rx2/2 embryosdemonstrates that the initial activation of Six3 is not Rxdependent.

Pax6

The homeobox gene Pax6 plays an important role ineye formation, and has been postulated to be the master

FIG. 1. Analysis of Rx deficient embryos. (A) Transverse section of the forebrain of an E12 wild-type embryo. Black arrow points to theventral neural tube and white arrow to the head mesenchyme separating the brain and the oral cavity. (B) Transverse section of a Rxdeficient embryo demonstrating rudimentary nature of the ventral neuroectoderm and the absence of the head mesoderm between theventral neural tube and oral cavity. (C) Sagital section of an E12 wild-type embryo. (D) Sagital section of an E12 Rx2/2 embryo. Blackarrows point to ventral neuroectoderm and palate mesenchyme and white arrows to Rathke’s pouch.

137GENE EXPRESSION IN RX DEFICIENT EMBRYOS

gene of eye development. If this were the case, onemight expect Pax6 to be upstream of Rx and thereforeexpressed in the presumptive eye field of Rx deficientembryos. In situ analysis of Pax6 expression at E10.5shows strong expression in the spinal cord and forebrainof wild-type and mutant embryos, but the upregulationin the presumptive optic area that is visible in the wild-type embryos is absent in the Rx deficient embryos (Fig.4A and B). As with the other markers, the differences ingene expression can already be observed at E9.0. At thisstage, Pax6 is beginning to be upregulated in the pre-sumptive eye region of the wild-type embryos (Fig. 4C),but not in the Rx2/2 embryos (Fig. 4D). Pax6 expres-sion in the brain and spinal cord is not affected in mutantembryos and is comparable to that of wild type embryos.Thus, Pax6 upregulation in the eye field is dependent onfunctional Rx expression.

Expression of Rx in Small eye Mutant

Our finding that Pax6 shows abnormal expression inRx deficient embryos suggested that Pax6 expression inthe eye field is dependent on Rx function. To test this,we examined Rx expression in Small eye embryos thatlack Pax6 function. Pax6 deficient embryos, like Rxdeficient embryos, do not develop eyes. However, un-like Rx2/2 embryos, Pax6 deficient embryos form op-tic vesicles that do not develop normally at later stages.In situ analysis shows that homozygous and heterozy-gous Small eye embryos express Rx RNA in their opticvesicles (Fig. 5). This demonstrates that neither the ini-tial activation of the Rx in the anterior neural plate norits subsequent upregulation in the optic vesicle is depen-dent on Pax6 function.

DISCUSSION

We have previously described a paired-like homeoboxgene Rx that is essential for vertebrate eye formation.Mouse embryos lacking a functional copy of this gene do

FIG. 2. Expression of Otx2 in Rx2/2 mutant embryos. (A) In situhybridization of digoxygenin labeled Otx2 probe to a E10.5 wild-type embryo. (B) In situ hybridization of Otx2 to a Rx deficient E10.5embryo demonstrating the lack of expression in the putative opticarea. Expression in the other parts of the brain is relatively normal.(C) In situ hybridization of Otx2 to a E9.0 wild-type embryo. (D) Insitu hybridization of Otx2 to a Rx2/2 E9.0 embryo showing a lackof upregulation of Otx2 in the presumptive optic area, but normallevels of expression in the other parts of the head.

FIG. 3. Expression of Six3 in Rx2/2 mutant embryos. (A) In situhybridization of digoxygenin labeled Six3 probe to an E10.5 wild-type embryo. (B) In situ hybridization of Six3 to a Rx deficient E10.5embryo demonstrating the lack of expression in the putative opticarea. Expression in the other parts of the brain is relatively normal.(C) In situ hybridization of Six3 to a E9.0 wild-type embryo. Arrowpoints out the presumptive optic area. (D) In situ hybridization ofSix3 to a Rx2/2 E9.0 embryo showing a lack of upregulation of Six3in the presumptive optic area (arrow), but normal levels of expres-sion in the other parts of the head.

