Pax2 Expression and Retinal Morphogenesis in the Normal ... · surecan besubdividedinto twocontiguousparts: theretinal congenital retinal and optic disc anomalies collectively re-

DEVELOPMENTAL BIOLOGY 193, 209–224 (1998)ARTICLE NO. DB978794

Pax2 Expression and Retinal Morphogenesisin the Normal and Krd Mouse

Deborah C. Otteson,* Eric Shelden,* Julie M. Jones,†Jiro Kameoka,‡ and Peter F. Hitchcock*,‡,1

*Department of Anatomy and Cell Biology, †Department of Human Genetics, and‡Department of Ophthalmology, University of Michigan, Ann Arbor, Michigan 48105

The Kidney and retinal defects (Krd) mouse carries a 7-cM transgene-induced deletion on chromosome 19 that includesthe Pax2 locus. Adult mice heterozygous for the Krd deletion (Krd//) are haploid for Pax2 and have a variable, semidomi-nant phenotype characterized by structural defects of the kidney, retina, and optic disc. Renal and ocular anomalies presentin heterozygous Pax2 mutants in both mice and humans support the hypothesis that haploinsufficiency of Pax2 underliesthe Krd phenotype. To understand the embryonic basis of ocular defects observed in adult Krd//mice, we used immunohis-tochemistry, digital three-dimensional reconstructions, and quantitative morphometry to examine Pax2 protein distributionand ocular development in normal and Krd//mice from E10.5 to P2. In /// embryos, Pax2 immunopositive (Pax2/) cellsdemarcate the embryonic fissure as it forms in the ventral optic cup and optic stalk. After closure of the embryonic fissure,Pax2 immunostaining disappears from the ventral retina, but persists in a cuff of cells encircling the developing optic disc,the site where ganglion cell axons exit the retina. In Krd// embryos, Pax2/ cells in the posterior optic cup and the opticstalk undergo abnormal morphogenetic movements and the embryonic fissure fails to form normally. This results in anabnormal organization of the Pax2/ cells and ganglion cell axons at the nascent optic disc. The abnormal morphogeneticmovements of the Pax2/ cells in the embryonic retina and optic stalk and the initial misrouting of the ganglion cell axonsgive rise to retinal and optic disc defects observed in the adult Krd// mice. q 1998 Academic Press

Key Words: development; embryo; retina; optic cup; optic disc; optic stalk; embryonic fissure; gene expression; immuno-histochemistry; reconstruction.

INTRODUCTION progresses, the two sides of the retinal fissure make contactand fuse, leaving no trace in the adult retina (Mann, 1964;Silver and Robb, 1979; Hero, 1990). At the back of the opticMorphogenetic movements of embryonic epithelial lay-cup, there is a transition between the retinal fissure anders establish the basic architecture of many tissues. This isoptic groove. Here, cells of the ventral neuroepithelium in-true for the eye, where the neuroepithelium undergoes avaginate and encircle the nascent optic disc, the site whereseries of complex movements as it transforms from a simpleganglion cell axons will exit the optic cup to form the opticballoon-like optic vesicle into a bilayered optic cup. Innerve (Silver and Robb, 1979; Silver and Sapiro, 1981; Brownmammals, the ventral epithelium of the optic vesicle andand Tasman, 1983). Abnormal formation and/or closure ofoptic stalk invaginates to form the embryonic fissure (Lo-the embryonic fissure is believed to result in an array ofpashov and Stroeva, 1964; Mann, 1964). The embryonic fis-congenital retinal and optic disc anomalies collectively re-sure can be subdivided into two contiguous parts: the retinalferred to as colobomas (Mann, 1957, 1964; Lopashov andfissure, which forms a transient cleft within the ventralStroeva, 1964; Kindler, 1970; Brown and Tasman, 1983;optic cup, and the optic groove, which extends proximallyApple, 1984; Silver et al., 1984; Schimmenti et al., 1995).toward the diencephalon as an invagination of the ventral

The growing array of murine mutations affecting ocularoptic stalk epithelium (Lopashov and Stroeva, 1964; Silverdevelopment has begun to provide new insight into the ge-and Robb, 1979; Silver and Sapiro, 1981). As developmentnetic and molecular basis of congenital defects of the eyeand the retina (reviewed by Beebe, 1994; Reh and Cagan,1994; Graw, 1996; MacDonald and Wilson, 1996). One ex-1 To whom correspondence should be addressed at Departmentample is Kidney and retinal defects (Krd) mice which carryof Ophthalmology, 418 Kellogg Eye Center, 1000 Wall Street, Uni-

versity of Michigan, Ann Arbor, MI 48105. Fax: (313) 647-0228. a 7-cM transgene-induced deletion on chromosome 19 (Kel-

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0012-1606/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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210 Otteson et al.

ler et al., 1994). These animals have a variable, semidomi- ment for full diploid expression of Pax2 for normal morpho-genesis of a portion of the embryonic fissure, the opticnant phenotype characterized by structural defects within

the kidney and retina. Retinal anomolies in adult Krd// groove, and provide insight into the origins of the retinaland optic disc defects observed in Krd// mice and othermice include laminar defects, photoreceptor rosettes, pan-

retinal hypocellularity, reduced electroretinograms, and op- Pax2 heterozygotes.tic disc malformations (Keller et al., 1994; Hitchcock et al.,1995; Otteson et al., 1996; Green et al., 1997). The develop-mental regulatory gene, Pax2, maps within the Krd deletion MATERIALS AND METHODS(Keller et al., 1994).

Pax2 is a member of the Pax gene family of transcription Animalsfactors that are characterized by the presence of a highly

Wild-type and Krd// embryos were collected from timed mat-conserved DNA binding domain, the paired box (Stuart etings of C57BL/6J females with Krd// males. Twenty-two litters ofal., 1993; Strachan and Read, 1994; Stuart and Gruss, 1995,embryos (7–10 embryos per litter) were collected at ages between1996). Several Pax genes share an uncommon genetic trait,E9.5 and P2. Three litters were analyzed at E11.5 and E16.5, and 4haploinsufficiency, wherein the presence of one wild-typewere analyzed at E10.5 and E12.5; one litter was examined forallele cannot compensate for a second mutated or deletedeach other time point. Embryos were dissected from the uterus in

allele (Gruss and Walther, 1992; Stuart et al., 1993; Stuart phosphate-buffered saline (PBS), and their predicted developmentaland Gruss, 1995). Krd// mice, which are heterozygous for age was verified using crown to rump length, heart development,the Krd deletion, are haploid for Pax2. limb bud morphology, and extent of ocular melanization (Kaufman,

