Postnatal Development of the Eye inthe Naked Mole Rat

(Heterocephalus glaber)NATALYA V. NIKITINA,1 BRONWEN MAUGHAN-BROWN,1

M. JUSTIN O’RIAIN,2 AND SUSAN H. KIDSON1*1Department of Human Biology, Faculty of Health Sciences, University of Cape

Town, Cape Town, South Africa2Department of Zoology, University of Cape Town, Cape Town, South Africa

ABSTRACTThe naked mole rat (Heterocephalus glaber) is a subterranean rodent

whose eyes are thought to be visually nonfunctional and as such is an idealanimal with which to pursue questions in evolutionary developmental biology.This report is the first in-depth study on the development and morphology ofthe naked mole rat eye. Using standard histological analysis and scanning andtransmission electron microscopy, we describe the structural features of theeye. We further report on the morphological changes that accompany thedevelopment of this eye from neonate to adult and compare them with thosethat occur during mouse eye development. We observed numerous abnormal-ities in the shape and cellular arrangement of the structures of the anteriorchamber, with notable malformations of the lens. Cell proliferation and celldeath assays were conducted to investigate the possible causes of lens malfor-mation. We found that neither of these processes appeared abnormal, indicat-ing that they were not responsible for the lens phenotype of the mole rat. Inorder to investigate the process of lens differentiation, we analyzed the expres-sion of �-crystallins using Western blots and immunocytochemistry. At birth,levels of �-crystallin appear normal, but soon thereafter, the �-crystallin ex-pression is terminated. Absence of detectable �-crystallins in adults suggeststhat there is a gradual degradation and loss of these proteins. The evolutionaryfactors that could be responsible for the eye morphology of the naked mole ratare discussed. A model for abnormal lens differentiation and the role it plays inthe morphogenesis of the rest of the eye in the naked mole rats is proposed.Anat Rec Part A 277A:317–337, 2004. © 2004 Wiley-Liss, Inc.

Key words: mole rat; Heterocephalus glaber; eye development;lens; �-crystallins

The eyes of fossorial mammals provide an excellentmodel for studies of eye malformations during develop-ment and for research into the evolution of blindness. Thebasic process of eye development is similar in all verte-brates and has been extensively reviewed (Graw, 1996;Oliver and Gruss, 1997; Jean et al., 1998; Chow and Lang,2001). In brief, the eye forms as a lateral outgrowth of thediencephalon (forebrain) neuroectoderm, which enlargesto become the optic vesicle. When the optic vesicle contactsthe overlying surface ectoderm, an exchange of inductivesignals between these tissues is thought to take place,which results in their coordinated invagination to formthe lens vesicle and the optic cup (Chow and Lang, 2001).The lens vesicle formed during this process is sphericaland initially hollow, but soon becomes filled by the pri-

mary lens fibers, which form by elongation of the epithe-lial cells located at the posterior of the lens vesicle. There-

Grant sponsor: South African Medical Research Council; Grantsponsor: University of Cape Town; Grant numbers: 415634 and435971.

*Correspondence to: Dr. Susan H. Kidson, Department of Hu-man Biology, Faculty of Health Sciences, University of CapeTown, Observatory, 7925, Cape Town, South Africa. Fax: 27-21-448-7226. E-mail: suekid@cormack.uct.ac.za

Received 20 November 2003; Accepted 18 December 2003DOI 10.1002/ar.a.20025

THE ANATOMICAL RECORD PART A 277A:317–337 (2004)

© 2004 WILEY-LISS, INC.

after, new fibers are constantly added to the body of thelens from a population of proliferating cells located in theequatorial region. These new secondary lens fibers elon-gate and form the outer layers of the lens. As the lensgrows, the primary fibers become compacted, and eventu-ally become dissociated from the lens epithelium and thelens capsule and are pushed into the center of the lens,forming the embryonic lens nucleus. In the mouse, matu-ration of these primary fibers, which involves the loss ofnuclei and organelles and synthesis of lens-specific pro-teins �-, �-, and �-crystallins, is completed by postnatalday 14 (P14), when the eyelids open. The denucleationprocess is largely completed by P1, so that at birth, nonucleated fibers are found in the center of the lens(Vrensen et al., 1991). There is no turnover of proteins inthe mature lens, but because the new fibers are added toit continuously, the expression of �-crystallins can be de-tected in the maturing secondary fibers throughout thelife of an animal. In the mouse, �-crystallin expressionincreases until it reaches adulthood (about P40), and thengradually declines (Goring et al., 1992).