138 ZHANG ET AL.

not develop eyes, optic sulci, or optic cups. To under-stand the regulatory mechanism of Rx action, we haveanalyzed the expression of genes activated at the initialstages of eye development in Rx deficient embryos.

The Rx deficient embryos can be roughly divided intotwo categories. Although all of the embryos lack eyes,the first group of embryos shows significant deficienciesin anterior brain formation (Mathers et al., 1997). Thesecond group shows relatively normal brain develop-ment with deficiencies mostly limited to the eye andventral hypothalamus. A possible explanation for thesetwo phenotypes can be found in the expression patternof the Rx gene. Initial expression is in the anterior neuralplate in a region that is larger than the presumptiveretinal area and encompasses a large part of the fore-brain. Only later does its expression become limited tothe developing retina and ventral hypothalamus. Al-though the broad initial expression domain is only ap-

parent for a short time, it does include areas that aredeleted in the severely affected embryos. This need fortransient Rx expression in the presumptive forebrainarea can be compensated in some embryos presumablyby other paired homeobox genes expressed in overlap-ping fashion. In contrast, expression of Rx in the devel-oping eyes and ventral forebrain is absolutely requiredfor normal development of these regions, because all ofthe Rx deficient embryos lack eyes and have defects inthe hypothalamic region. The optic cup and the ventralhypothalamus map to adjacent positions on the verte-brate fate map at open neural plate stage (Couly andLeDouarin, 1988; Eagleson and Harris, 1990; Osumi-Yamashita et al., 1994). Thus, the progenitor cells ofthese two tissues have common origins and might rep-resent a common domain that requires Rx for specifica-tion.

In our analysis of gene expression in Rx deficientembryos, we have selected embryos that show only amild forebrain phenotype. Sections of these embryosshow that the dorsal and lateral areas of the neural tubeare normal in appearance, but the ventral neuroecto-derm that forms the optic vesicles and ventral hypothal-amus is thinner and abnormal in appearance.

To determine whether there is any specification ofretinal precursor cells in the forebrain area of Rx defi-cient embryos, we have analyzed the expression patternof the homeobox genes Otx2, Six3, and Pax6 by in situhybridization. Like Rx, in normal embryos, Otx2, Six3,and Pax6 are expressed in the anterior neural plate.Later, their expression is upregulated in the primordiumof the optic vesicle, the retinal progenitor cells of thedeveloping eye.

We have found that Otx2, Six3, and Pax6 are ex-pressed normally in the anterior neural plate of Rx2/2embryos, demonstrating that the initial activation ofthese genes in the anterior neural plate is not Rx depen-dent. However, although expression of all three of thesegenes is upregulated or stabilized in the optic vesicles/cups of wild-type embryos, none of these genes are

FIG. 4. Expression of Pax6 in Rx2/2 mutant embryos. (A) In situhybridization of digoxygenin labeled Pax6 probe to a E10.5 wild-type embryo. (B) In situ hybridization of Pax6 to a Rx deficient E10.5embryo demonstrating the lack of expression in the putative opticarea. Expression in the other parts of the brain and spinal cord isrelatively normal. (C) In situ hybridization of Pax6 to an E9.0 wild-type embryo. (D) In situ hybridization of Pax6 to a Rx2/2 E9.0embryo showing a lack of upregulation of Pax6 in the presumptiveoptic area, but normal levels of expression in the other parts of thehead and spinal cord. Arrows in (C) and (D) point to the presumptiveoptic area.

FIG. 5. Expression of Rx in Small eye mutant embryos. In situanalysis of expression of Rx in Small eye embryos. (A) Expression ofRx in E10.5 wild-type embryo demonstrating the Rx expression inthe optic cup. (B) Expression of Rx in a heterozygous Small eyeembryo at E10.5. (C) Homozygous Small eye E10.5 embryo showingRx expression in the optic cup. The optic cup is thinner in appear-ance, but it clearly does express Rx mRNA.