Based on concordance of the known expression of Pax2 1992). Heads were removed and placed directly into fixative (seein the developing kidney, retina, and optic stalk (Nornes et below). For embryos older than E15, the head ectoderm and skull

overlying the brain were removed prior to fixation, and heads wereal., 1990; Puschel et al., 1992; Stoykova and Gruss, 1994;hemisected along the midline prior to further processing (see be-Torres et al., 1995, 1996) with the sites of congenital anoma-low). The posterior portion of each embryo was frozen at 0807Clies in Krd//mice, haploinsufficiency of Pax2 was proposedfor subsequent DNA isolation and genotyping.as the molecular genetic basis for the Krd phenotype (Keller

et al., 1994). This proposal is consistent with reports ofsevere renal and retinal defects resulting from both hetero- DNA Isolation and Genotypingzygosity and homozygosity for induced and spontaneousPax2 mutations in mice (Torres et al., 1995, 1996; Favor et Krd// embryos were identified by PCR amplification of genomic

DNA using primers complementary to the transgene, as previouslyal., 1996). Likewise, congenital renal-coloboma syndromedescribed (Keller et al., 1990). DNA was isolated from embryonicin several human families has been shown to result fromtissue by proteinase K (Boehringer Mannheim, Indianapolis, IN)heterozygosity for PAX2 mutations (Sanyanusin et al.,digestion, phenol/chloroform extraction, and ethanol precipitation.1995a,b; Schimmenti et al., 1995, 1997).The transgene was detected in 47% of the embryos and pups, con-Based on the hypothesis that ocular abnormalities ob-sistent with the expected Mendelian inheritance of 50%.

served in adult Krd// mice arise from haploinsufficiencyof Pax2 during early embryogenesis, we characterized Pax2expression and ocular morphogenesis in normal and Krd// Tissue Preparationembryos beginning with the period of embryonic fissure

Tissues were fixed and embedded using routine procedures.formation. We confirm and extend the reported expressionHeads for immunostaining were fixed in 4% paraformaldehyde inof Pax2 based on in situ hybridization (Nornes et al., 1990;0.08 M phosphate buffer, pH 7.2, for 90 min and washed in phos-Torres et al., 1996) and immunohistochemistry (Puschel etphate buffer. Following overnight cryoprotection at 47C in 20%al., 1992). Within the optic cup, Pax2/ cells form the lipssucrose, heads were equilibrated and frozen in a 2:1 mixture ofof the retinal fissure as they come together and fuse in the20% sucrose:OCT (TissueTek, Miles, Inc., Elkhart, IN). Cryosec-

ventral optic cup. Pax2 cells of the ventral optic stalk tions were cut at 10 mm in the coronal, horizontal, or sagittal planeinvaginate to form the optic groove. After closure of the and air-dried onto microscope slides coated with 3-aminopropyl-retinal fissure, Pax2 immunoreactivity is lost from the ven- triethoxysilane (TESPA, Sigma, St. Louis, MO). All tissues and sec-tral retina; however, cells that encircle the nascent optic tions were stored at 0807C prior to use.

Heads processed for embedding in glycomethacrylate were fixeddisc continue to express Pax2/ into late embryogenesis. Inin 2.5% glutaraldehyde, 2% paraformaldehyde in 0.08 M phosphateKrd// embryos, although there is apparently normal forma-buffer, pH 7.2, overnight at 47C. Following multiple washes in 0.08tion and closure of the retinal fissure, abnormal morphogen-M phosphate buffer, heads were dehydrated in ethanol, infiltratedetic movements of Pax2/ cells within the posterior retinawith catalyzed glycomethacrylate (JB4, Polysciences Inc., War-and ventral optic stalk lead to a failure of optic groove for-rington, PA), and cast in plastic molds. Sections were cut at 5mation and malformation of the optic disc and optic stalk.mm in either the coronal or parasagittal plane with a Leitz rotary

This is seen as a spatial disorganization of Pax2/ cells that microtome, collected at variable intervals onto microscope slidesnormally encircle the developing optic disc and is associated coated with poly-L-lysine or gelatin, and stained with toluidinewith anomalies in the organization and trajectory of gan- blue. For eyes used for digital reconstructions and morphometricglion cell axons at the disc and subsequent laminar defects analysis, the interval between each section was tabulated during

sectioning.within the adjacent retina. These results indicate a require-

Copyright q 1998 by Academic Press. All rights of reproduction in any form reserved.

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211Pax2 and Retinal Morphogenesis

Immunostaining Quantitative Morphometric Analysis

For quantitative morphometric analysis, a series of 5-mm glyco-Tissue sections were warmed to room temperature and air-driedmethacrylate sections were traced using a drawing tube attachedfor 15 min prior to immunostaining. All reagents were appliedto an Olympus compound microscope. In each eye, the posteriordirectly to the sections on slides in a humidified chamber. Initialpole of the lens was used as a landmark, allowing the identificationPBS washes were followed by blocking of nonspecific binding ofof comparable regions through the posterior optic cup and the opticsecondary antibodies by incubation in PBS/0.2% Triton X-100 withstalk for analysis. For each eye at E10.5, 16 sections were traced at20% normal goat serum (NGS) for 30–90 min at room temperature.10-mm intervals spanning 2

3 of the optic cup and the distal 23 of thePolyclonal Pax2 antibodies (a gift of G. Dressler, HHMI, University

optic stalk (///, n Å 4 eyes; Krd//, n Å 6 eyes). For each eye atof Michigan), diluted 1:50 in PBS/0.2% Triton X-100/2% NGS,E11.5, 13 sections were traced at 20-mm intervals spanning 1

2 of thewere applied to sections and incubated for 2 h at room temperature.optic cup and the distal 2