At the same time, and closely coordinated with theformation of the lens, morphogenesis of other eye struc-tures takes place. The optic cup differentiates to form theneural retina and the retinal pigmented epithelium(RPE), and its anteriormost tips begin to form the iris andthe ciliary body under the influence of the signals from thelens (Beebe, 1986). The ciliary epithelium folds to form theciliary processes, and the connective tissue of the ciliarybody and the ciliary muscle are formed from the mesen-chymal cells that have immigrated from the neural crest(Johnston et al., 1979). In the mouse, the first indication ofciliary folding and the appearance of the iris primordiumare observed around E17, and by birth there are clearborders between the ciliary body and the retina posteri-orly and between the ciliary body and the iris anteriorly(Pei and Rhodin, 1970; Theiler, 1989). The differentiationof neural retina begins around E17.5, with the appearanceof the ganglion cell layer. By P7, the outer plexiform layerappears, separating inner and outer nuclear layers. Thedifferentiation of the retina is completed by P14 (Pei andRhodin, 1970; Young, 1984; Theiler, 1989). Much of ourcurrent knowledge on eye morphogenesis is derived fromthe studies of mutant animals that exhibit various eyedefects (Jean et al., 1998). Naturally blind animals canprovide an additional model for such studies and allowresearchers to circumvent the problems associated withinducing blindness in sighted animals. Since all naturallyblind vertebrates must have evolved from sighted ances-tors, investigations into their eye structure and develop-ment can also provide a better understanding of the evo-lutionary mechanisms operating during the atrophy of anorgan that has become obsolete.

About 3.5% of all mammals are adapted to living under-ground and reduced visual systems are common amongvertebrates adapted to such subterranean habitats (Nevo,1979). The loss of eyesight is postulated to be the result ofthe decrease in the evolutionary pressure that is respon-sible for the maintenance of functional organs of visualperception. Such a decrease in selection pressure wouldallow for the accumulation of mutations in genes involvedin eye development (with the exception of developmentalregulatory genes, which are also involved in the pattern-ing and morphogenesis of other important structures inthe body). As a result, various eye phenotypes can be

observed among underground animals, ranging from rel-atively normal to completely malformed or microphthal-mic. The smallest eyes, completely covered by skin, areseen in Notorychtes (marsupial moles), and in the goldenmoles Chrysochloris and Eremitalpa (Chrysochloridae; In-sectivora). The eyes of Notorychtes are vestigial and lacklenses (Sweet, 1906; Vaughan, 1978), while those of Er-emitalpa consist of a mass of disorganized cells represent-ing the lens, surrounded by a well-differentiated retina(Gubbay, 1956). The true moles of the family Talpidaehave larger eyes with more typical eye architecture, in-cluding the iris, ciliary body, anterior and posterior cham-bers, and the retina with clearly distinguishable layers.The lens is present, but consists of irregularly shapednucleated cells, which cannot be called true fibers(Slonaker, 1902; Quilliam, 1966). An interesting eye phe-notype is observed in Spalax ehrenbergi (Spalacidae; Ro-dentia): both the pupil and the anterior chamber of thisanimal’s eye are completely obliterated by an overgrowthof highly pigmented iris-ciliary body complex. The lens isvery small, undifferentiated, and seems to undergo necro-sis (Sanyal et al., 1990). These various eye phenotypes arethought to have appeared as independent evolutionaryevents, and their superficial similarity is postulated to bedue to convergent evolution (Nevo, 1979). For instance,members of the two rodent families Spalacidae and Bathy-ergidae have similarly reduced eyes, but they are morephylogenetically disparate than other families withsighted species within the order (Eisenberg, 1981). Whilesome of the structural and molecular aspects of the eye ofthe blind mole rat Spalax have been investigated (Quax-Jeuken et al., 1985; Hendriks et al., 1987; Avivi et al.,2001; Hough et al., 2002), very little is known about theBathyergid eye. Previous descriptions of the structure ofthe adult naked mole rat eye are superficial and oftencontradictory (Cei, 1946b; Hill et al., 1957). For instance,Cei (1946b) describes the mole rat lens as poorly differen-tiated with primitive characteristics, while Hill et al.(1957) state that the lens is differentiated; similarly, thereis a disagreement between these two authors concerningthe structure of the iris. To our knowledge, no descriptionof the structure of the neonate mole rat eye or its devel-opment to adulthood has been published.