139GENE EXPRESSION IN RX DEFICIENT EMBRYOS

upregulated in Rx deficient embryos. Thus, Rx is genet-ically, as well as temporally, upstream of these genes ineye development. This does not necessarily imply thatthere is a direct regulation of these genes by Rx. It ismore likely that Rx is essential for the formation of thepresumptive eye field, and because eye progenitor cellsare not formed, eye-specific genes are not expressedeither. Although it is difficult to completely exclude thepossibility that eye-specific progenitors exist in theseembryos, we have failed to find any evidence of theirexistence by either morphological or molecular criteria.We have reported previously that Rx deficient embryosshow no morphological signs of eye formation. In thisstudy, we show that three important homeobox genesthat are normally expressed in retinal progenitor cellsare not expressed in the topographical location wherethe optic vesicle primordium/retinal progenitor cellswould be located. This suggests strongly that the ab-sence of optic structures in Rx deficient embryos is notdue to the lack of morphogenesis of the optic cup, butrather due to a true absence of specified retinal progen-itor cells.

Rx Is Expressed in Embryos Missing a FunctionalCopy of Pax6

Because embryos missing a functional copy of thePax6 gene have also an eyeless phenotype, we haveanalyzed Rx expression in the Small eye (Sey) embryos.We have found that Rx is expressed in the optic vesicleof heterozygous and homozygous Small eye embryos,demonstrating that Rx expression in the eye is not de-pendent on Pax6 gene function.

The analysis of gene expression in Rx2/2 embryosillustrates that the initial expression of Otx2, Six3, andPax6 in the anterior neural region is not dependent onRx activity. Only the eye-specific upregulation of thesegenes is Rx dependent. Similar to our results withRx2/2 mutant, the initial activation of Rx, Six3, andOtx2 in the anterior neural region of Small eye embryosis independent of Pax6 activity (Oliver et al., 1995;Stoykova et al., 1996). These observations open thepossibility that these four genes initially are not activatedby each other, but rather by another unknown factor.

What Is the Role of Rx and Pax6 During EyeDevelopment?

Pax6 and Rx are two homeobox genes that haveessential roles in eye development. Mutations in Pax6result in eye malformations in many species, and misex-pression of murine Pax6 can lead to formation of ec-topic eyes in Drosophila. (Halder et al., 1995). Based onthese observations, Pax6 was proposed to be the “mas-ter gene” of eye development. Our observations do notsupport the hypothesis that Pax6 is the “master gene” ofvertebrate eye development.

Although it is true that homozygous Small eye em-bryos do not develop eyes, they develop optic vesicles.These vesicles form optic stalks with structures thatresemble optic cups (Grindley et al., 1995). Although

these cups do not have a normal morphology, it isimportant to remember that they are formed in theabsence of the lens placode and lens vesicle. In thispaper, we show that homozygous Small eye embryosexpress Rx, one of the earliest markers of retinal forma-tion, in their optic vesicles. But Rx is not the onlyeye-specific gene expressed in these vesicles. Jean et al.(1999) have shown that Six6 is expressed in the opticprimordia of Small eye embryos. Grindley et al. (1995)found that these optic primordia not only express Pax6in a region specific manner, but also the tyrosine-relatedprotein gene Tyrp2, a marker of the retinal pigmentepithelium (RPE). This expression of eye-specific genesin this rudimentary optic cup suggests that retinal pro-genitor cells are not only formed in the absence of Pax6function, but also differentiate to some degree. Thesefindings argue against the possibility that Pax6 is the“master gene” of eye development in higher vertebrates.Rather, it seems that Pax6 is responsible for some func-tion that is needed for a correct differentiation of retinalprogenitor cells.

In contrast to Small eye embryos, homozygousRx2/2 embryos do not develop optic cups, optic vesi-cles, or optic sulci. They do not show any signs ofeye-specific gene expression. This demonstrates that Rxis acting earlier during eye development, and is requiredfor the initial specification of the entire optic primordia.