3 of the optic stalk (///, n Å 5 eyes; Krd//,Prior to the application of secondary antibodies, endogenous peroxi-n Å 6 eyes). All tracings were digitized using a drawing tabletdase activity was eliminated, in most cases, by application of 3%(Kurta Corp., Phoenix, AZ) and NIH Image 1.60 software. Individualhydrogen peroxide in 70% methanol. Antibodies against Pax2 werecomponents of the eye (lens, retinal neuroepithelium, pigmenteddetected using peroxidase-conjugated secondary antibodies (goatepithelium, subretinal space) and optic stalk (optic stalk epithe-anti-rabbit (IgG); Sigma), with a diaminobenzidine (SigmaFast-lium, optic stalk lumen) were identified by the morphology of indi-DAB, Sigma) color substrate. For fluorescence immunostaining,vidual cellular elements and/or position. The area (mm2) of eachgoat anti-rabbit (IgG)–TRITC (Sigma) (diluted 1:64 in PBS/0.2%digitized image was measured using NIH Image 1.60. Measure-Triton X-100/2% NGS) was substituted for the peroxidase-conju-ments of individual components (i.e., lens, subretinal space) weregated secondary antibodies and DAB. Slides were coverslipped us-summed for each eye and mean values for total areas were com-ing Gelmount (Biomedia, Foster City, CA) and photographed usingpared by independent sample t tests, using SPSS for Windows 6.1.differential interference contrast or epifluorescence illumination.This method tests the null hypothesis that the mean of the individ-ual ocular component measurement from the Krd// embryos isequal to the mean of the corresponding measurement from age-matched/// embryos. Values for P° 0.05 were considered statisti-Imagingcally significant.

Digital images were captured with an Optronics CCD camera(Optronics Engineering, Goleta, CA) attached to an Olympus Van-nox compound microscope and Capture/ software (TrueVision, RESULTSIndianapolis, IN) running on a Macintosh PowerPC. Adobe Pho-toshop 3.0 software was used to prepare all photomontages from

We use the convention of describing anatomical relation-either the original digital images or from digitized 35-mm slides.ships within the eye in terms of the longitudinal axis of theeye itself, rather than that of the embryo. Thus, structuresthat lie closest to the overlying head ectoderm, including

Three-Dimensional Reconstructions the cornea, lens, and iris, form the anterior structures ofthe eye, and those near the optic stalk and nerve, including

Three-dimensional reconstructions were generated from digitalthe retina and optic disc form the posterior structures. Theimages of a continuous series of 5-mm glycomethacrylate sectionsterms proximal and distal are also used to describe relativecut in the sagittal plane. The intervals between sections variedposition of histological sections within the optic cup andwithin the eye, with smaller intervals in regions of rapid change,optic stalk. In this context, a proximal section lies closer tosuch as the transition from optic cup to optic stalk. One eye wasthe embryonic midline and diencephalon, whereas a distalreconstructed from a normal and a mutant embryo from litters at

E10.5, E11.5, and E12.5 and, in the final reconstructions, each eye section lies closer to the anterior segments of the eye andincluded multiple ocular components as separate objects: E10.5, the overlying head ectoderm (see diagram, Fig. 2G).the optic cup (retina / pigmented epithelium), the lens, and thesubretinal space; E11.5, the optic cup and lens; and E12.5, the opticcup, lens, and optic nerve fibers. A more detailed description of Pax2 Protein Expression in the Normal Optic Cupthe methodology for the three-dimensional reconstructions will be and Stalkpublished separately. Briefly, captured images were exported into

We characterized Pax2 protein expression in sectionsAdobe Photoshop 3.0, aligned manually as layers of a single file, andexported to NIH Image 1.60 (available on the Internet by accessing through the optic cup and optic stalk from embryos athttp://rsb.info.nih.gov//nih-image/) to create a multi-image stack. E10.5, 11.5, 12.5, 14.5, 16.5, and adults, confirming and ex-Ocular components were isolated by removing unwanted portions tending the previous reports (Nornes et al., 1992; Puschelof the original images from duplicate copies of the stack file. Using et al., 1992; Torres et al., 1996). At E9.0, Pax2 expressioncustom programs (written by E. S. using Metrowerks Code Warrior), presages the sites within the optic vesicle where the neuro-X, Y, and Z coordinates, defining the perimeter of each image and epithelium will invaginate to form the double-layered opticthe intervals between sections, were used to generate contours

cup and ventral embryonic fissure (Nornes et al., 1990;which were subsequently transformed into 3D-DXF meshes usingPuschel et al., 1992). At E10.5 (data not shown) and E11.5,Nuages, a freeware program running on a Sun Sparc 2 workstation.Pax2/ cells in the ventral optic cup form the lips of theStrata Studio Pro software was used to align and smooth the 3D-retinal fissure, with the most darkly stained cells, presum-DXF mesh images, to assign color and opacity, and to generate final

surface renderings. ably those expressing the highest levels of Pax2, aligned at


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the ventral midline of the optic cup where the two sides of pattern beginning in the dorsal optic cup and progressingventrally toward the ventral midline and proximally towardthe retinal fissure meet (Figs. 1A and 1B). Pax2 immunoreac-

tivity continues uninterrupted into the optic stalk where, and into the optic stalk (Silver and Sapiro, 1981; Colello andJeffery, 1991). In the optic cup, there is a distinct junctionby E11.5, all cells express Pax2 and cells of the ventral optic

stalk epithelium have invaginated to form the optic groove between melanin-containing cells and those lacking mela-nin, but expressing Pax2 (Fig. 1D). Likewise, during the tran-(Fig. 1C; see also Fig. 3E). In the posterior optic cup, at

the transition between the retinal fissure and optic groove, sient melanization of the dorsal optic stalk (Silver andSapiro, 1981; Colello and Jeffery, 1991), melanin-containingPax2/ cells invaginate and encircle the site within the ret-

ina where the optic disc will form. In cross section, these cells do not express Pax2 and vice versa (Fig. 1H), although,in unpigmented regions of the optic stalk, all cells arecells form a wedge within the retinal neuroepithelium

flanking the fissure (Figs. 1C and 1D). From E11.5 to 12.5, Pax2/ (Fig. 1I).as the retinal fissure closes, the Pax2 immunostaining inthe ventral optic cup decreases, and by E16.5, Pax2/ cells

Ocular Development and Pax2 Immunostaining inare no longer found in the ventral retina (data not shown).Krd// EmbryosIn contrast, Pax2 immunostaining persists in cells at the

posterior pole of the retina, so that by E12.5, Pax2/ cells Sectioned optic cups and optic stalks/nerves from 55 Krd//animals at E10.5 to P2 were examined and compared to 59encircle the ganglion cell axons and hyaloid artery at the

nascent optic disc (Figs. 1E and 1F). Similarly, at E14.5 (data normal littermates. A single litter of animals at E9.5, theonly litter not genotyped, was also examined, and none ofnot shown) and at E16.5 (Fig. 1G), Pax2/ cells remain as a

thin cuff of cells that surround the optic disc, now filled these embryos exhibited malformations in either the opticvesicles or optic stalks. In contrast, in embryos E10.5 andwith the axons of ganglion cells.