The naked mole rat (Heterocephalus glaber) is found inthe hot, arid regions of Kenya, Somalia, and Ethiopia.Large colonies, usually composed of 75 to 80 related indi-viduals, live in extensive burrow systems that can be up to3 km long and occupy an area greater than 100,000 m2

(Sherman et al., 1991). Naked mole rats are eusocial,exhibiting a truly social structure with a reproductivedivision of labor, cooperative care of young, and an overlapof generations (Jarvis, 1981). Since most naked mole ratswithin a colony do not reproduce, the definition of adult-hood as reproductive maturity is difficult to apply. Theyoungest captive mole rats reported to be reproductivelyactive were 8–12 months old (Jarvis, 1991); therefore, forthe purposes of this study, we have designated the adultas being over 12 months old, regardless of its reproductivestatus. The peculiar social system of the naked mole ratsand their tendency to establish new colonies by fissionresult in extremely high levels of inbreeding both within asingle colony and between the colonies of a particulargeographic region. The inbreeding coefficient of these an-imals is the highest recorded among wild mammals and issimilar to that for the inbred strains of laboratory mice

318 NIKITINA ET AL.

(Reeve et al., 1990). This genetic homogeneity provides anadditional advantage for the use of this animal in devel-opmental studies, because there would be less develop-mental variation due to genetic background.

Here we provide a detailed description of the morphol-ogy of the adult naked mole rat eye and the developmentalchanges that accompany the growth and maturation of theocular structures from birth into adulthood. We then ex-amine the respective roles that cellular proliferation, pro-grammed cell death, and fiber differentiation play in lensmorphogenesis. Based on our results, a model for abnor-mal lens differentiation, and the role it plays in the mor-phogenesis of the eye in the naked mole rats, is proposed.

MATERIALS AND METHODSAnimals

Juvenile and adult naked mole rats were obtained fromthe Department of Zoology, University of Cape Town,where a number of successfully breeding colonies havebeen established (Jarvis, 1981). Adult naked mole ratswere sacrificed with chloroform or halothane and pups bydecapitation. Eyes were harvested from mole rats at post-natal days 0, 1, 5, 14, 21, 32, and 34 and from 10 adults ofvarious ages. All studies were carried out in compliancewith the guidelines of the Animal Ethics Committee of theUniversity of Cape Town.

Histology and Eye MeasurementsThe eyes of 19 mole rats, aged 1 day to 12 years, were

analyzed using standard histological techniques. Afterdissection, the eye globe was placed in ice-cold fresh 4%paraformaldehyde (PFA) in phosphate-buffered saline. Asharpened tungsten needle was used to pierce the poste-rior hemisphere of the eyeball, allowing a rapid penetra-tion of fixative into the eye. Eyes were fixed in PFA over-night, then dehydrated through a series of increasingethanol concentrations and embedded in paraffin wax.The eyes were embedded so that sectioning would bethrough the vertical meridian of the eye. Sections 4 or 5�m thick were stained with hematoxylin and eosin usingstandard procedures. Images were captured using a CarlZeiss AxioCamHR digital camera mounted on an Axioskop2 microscope. The measurements of the diameter of theeye, lens, ciliary body, iris, and cornea were made usingthe Axiovision 2.4 software package. To ensure consis-tency in the results, measurements of various structureswithin the eye were taken of histological sections throughthe center of the pupil. We recognize that histologicalprocessing causes tissue shrinkage, but since all our sam-ples were processed in exactly the same way, we reasonthat this shrinkage would cause a consistent error in allmeasurements taken. The diameters of the whole eye andthe lens were separately measured along the anteroposte-rior axis (at the level of ora serrata in the mole rat) and thedorsoventral axis (along the line extending from the cor-nea to the retina and passing through the center of theeye). The average values of these two measurements, foreach eye and lens diameter, were then calculated. Thelens flattening ratio was obtained by dividing the lensdiameter, measured along the anteroposterior axis, bythat along the dorsoventral axis. The lens flattening ratioreflects the degree to which the shape of the lens deviatesfrom a perfect circle.

Electron Microscopy

Scanning electron microscopy (SEM). Whole eyeswere fixed overnight in Karnovsky’s fixative. The cornea,iris, and ciliary body were dissected out and returned tofixative for 1 hr. Samples were briefly washed in Soren-son’s phosphate buffer, postfixed for 90 min in 1% osmiumtetroxide, dehydrated, and dried at the critical point ofcarbon dioxide. Samples were splutter-coated with gold,mounted onto coated stubs, and examined with a LeoS440scanning electron microscope operating at 15 kV.