Before we reached this conclusion, we consideredseveral possibilities. We considered that in Rx2/2 em-bryos, eye-specific genes are activated in eye progenitorcells, but the optic vesicles do not undergo normalmorphogenesis. This situation is observed in the medakafish mutation eyeless, in which no morphologically visi-ble eyes can be observed, yet eye-specific gene expres-sion can be detected in the brain by in situ hybridization(Winkler et al., 2000). We have not observed any evi-dence of uncoupling of morphogenesis and gene expres-sion in the Rx2/2 mutant.

We considered the possibility that a gene other thanRx is determining the eye progenitor cells, and Rx iscontrolling the proliferation of these cells. In that case,the field could be significantly smaller than in the wildtype and not easily morphologically recognizable. If thatwere the case, we still should have seen some expres-sion of early eye-specific genes in the presumptive reti-nal precursor cells because, by in situ hybridization, it ispossible to recognize even a single positive cell. No suchexpression was found in Rx2/2 embryos. Simple reduc-tion of the retinal field in Rx2/2 embryos is a reason-able possibility because it was shown that interferencewith the homeobox gene Xoptx2 can severely reducethe size of the eye and the domain of Rx expression(Zuber et al., 1999). In addition, the injection of thedominant negative Rx-eng construct into Xenopus em-bryos can reduce the size of the eye field in injectedembryos (Andreazzoli et al., 1999). However, no eye-specific expression domain of any size was detected inour studies on Rx2/2 embryos.

140 ZHANG ET AL.

Our observations are somewhat difficult to reconcilewith the finding that in Xenopus Pax6 misexpressioncan lead to formation of ectopic eyes and activation ofRx (Chow et al., 1999). However, it is possible that inthese experiments Pax6 is activating Rx through a feed-back mechanism. This is a reasonable possibility becauseit has been shown in Drosophila that genes involved ineye development form a series of positive feedbackloops and can activate each other (Chen et al., 1997).Therefore, an overexpression of a gene that is normallydownstream of other genes can lead to activation ofupstream genes. Although it is an interesting observationthat eye formation can be achieved by overexpression ofseveral different genes, it does not necessarily demon-strate that this activation exactly recapitulates the pro-cess of normal development.

METHODS

Embryos and Genotype AnalysisRx2/2 and SeyNeu/SeyNeu mouse embryos were ob-

tained by naturally mating Rx1/- mice and SeyNeu/1mice, respectively. Genotypes of mouse embryos weredetermined by PCR using genomic DNA extracted fromextra-embryonic membranes. Two Rx gene-specific prim-ers MgRxF2(59-GGGAGTAGGGTTACTGGACTGAGGC-39),MgRxR2(59CGAGTATCCCTACTGCCTGGAAATC-39) and aneospecific primer NeoF(59-CAGCTCATTCCTCCCACT-CATGATC-39) were used to determine the genotype of theRx2/2 embryos. Genotype of SeyNeu/SeyNeu embryos wasdetermined as described previously (Xu et al., 1997). Prim-ers 10.5(59-GCATAGGCAGGTTATTTGCC-39) and PST-MSE(59-GGAATTCCTGAGGAACCAGAGAAGACAGGC-39)were used to amplify a 336 bp fragment. The single-base-pair change of SeyNeu allele within the Pax6 gene gives riseto a novel HindII site (Hill et al., 1991). The amplifiedfragment yields 114 bp and 222 bp fragments when di-gested with HindII. The digested DNA was analyzed byagarose gel electrophoresis.

Whole-Mount In Situ Hybridization

Whole-mount in situ hybridization was performed asdescribed (Conlon and Rossant, 1992).

Histology

Embryos were fixed in 4% paraformaldehyde, dehy-drated in ethanol, and cleared in xylene. After three 2-hincubations in paraplast, they were mounted and sec-tioned. The section were dewaxed, rehydrated, andstained with eosin and hematoxylin.

ACKNOWLEDGMENTS

We would like to thank Dr. Kathleen Mahon and IsaacBrownell for helpful suggestions and for a critical read-ing of this manuscript. This research was supported bygrant 1 RO1 EY12163-01A1 from the National Eye Insti-tute to M. Jamrich.

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