During these periods of retinal development, Pax2/ cells older, the ocular phenotype invariably matched the geno-type that was determined using PCR.are also present within the optic stalk (Figs. 1C, 1H, and 1I)

and the lateral wall of the diencephalon (Figs. 1A and 1C; In normal embryos at E10.5, parasagittal sections throughthe head, which cut the optic cup en face (see Fig. 2G),see also Nornes et al., 1990; Puschel et al., 1992). By E16.5,

after the stalk is transformed into the optic nerve, Pax2/ reveal the retinal fissure as a cleft in ventral retina filledwith migrating mesenchymal cells that are destined to formcells are present in the optic nerve and at the vitreal bound-

ary of the optic disc (Fig. 1G). A similar pattern of Pax2 the hyaloid vasculature (Figs. 2A and 2C). The ventral opticgroove confers a characteristic horseshoe shape to the opticmRNA expression has been observed at E18.0 (Nornes et

al., 1990). We did not determine the stage at which Pax2 stalk (Fig. 2E), leading to the eventual occlusion of the opticstalk lumen. In Krd// embryos at E10.5, the retinal fissureprotein expression ceases, but there were no Pax2/ cells

in the retina, optic disc, or nerve of adult mice (data not forms in the distal optic cup and mesenchymal cells arevisible migrating into the optic cup (Fig. 2B). However, theshown).

In both the optic cup and stalk, there is a reciprocal pat- retinal fissure has not progressed into the posterior opticcup, resulting in an alteration in the distribution of cells intern of melanin pigment and Pax2 expression (see also Tor-

res et al., 1996). Within the optic cup at E11.5, Pax2 is the ventral optic cup and a somewhat misshapen profile(Fig. 2D). The optic groove fails to form in the ventral opticexpressed in cells of the prospective pigmented epithelium

that do not yet contain melanin granules (Fig. 1D). Melani- stalk resulting in the retention of a round or oval shape anda patent lumen (Fig. 2F).zation of the pigmented epithelium follows a characteristic

FIG. 1. Photomicrographs of Pax2 immunostaining in the normal embryonic eye. Horizontal sections through the ventral (A, B) and midoptic cup (C, D) at E11.5; B and D are higher magnification views of A and C, respectively. (A, B) Pax2/ cells are present in the ventralretina, where they are aligned at the embryonic fissure (arrowheads in B). (C) Pax2/ cells extend from the neuroretina in the posterioreyecup (large arrows) through the optic stalk (arrowheads) and into the diencephalon (see arrowheads in A). (D) Unpigmented cells withinthe outer layer of the optic cup (solid arrowhead) are Pax2/, whereas adjacent pigmented cells (open arrowhead) are not. Intense stainingof erythrocytes (RBC in C; see also A, D) reflects endogenous peroxidase activity which was not quenched prior to application of thediaminobenzidine substrate. In parasagittal sections through the posterior optic cup (E, F) and optic stalk (H, I) of E12.5 embryos, Pax2/cells extend from the ventral margin of the optic cup, along the site of the erstwhile embryonic fissure (arrowheads in E), and encirclethe optic disc (F). Ganglion cell axons within the optic disc appear lightly stained because, in our hands, the Pax2 antibody shows weakcross-reactivity to all axons. In regions of the optic stalk just behind the optic cup (H), Pax2/ cells (solid arrowhead) are present in theventral unpigmented half, whereas the pigmented cells (open arrowhead) in the dorsal optic stalk show no immunostaining. In sectionsthrough the optic stalk closer to the diencephalon (I), there are no pigmented cells and all cells are Pax2/ (arrowheads). (G) A frontalsection through the posterior optic cup and optic disc at E16.5 reveals that Pax2/ cells form a boundary between the neuroretina andoptic nerve (large arrowhead) and Pax2/ cells, presumptive astrocyte precursors, are also present within the optic nerve (small arrowhead)and at the optic nerve head (small arrow). (J) Cross section through the optic stalk at E12.5 (similar to H), where the primary antibodywas omitted (Note: see Fig. 2G for orientation of sections in E, F, H–J). NE, neuroepithelium; L, lens; HA, hyaloid artery. Scale bar: 140mm in A and C; 70 mm in B and D; 150 mm in E; 50 mm in F, H, I, and J; 100 mm in G.


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214 Otteson et al.

FIG. 2. Photomicrographs of representative parasaggital sections through the optic cup and optic stalk of normal (A, C, E) and Krd// (B,D, F) embryos, E10.5. In the distal optic cup of both normal (A) and Krd// (B) embryos, the embryonic fissure (arrowheads) is present,whereas in more proximal portions of the optic cup (D) and in the optic stalk (F) of Krd// embryos, the embryonic fissure is absent. Notethe visible subretinal space (arrow) present in the dorsal optic cup of Krd// embryos (B, D). Because of the lateral orientation of the opticcup, parasagittal sections through the head cut the optic cup en face and the optic stalk in cross section, as illustrated by a cartoon (G),indicating the plane of section (gray) through the optic cup and posterior lens shown in C and D. The lens (L) lies in the anterior portionof the optic cup with the optic stalk at the posterior pole. The terms distal and proximal refer to the relative position of structures andsections, relative to the diencephalon (Di) and the embryonic midline, with distal structures farthest from and proximal structures nearestto the diencephalon. Scale bar, 100 mm in A–F.


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FIG. 3. Photomicrographs of parasaggital sections through the optic cup and optic stalk in normal (A, C, E) and Krd// (B, D, F) embryos atE11.5. Note the obvious retinal fissure and the overall similarity of the distal optic cups from normal (A) and Krd// (B) mice. Sections throughthe nascent optic disc show a well-defined ventral invagination in the normal embryo (arrowhead in C), that is absent in the Krd// littermate(D). Likewise, in the optic stalk of the normal embryos (E), there is a clear invagination of the optic groove (arrowhead), whereas the opticstalk in the Krd// embryo (F) shows no invagination. L, lens. Scale bar: 150 mm in A and B; 110 mm in C and D; 100 mm in E and F.