Transmission electron microscopy (TEM). Eyeswere fixed overnight in Karnovsky’s fixative and then cutin half equatorially and the anterior segment was re-turned to fixative for another hour. Samples were washedin Sorenson’s phosphate buffer, postfixed for 90 min in 1%osmium tetroxide, stained for 30 min in 2% uranyl ace-tate, dehydrated, and embedded in epon-araldite orSpurr’s resin. Sections 1 �m thick were stained for 5 secwith 1% toluidine blue in 1% borax. Ultrathin sectionswere stained in 8% saturated uranyl acetate and Reyn-old’s lead citrate, then viewed and photographed using aJEM109 transmission electron microscope at 120 kV.

ImmunohistochemistryApoptotic cells were identified using the in situ double-

strand DNA break detection protocol developed by Gavri-eli et al. (1992) with modifications. Wax sections, 6 �mthick, were placed on 3-aminopropyltriethoxysilane(APTES)-coated slides, dewaxed, and rehydrated accord-ing to standard procedures. The sections were then incu-bated in sodium sodium citrate (SSC, pH 7.0) for 20 min at75°C, washed in distilled water (3 � 5 min), rinsed interminal transferase buffer (1 � 5 min), and incubatedwith digoxygenin (DIG)-labeled dUTP and terminal trans-ferase (Roche) for 2 hr at 37°C. Afterward, the sectionswere washed in distilled water (3 � 5 min), blocked in 5%sheep serum in tris-buffered saline (TBS) for 2 hr at roomtemperature, and incubated for 1 hr with an alkalinephosphatase-conjugated anti-DIG antibody (Roche). Theantibody binding was visualized by the alkaline phospha-tase reaction with 4-nitroblue tetrazolium chloride (NBT)and 5-bromo-4-chloro-3-indolyl phosphate (BCIP; all fromBoehringer-Mannheim). The color reaction was allowed todevelop for 1.5 hr and was subsequently stopped withTris-EDTA.

Cell proliferation in eyes from animals of different ageswas examined by phospho-histone H3 and bromodeoxyuri-dine (BrdU) analysis. For the phospho-H3 histone analy-sis, paraffin wax sections were dewaxed and hydratedaccording to standard procedures. For antigen retrieval,sections were microwaved for 1 min in 10 mM sodiumcitrate, allowed to cool for 1 min at room temperature,microwaved for a further 25 sec, and cooled for 20 min atroom temperature. Sections were washed in buffer, per-meabilized for 10 min in 0.2% Triton X-100 in PBS,blocked for 1 hr in 5% sheep serum at room temperature,and incubated in rabbit polyclonal antiphospho-histoneH3 antibody (Upstate Biotechnology) overnight (1:500 in5% sheep serum) at room temperature. Sections werewashed in buffer, then incubated in secondary antibody(Cy-3-conjugated donkey antirabbit IgG in 5% sheep se-rum; 1:1,000; Amersham) in the dark overnight. All suc-cessive steps were conducted in the dark. Sections were

319EYE DEVELOPMENT IN NAKED MOLE RAT

counterstained for 10 min with 1:100 dilution of DAPI(Sigma) in PBS at room temperature, extensively washedin PBS, then mounted in Mowiol. Slides were stored at 4°Cin the dark and viewed on a Zeiss Axioskop fluorescentmicroscope in the red and UV channels. Images werecaptured on a Carl Zeiss AxioCam camera mounted on anAxioskop 2 microscope using the Axiovision 3.1 softwarepackage.

For BrdU incorporation assays, we injected a 14-day-oldmole rat with 300 �g/g body weight BrdU (Boehringer-Mannheim) for 2 days, and two adult mole rats, 4 and 12years old, with 15 �g/g body weight at weekly intervalsover a period of 14 weeks. All animals were sacrificed 24hr after the last injection. Eyes were removed, fixed in 4%PFA, and processed to paraffin wax as described above.Paraffin sections 5 �m thick were dewaxed and rehy-drated, washed in TBS, treated briefly with proteinase K(Roche), and denatured with 1.5 M HCl for 30 min at 37°C.Sections were blocked with 0.5% BSA in TBS for 30 minand incubated with a mouse monoclonal anti-BrdU anti-body (Roche) diluted 1/50 in blocking solution overnight at4°C. Sections were washed extensively in TBST and incu-bated with Alexa 488-conjugated goat antimouse second-ary antibody (1/1,000 dilution in blocking solution; Molec-ular probes, Eugene, OR) for 1 hr in the dark at roomtemperature. Sections were washed extensively in buffer,counterstained for 10 min with DAPI (1:100 in TBS) atroom temperature, then mounted in Mowiol. The slideswere viewed and analyzed as described above.