At E11.5, the anterior regions of the optic cup of both malformations within the posterior optic cup and opticstalk at E10.5 (Figs. 2D and 2F) persist and are relativelynormal and Krd animals are morphologically similar, with

a well-formed retinal fissure (Figs. 3A and 3B). However, unchanged at E11.5 (Figs. 3D and 3F). Three-dimensional


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FIG. 4. Three-dimensional reconstructions of representative normal (A) and Krd// (B) optic cups at E11.5, viewed from a perspectivethat shows the ventral surface of the optic cup and optic stalk. Although the retinal fissure is present in the optic cup of Krd// embryos(B), it fails to extend through the posterior pole of the retina into the optic stalk. Arrows indicate the approximate location of sectionsillustrated in Fig. 3.

reconstructions of optic cups from normal and Krd// em- Quantitative differences observed at E10.5 are transientand differences in the amount of retina, pigmented epithe-bryos at E10.5 (data not shown) and at E11.5 (Figs. 4A and

4B) confirm that the apparent difference in optic cup mor- lium, and subretinal space are no longer present by E11.5(Fig. 5B), although the posterior optic cup and optic stalkphogenesis between normal and Krd// embryos is a failure

of the embryonic fissure to progress proximally beyond the continue to be visibly malformed (see Figs. 3D and 3F).During early embryogenesis, there is significant dailyoptic cup and into the optic stalk.

Differences between normal and Krd// embryos are not growth of the optic cup, and by E11.5, the area of the malfor-mation within the posterior optic cup of Krd// embryosa consequence of an overall developmental delay in Krd//

embryos. This is based on two sets of observations. First, constitutes a much smaller portion of the total optic cupthan it did at E10.5. Thus, in this analysis, the overall simi-differences in the size and apparent developmental stage of

the embryos (e.g., crown to rump length; extent of melaniza- larity of the optic cups from normal and mutants embryosat E11.5 outweighs the differences associated with the mal-tion of the optic cup) did not correlate with the genotype

of the animals. Second, quantitative comparisons of optic formation at the forming optic disc.There is apparently normal closure and fusion of the reti-cups at E10.5 (Fig. 5A) and E11.5 (Fig. 5B) indicate there are

no differences in the average size of the optic cup or lens nal fissure in Krd// embryos (data not shown), and inadults, defects are not observed at the former site of thein normal and Krd// embryos. At E10.5, however, there is

a statistically significant difference in the relative propor- retinal fissure within the ventral retina (Keller et al., 1994).In contrast, within the posterior optic cup, the failure oftions of inner retinal neuroepithelial and outer pigmented

epithelial layers. In the optic cups of Krd// mice, there is the optic groove to form has consequences for the growthof ganglion cell axons out of the retina. In the retinas ofless tissue within the inner layer and more in the outer

layer of the optic cup (Fig. 5A) when compared to their normal embryos at E12.5, Pax2/ cells encircle the fasciclesof ganglion cell axons which form a crescent dorsal to thenormal littermates. Because in our analysis, epithelial lay-

ers were identified by position only, we believe that these hyaloid artery (Figs. 6A–6C; see also Figs. 1E and 1F). Asdevelopment proceeds, the number of axons exiting the eyedifferences result from the abnormal formation of the em-

bryonic fissure in Krd// mice; neuroepithelial cells des- increases and the dorsal crescent of axon fascicles expandsventrally to surround the artery (Silver and Robb, 1979), alltined for the inner retinal layer are present in the outer

pigmented epithelial layer. In addition, at E10.5, the de- the while remaining within the cuff of Pax2/ cells (see Fig.1G). In the Krd// embryos, Pax2/ cells are present in thecreased size of the retinal neuroepithelium and concomitant

increase in the size of the pigmented epithelium creates a approximate location of the nascent optic disc, but do notform a coherent annulus surrounding the ganglion cell ax-spatial mismatch between the two layers of the optic cup

resulting in an increase in the amount of subretinal space ons (Figs. 6D–6F). With the failure of optic groove forma-tion, Pax2/ cells that do not invaginate to surround the(Fig. 5A; see also Figs. 2A–2D).


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Despite the failure of the embryonic fissure to invaginatein the posterior optic cup and optic stalk of Krd// embryos,ganglion cell axons are able to exit the optic cup, as evidencedby the presence of axon fascicles in sections through the opticstalk of embryos at E12.5 (Fig. 6L) and later (Figs. 7B–7D). Inthe distal optic stalk of normal embryos at this stage, Pax2/cells are confined to the ventral aspect of the optic stalk and,as in the retina, are associated with fascicles of ganglion cellaxons (Figs. 6G, 6H, and 6K). Similarly, in Krd// embryos,Pax2/ cells are also present in the ventral optic stalk in closeassociation with ganglion cell axons, although the failure ofthe embryonic fissure to extend proximally results in a stalkwith an abnormal morphology (Figs. 6I, 6J, and 6L).

In embryos older than E12.5, although there are no appar-ent defects in other regions of the optic cup, malformationsof the optic nerve, optic disc, and the adjacent retina persistas features diagnostic of the Krd// genotype. In the Krd//animals, the ganglion cell axons appear to penetrate the eye,not as a coherent bundle, but as isolated fascicles. At theoptic disc, ganglion cell axons traverse the retinal neuroepi-thelium, creating ‘‘islands’’ of neuroepithelial cells amongthe axons (Fig. 7B). In areas of the retinal neuroepitheliumadjacent to the optic disc, there are clefts and folds withinthe neuroepithelium (Fig. 7D). These neuroepithelial ‘‘is-lands’’ and folds are likely the origins of the rosettes ofphotoreceptor cells and laminar defects seen in the adultFIG. 5. Graphs illustrating the quantitative morphometric analy-retina (Keller et al., 1994). In addition, although not quanti-sis of optic cups from Krd// embryos at E10.5 (A) and E11.5 (B).

The data are expressed as a percentage difference from age-matched fied, there appear to be fewer cell bodies present among thenormal controls. Data in A show that there are no statistically ganglion cell axons of the optic disc and nerve (Figs. 7B andsignificant differences in the overall size of the optic cup, lens, 7C). In the eyes of normal mice at E16.5 and later, there isor optic stalk in normal and Krd// embryos, although there are a distinct boundary between the pigmented epithelium andsignificant differences in the amount of retinal neuroepithelium the unpigmented optic nerve (Fig. 7A). In the eyes of Krd//and subretinal space; n Å 4 (///), n Å 6 (Krd//). (B) At E11.5, these littermates, pigmented cells extend along the surface of thesame measures show no statistically significant differences. The

forming optic nerve (Figs. 7B–7D) and the retinal neuroepi-large numerical difference in the amount of subretinal space isthelium often extends into the optic nerve just beneathnot statistically significant because of large variance across boththese pigmented cells (Fig. 7B). In homozygous Pax2 mu-normal and mutant eyes; n Å 5 (///), n Å 6 (Krd//). *P õ 0.05;tants, similar but more extensive pigmentation defects have**P õ 0.01; OC, optic cup; L, lens, R, retinal neuroepithelium,

RPE, retinal pigmented epithelium; SR-S, subretinal space; OS, op- been reported (Torres et al., 1996).tic stalk epithelium. At all time points examined, there is considerable varia-

tion in the extent of ocular defects between littermates andoften between eyes of the same animal. However, we findthat all Krd// embryos at E10.5 and older show at least anascent disc remain outside the retina, within the ventralsubset of these defects. The defects which most consistentlyoptic stalk. Consistent with this, although not quantified,predicted the mutant geneotype were the failure of opticwe observe an increase the number of Pax2/ cells presentgroove formation and patent optic stalk lumen during mor-in the ventral optic stalk in areas immediately adjacent tophogenesis and the abnormal pigmentation of the opticthe optic cup (compare Figs. 6H and 6J; see also Figs. 6Knerve during later development.and 6L). Thus, although there is an apparent reduction in

the numbers of Pax2/ cells in many sections through thedeveloping optic disc (Fig. 6E), we believe this likely reflectsaltered morphogenetic movements of Pax2/ cells rather DISCUSSIONthan an overall reduction in cell numbers. Axon fasciclesare present and identifiable the retinas of Krd// embryos, Pax2 Haploinsufficiency and Ocular Defects inbut are generally reduced in overall number, often forming Krd// Micea single fascicle (Fig. 6F) or a scattering of smaller fascicles

Krd mice carry a 7-cM deletion that includes the Pax2within the retina (data not shown), rather than the normalgene and may encompass as many as 400 other loci (Kellercrescent-shaped array (Fig. 6C). In Krd// embryos, axon fas-et al., 1994). Although we cannot dismiss the possibilitycicles are nearly always associated with cells with an elon-

gated morphology typical of the Pax2/ disc cells (Fig. 6F). that hemizygosity of other genes within this large deletion


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FIG. 6. Pax2 immunostaining and cellular morphology in photomicrographs of parasagittal sections through the optic disc and opticstalk of normal (A–C, G, H, K) and Krd// (D–F, I, J, L) embryos at E12.5. (A, B) Pax2/ cells surrounding the optic disc in a normalembryo. (C) Comparable section from a glycomethacrylate-embedded eye illustrating the elongated morphology of Pax2/ cells (arrowheads)arrayed about the hyaloid artery and ganglion cell axons (arrow). (D, E) Pax2/ cells in the retina of a Krd// embryo. (F) Comparablesection from a glycomethacrylate-embedded eye. Note the relative spatial disorganization of the Pax2/ cells in the area of the optic disc(D, E; arrowheads in F), and the paucity of axon fascicles (arrow in F). (G, H) Pax2/ cells in the optic stalk of a normal embryo. (K)Comparable section from a glycomethacrylate-embedded eye illustrating the fascicles of axons at the ventral surface of the optic stalk.(I, J) Pax2/ cells in the optic stalk of a Krd// embryo. Note that, as in the normal embryo, Pax2/ cells are present in the ventral opticstalk, although the lumen remains patent. (L) Comparable glycomethacrylate section illustrating the presence of axon fascicles (arrow) atthe ventral surface of the optic stalk. (A, D, G, I) Double exposures using differential interference contrast and epifluorescence illumination.Pax2 immunostaining was consistently less intense on sections from Krd// embryos, and exposure time for Pax2 immunofluorescenceof sections from Krd// embryos was twice that of the normal controls. HA, hyaloid artery. Scale bar: 100 mm in A and D; 50 mm in B,C, E, and F; 80 mm in G, H, I, and J; 60 mm in K and L.

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FIG. 7. Photomicrographs of frontal sections through the optic discs and nerves from normal (A) and Krd// (B–D) mice at E16.5. B–Dillustrate representative optic disc malformations observed in Krd// mice: Arrowheads indicate islands of neuroepithelial cells withinthe optic nerve (B) and laminar defects in the retinal neuroepithelium (D). Pigmented cells, continuous with those of pigmented epithelium(arrowhead in C, see also B and D), and retinal neuroepithelial cells (arrow in B) extend along the optic nerve, beyond their normalboundary at the junction of the optic cup and optic stalk (arrowhead in A). Scale bar, 150 mm.

contributes to the Krd phenotype, we believe haploinsuffi- defects observed in the retina are specifically associatedwith abnormal morphogenetic movements of Pax2-express-ciency of Pax2 is primary in the etiology of the congenital

anomalies found in Krd// mice. First, we find no evidence ing cells. Third, the congenital anomalies present in theKrd//mice, including aplastic, hypoplastic, and cystic kid-for generalized developmental delay in Krd// embryos that

might indicate nonspecific deletion effects. Second, Pax2 is neys, pan-retinal hypocellularity, reduced electroretino-gram, and optic disc malformations (Keller et al., 1994;expressed during embryogenesis in precisely those tissues

affected in the mutants. Indeed, we find that the initial Hitchcock et al., 1995; Otteson et al., 1996), are remarkably


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220 Otteson et al.

similar to those found in other Pax2 heterozygotes, both in the promoter region of the cell adhesion molecule, L1(Chalepakis et al., 1994b), and Pax3 (Chalepakis et al.,murine (Torres et al., 1995, 1996; Favor et al., 1996) and

humans (Sanyanusin et al., 1995a,b; Schimmenti et al., 1994a), Pax6 (Holst et al., 1997), and Pax8 (Holst et al.,1994) can regulate in vitro transcription of the neural cell1995, 1997). Although ocular malformations of the optic

disc and/or optic stalk were present in all Krd// animals adhesion molecule, N-CAM. Several cell adhesion mole-cules are known to be expressed in the developing eye, in-examined, defects were reported to be variable in their oc-

currence in mice heterozygous for induced Pax2 mutation cluding R-cadherin and N-cadherin (Redies and Takeichi,1993; Riehl et al., 1996). Interestingly, a novel cadherin, K-(Torres et al., 1996). This may reflect differences in genetic

background or a failure to identify the full spectrum of reti- cadherin, has been shown to be expressed in many tissuesthat express Pax2, including the developing kidney andnal defects present. Alternatively, hemizygosity for other

genes within the Krd deletion may compound or enhance brain (Xiang et al., 1994) and, within the embryonic eye, thedeveloping optic disc and nerve (P. Hitchcock, unpublishedthe retinal defects associated with hemizygosity of Pax2.data).