For anti-�-crystallin immunocytochemistry, 5 �m par-affin wax sections, cut through the center of the pupil,were placed on APTES-coated slides, dewaxed and rehy-drated, rinsed with TBS, and incubated with 0.3% H2O2.Sections were blocked in 3% BSA in TBS for 1–1.5 hr atroom temperature prior to incubation with 1/500 dilutionof rabbit anti-�-crystallin antiserum (generously providedby Dr. Linlin Ding, Joseph Horwitz Laboratory, JulesStein Eye Institute, University of California, Los Angeles,CA) overnight at 4°C. The next day, the sections werewashed extensively in TBST and incubated with peroxi-dase-conjugated swine antirabbit antiserum (Dako) for 2hr. Antigen-antibody complexes were detected by 3,3�-diaminobenzidine tetrahydrochloride (DAB; Sigma) colorreaction. Sections were lightly counterstained with hema-toxylin, dehydrated through ethanol series, cleared in xy-lol, and mounted in Entellan (Merck).

Gel Electrophoresis and Western Blot AnalysisLenses were dissected from four 2-day-old naked mole

rat pups, homogenized in 20 �l extraction buffer (0.1 MTris-HCl, 1% Nonidet P40, 0.01% SDS, 1 �g/ml aprotonin,and 0.1 mM phenylmethylsulfonyl fluoride) at pH 7.2 toextract the water-soluble proteins, and centrifuged at12,000 g for 15 min. The water-insoluble proteins from thepellet were resuspended in 5 �l of 4 � sample dye (4%SDS, 20% glycerol, 10% �-mercaptoethanol, 0.125 M Tris,0.03% bromophenol blue) and then diluted to 10 �l withextraction buffer. The supernatant (10 �l) was boiled todenature proteins, electrophoresed on a 12% sodium do-decyl sulfate polyacrylamide gel (SDS-PAGE), along with5 and 1 �g samples of 2-day-old mouse lens protein ex-tract, and transferred to a nitrocellulose membrane (Hy-bond-C, Amersham Life Science) at 8 V in 25 mM Tris-HCland 5% methanol overnight. The membrane was blockedwith 10% fat-free milk solution for 1.5 hr, and �-crystal-

lins were detected with 1/1,000 dilution of rabbit anti-�-crystallin antiserum (provided by Dr. Linlin Ding). Pri-mary antibodies were detected with horseradish-peroxidase-conjugated swine antirabbit polyclonalantibody using ECL fluorescent detection system (Amer-sham Life Science). After transfer, the gel was fixed in20% methanol and 7% acetic acid solution overnight withconstant agitation, washed three times in distilled water,and silver-stained for 15 min with 0.05 M solution ofAgNO3. The protein bands were visualized after additionof the developer (0.005% citric acid, 0.02% formaldehyde).The reaction was stopped with 1% acetic acid solution, andthe gel was photographed using ChemiImager.

RESULTSEye of Naked Mole Rat Is Microphthalmic

In this study, the structure and development of thenaked mole rat eye from birth to adulthood were investi-gated in detail. The eyes from mice of corresponding ageswere used for morphological and developmental compari-sons. The mouse was chosen for this comparison because itbelongs to the same order as the mole rat (Rodentia), hasa comparable body size, good visual acuity, and well-stud-ied eye structure. At birth, the mole rat greatly resemblesthe mouse in body size and general appearance (Fig. 1A).The eyelids of both species are closed at birth and firstopen 2 weeks later in the mouse (Graw, 1996) and between21 and 30 days after birth in the naked mole rat (O’Riain,1996). The eyes of the adult mole rat are deeply sunk intothe head, and the eyelids are thickened and generally keptclosed unless the animal is alarmed (Fig. 1B) (Jarvis,1991). The mole rat eye is typically round in shape, butlacks the turgidity characteristic of the mouse eye. Thisfeature was particularly evident after dissection, and wenoticed that the eyeball could be easily pinched or in-dented with forceps (much like squashing a football with-out air). The eye has an outwardly visible cornea and lensand a highly pigmented iris (Fig. 1C and D). The pupillaryopening is somewhat irregular in shape. The posteriorhemisphere of the adult mole rat eyeball, unlike that ofthe mouse, is always covered with a thick layer of fat (Fig.1D). The normal extraocular muscles are present.