Pax2/ Cells and Optic Groove MorphogenesisPax2/ Cells and Axon Guidance at the FormingThe analysis of adult phenotypes provides only indirectOptic Discinsight into the origins of congenital defects; therefore, we

undertook a detailed comparative analysis of early ocular Based on morphology and position, we believe that thePax2/ cells at the optic disc play a role in guiding ganglionmorphogenesis and Pax2 protein expression in normal and

Krd// mice. It has previously been shown that Pax2 is ex- cell axons out of the retina. The Pax2/ cells that encirclethe optic disc are a subset of the channel-forming cells firstpressed in cells of the ventral retina and optic stalk at the

site where the embryonic fissure forms (Nornes et al., 1990; described by Silver and Robb (1979). These cells create ex-tracellular spaces or channels among their endfeet at thePuschel et al., 1992; Torres et al., 1996). Our results confirm

and extend these results to show that within the posterior inner surface of the pia that are believed to play a role inguiding ganglion cell axons out of the retina into the opticretina, Pax2/ cells of the ventral neuroepithelium invagi-

nate to demarcate the retinal site of the optic disc. Here, nerve (Silver and Robb, 1979; Silver and Sidman, 1980; Hors-burgh and Sefton, 1986; Colello and Guillery, 1992; Brittisthe Pax2/ cells persist into late embryogenesis as a cuff of

cells that encircles the forming optic disc, creating a bound- and Silver, 1995). In addition, Netrin-1, a secreted proteinthat is required for axon guidance of commissural neuronsary between the ganglion cell axons and the retinal neuro-

epithelium. Pax2-expressing cells that encircle the disc are within the developing brain (Serafini et al., 1996; see alsoSerafini et al., 1994), is expressed by the Pax2/ cells atcontinuous with those of the optic stalk and may represent

an extension of the optic stalk into the optic cup. Both the the optic disc and optic nerve, and Netrin-1 mutants showsignificant axon pathfinding defects at the optic disc (DeinerPax2/ cells that encircle the optic disc and those present in

the developing optic nerve have been shown to be astrocyte et al., 1997). In the Krd// embryos, the disorganization ofthe Pax2-expressing cells at the optic disc would be pre-precursors that derive from cells of the optic stalk (Edwards

et al., 1990; Huxlin et al., 1992; see also MacDonald et al., dicted to result in both an abnormal organization of thenetwork of extracellular channels and a dispersion of the1997). In the Krd// embryos, although the retinal fissure

forms within the ventral optic cup, the ventral optic stalk associated chemoattractant guidance cues to ectopic loca-tions. Either or both of these events could give rise to theepithelium does not invaginate to form the optic groove. At

the junction between these two portions of the embryonic intraretinal defects in axon pathfinding observed in Krd//embryos.fissure, the lack of invagination of the ventral epithelium

is associated with malformation of the optic disc. Specifi- We observe an initial reduction in the number of identi-fiable axon fascicles at E12.5 in Krd// mice (Fig. 6L). How-cally, it is the Pax2/ cells that fail to invaginate and, as a

consequence, fail to form a coherent annulus surrounding ever, at E16.5 (Fig. 7D) and E18.5 (data not shown), size ofthe optic nerve as it exits the cup does not appear qualita-the nascent optic disc.

The early defects associated with reduction or loss of tively different than in normal littermates. This is some-what surprising since adult Krd//mice have reduced num-Pax2 expression in mice result from altered movements of

epithelial cells (see Keller et al., 1994; Torres et al., 1995, bers of ganglion cells within the retina and correspondinglyreduced numbers of axons within the optic nerve (Green et1996; Favor et al., 1996). It is well known that precise regu-

lation of the expression of cell adhesion molecules is re- al., 1997). However, this is a highly variable phenotype andthe cause and time course of ganglion cell loss is not yetquired for the morphogenetic movements of simple epithe-

lia as they form the complex shapes of mature organs (re- known (see also below).viewed by Hynes, 1994; Fagotto and Gumbiner, 1996; Marrsand Nelson, 1996; Redies and Takeichi, 1996; Ruoslahti and

Ocular Anomalies in Later DevelopmentObrink, 1996; Steinberg, 1996). Although it is unknownwhich genes are regulated by Pax2 expression in the eye, Defects observed during ocular morphogenesis in Krd//

embryos provide insight into the origin of the ocular defectsthere is evidence that other Pax genes regulate the expres-sion of cell adhesion molecules. Pax6 binds to a sequence observed adult Krd//mice and, by extension, in other Pax2


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FIG. 8. Proposed model of optic disc formation in Krd// and Pax2 heterozygous mice. Diagrams based on three-dimensional reconstruc-tions of the optic cup and forming optic nerve at E12.5 and on histological preparations of later stages of development in /// (A, B) andKrd// (C, D) mice. Ganglion cell axons (yellow) follow the surface of the retina (gray) toward the posterior pole of the optic cup wherethey exit the eye among Pax2/ cells (red). (A) In normal mice, prior invagination of the optic groove has created a cuff of Pax2/ cellsthat encircle the axons and guide them to the optic stalk. (The optic stalk (OS) is shown as a cutaway to illustrate the formation of theventral optic groove.) (B) Later in development, continued addition of axons fills the optic disc and Pax2/ cells are no longer detected inthe retina. The former optic stalk, now filled with axons, has become the optic nerve. (C) In Krd// and Pax2 heterozygous mice at E12.5,the failure of optic groove formation alters the position of the posterior retina and the Pax2/ cells. Axons exit the eye along the retinalsurface, traveling obliquely to reach the ventral optic stalk. The transected optic stalk shows the lack of optic groove formation and thepatent optic stalk lumen (OSL) which creates a space behind the retina. Subsequent addition of ganglion cell axons at the disc pushes theadjacent retina into this space (arrow in C). (D) Displacement of retina into the optic stalk may give rise to laminar defects and theconcave morphology of the optic disc observed in Krd// mice and Pax2 heterozygotes, both murine and human. Note that diagramsdepicting different stages of eye development are not drawn to scale. The pigmented epithelium is indicated by the thick black line. Inboth normal (A) and mutant (C) eyes, pigmentation extends along the dorsal optic stalk at E12.5. Only mutant mice (D) show pigmentationin the optic nerve at later stages of development. ON, optic nerve; L, lens.

heterozygotes. Both adult Krd// mice and adult mice het- cup and optic nerve. Reduced electroretinograms corre-spond with the retinal hypocellularity (Green et al., 1997),erozygous for other Pax2 mutations show ocular pheno-

types characterized by rosettes of displaced retinal cells and although the basis for the reduction in cell number withinthe retina is less obvious. Retinal hypocellularity is alsolaminar defects of the retina, reduced electroretinogram,

pan-retinal hypocellularity, abnormal patterns of vasculari- observed in heterozygous Pax21Neu mice (Favor et al., 1996),suggesting that this may be related to reduced Pax2 expres-zation, and the presence of an enlarged cavitated optic disc

(Keller et al., 1994; Favor et al., 1996; Torres et al., 1996; sion rather than a nonspecific effect of the Krd deletion.Early defects in axonal organization and the subsequentGreen et al., 1997). Photoreceptor rosettes and laminar de-

fects observed in Krd// mice are generally found within malformations at the optic disc appear to arise, at least inpart, from the failure in optic groove formation. Duringthe retina adjacent to the optic disc and presumably arise

from the ‘‘islands’’ of retinal cells and from folding of the morphogenesis of the optic cup, the retina maintains physi-cal continuity with the ventral optic stalk at the site of theretinal neuroepithelium at the junction between the optic


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222 Otteson et al.

forming optic disc. The first ganglion cells are born in the to optic groove formation, haploid expression of Pax2 issufficient to mediate closure of the retinal fissure. Bilateraldorsal retina immediately adjacent to the forming optic disc

(Mann, 1964; Sidman, 1961; Kahn, 1973; Silver and Robb, retinal colobomas, resulting from failure of retinal fissureclosure, are present in homozygous, but not heterozygous1979) and their axons are thought to be constrained from

projecting away from the forming disc by the presence of Pax2 mutant mice (Favor et al., 1996; Torres et al., 1996).In seven independent human pedigrees, patients hetero-inhibitory cues within the peripheral retina (Snow et al.,

1991; Brittis et al., 1992). They must therefore project to- zygous for mutations in PAX2 have been identified, all withautosomal dominant renal anomalies and bilateral opticward posterior pole of the optic cup and the site of the

forming optic disc where they encounter the Pax2/ cells disc colobomas. This is a relatively uncommon type of colo-boma, similar to that present in Pax2 heterozygous mice,and their associated guidance cues (Fig. 8A; see discussion

above). In Krd// embryos, ganglion cell axons are appar- that is characterized primarily by an enlarged, funnel-shaped optic disc extending into the retina (Sanyanusin etently still guided toward the posterior pole of the optic cup,

but at the optic disc, they encounter a disorganized array al., 1995a,b; Schimmenti et al., 1995, 1997). The develop-mental mechanisms underlying this type of coloboma haveof Pax2/ cells. Despite the failure of the optic groove to

form, the retina maintains its continuity with the ventral not been directly examined in humans, although it has gen-erally been thought that both retinal and optic disc colobo-optic stalk epithelium allowing the ganglion cell axons to

follow the surface of the retina to reach the Pax2/ cells mas arises from a failure of closure of the embryonic fissure(Mann, 1957, 1964; Lopashov and Stroeva, 1964; Brown andand the ventral optic stalk (Fig. 8C). During subsequent

development, ganglion cell axons fill the disc and may force Tasman, 1983; Apple, 1984; Silver et al., 1984; Schimmentiet al., 1995). The early development of the optic vesicle andthe adjacent retinal neuroepithelium into the occult space

created by the persistent optic stalk lumen. Such epithelial eye is highly conserved among mammalian species (Mann,1964; Lopashov and Stroeva, 1964) and phenotypes presentdisplacement could cause the retinal folding observed near

the concave optic disc (Fig. 8D; see also Fig. 7D). in humans and mice heterozygous for Pax2 mutations areremarkably similar. This suggests that the murine Pax2Apart from the optic disc anomalies and the retinal hypo-

plasia observed in the retinas of Krd// and Pax21Neu mice mutations provide a model for understanding the develop-ment of these malformations in humans. Based on our anal-(Keller et al., 1994; Favor et al., 1996; Green et al., 1997),

the remainder of the optic cup is similar to that of age- ysis of the development of optic disc malformations inKrd//mice, we propose that optic disc anomolies observedmatched controls (data not shown). This is consistent with

the apparently normal growth of the optic cup and morpho- in Pax2 heterozygotes, both murine and human, representa unique subclass of coloboma that does not result fromgenesis of the retinal fissure in these mutants, and with the

limited domain of Pax2 expression within the developing abnormal closure of the retinal fissure, but rather from theabnormal formation of the optic groove.optic cup. With the failure of optic groove formation, it is

possible that the optic disc may be displaced toward mal-formed fissure. However, we were unable to discern if thiswas true or not in Krd//mice, suggesting that any malposi- ACKNOWLEDGMENTStioning of the disc is likely to be subtle.

The authors thank the following individuals who contributed tothis study: Dr. Gregory Dressler for his kind gift of the Pax2 anti-Pax2 Gene Expression and Colobomabody; Dr. Miriam Meisler for generously providing the Krd mice

Pax2 is important in the morphogenesis of the embryonic used in this study; Dr. David Sretavan for communicating resultsfissure, and reduction or loss of Pax2 expression results in concerning Netrin-1 prior to publication; Julie Pearlman, Jeff Van-

DeRyt, and Mitch Gillett for providing technical assistance; Drs.coloboma of the optic disc and/or retina (Hitchcock et al.,Kate Barald and Miriam Meisler and members of the Hitchcock1995; Sanyanusin et al., 1995a,b; Schimmenti et al., 1995,lab for critical readings of the manuscript. This research was sup-1997; Favor et al., 1996; Otteson et al., 1996; Torres et al.,ported by NIH Grants EY07003 and EY07060 (CORE) to P.F.H.,1996; see also MacDonald et al., 1997). A comparison ofGM24872 to Miriam Meisler, and NSF-Research Training Grant tophenotypes observed in heterozyous and homozygous Pax2D.C.O. P.F.H. is a Research to Prevent Blindness William and Mary

mutant mice shows different requirements for Pax2 during Greve International Research Scholar.morphogenesis of the embryonic fissure. Full diploid ex-pression of Pax2 is required for formation of proximal por-tions of the embryonic fissure and the lack of one copy of

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Pax2 Expression and Retinal Morphogenesis in the Normal ... · surecan besubdividedinto twocontiguousparts: theretinal congenital retinal and optic disc anomalies collectively re-