In comparison to the mouse, the adult mole rat hassignificantly smaller eyes (Fig. 1C). In order to comparethe differences in size, we measured the diameter of boththe unfixed whole eyes and the histological eye sections asdescribed above. We found that, on average, the eye of theadult mole rat is two times smaller in diameter, andtherefore eight times smaller in volume than the mouseeye (n � 9; Table 1). Thus, the naked mole rat eye canjustifiably be termed microphthalmic.

We next examined the histological and ultrastructuralfeatures of the adult mole rat eye, using specimens fromsix mole rats aged 3 to 12 years, and four mole rats ofunknown age. Samples of a variety of specimens of differ-ent ages are shown in Figure 2. The internal organizationof the adult eyes is similar to that of the mouse, with themain structures—cornea, lens, retina, anterior chamber,iris, and ciliary body—being discernible. We did, however,find a number of significant differences related to thestructure and cellular features of the cornea, iris, ciliarybody, lens, and retina.

The mouse cornea is made up of a distinct corneal epi-thelium, stroma, and endothelium (Fig. 3A). The adultmole rat cornea has an epithelial layer, a proportionately

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TABLE 1. Comparative measurements of the body length and eye size of the mouse and the naked mole-rat

Body length, cm(tail not included)

Eye diameter(wet specimen),

mm

Eye diameter(histological sections),

mmAverage volume*of the eye, mm3

Ratio of eye diameterto body length*

Mouse neonate(n � 3) 2.8 2.2 2.0 4.2 0.071

Mole-rat neonate(n � 3) 3.2 1.0 0.9 0.39 0.028

Mouse adult(n � 4) 10.5 3.2 2.7 10.3 0.026

Mole-rat adult(n � 9) 10.5 1.9 1.2 0.90 0.011

*These values are calculated using the eye diameter measurements from histological sections.

Fig. 1. The eye size and gross morphology in the mole rat. A: The head of a neonate mole rat. Eyelids stillfused. B: Adult mole rats. Eyes open because alarmed. D: Comparison of the size of an eye from the adultmouse (right) to that from the adult mole rat (left). D: The eye of an adult mole rat. Cornea (c), lens (l), highlypigmented irregularly shaped iris (i), and the layer of fat (f) covering the posterior hemisphere of the eye areall visible.

321EYE DEVELOPMENT IN NAKED MOLE RAT

Figure 2.

322 NIKITINA ET AL.

thinner stromal layer, and, in normal histological sec-tions, there appears to be no identifiable corneal endothe-lium present (Fig. 3B). To establish whether corneal en-dothelium is indeed absent, we carried out anultrastructural examination using SEM and TEM. A cor-neal endothelial cell layer was clearly visible by SEM in a5-year-old mole rat cornea (Fig. 4C). The cell morphologyof this specimen appeared very different to that seen inthe mouse. In the mouse, the corneal endothelium is amonolayer of tightly adhering hexagonal cells approxi-mately 13.5 �m in diameter and forming a regular cobble-stone pattern (Fig. 4A). In the 5-year-old mole rat, theendothelial cells are seen as a more rough layer, with thetypical hexagonal pattern not evident (compare Fig. 4C toA). The cells are irregular and rounded in shape, with anaverage diameter of about 9.5 �m, and have many extend-ing and overlapping processes. Many of the cells appear tohave burst and secretory vacuoles are visible. The shape ofthese cells and their arrangement suggests that the cellsare not joined together by junctional complexes, which aretypically present in the mouse corneal endothelium (Kid-son et al., 1999). Transmission electron microscopic exam-ination of a cross-section through a 4-year-old mole ratcornea revealed the presence of large rounded vacuoles,enclosed between two neighboring cells, which were flat-tened and joined by tight junctions (Fig. 5A). Because suchtranscellular transport vacuoles and tight junctions aretypical features of mouse corneal endothelium, we con-clude that the true corneal endothelium cells are liningthe anterior chamber of the naked mole rat eye.

When the mole rat eyes were bisected, we observed thelack of structured gel-like vitreous body. Instead, the eye-ball was filled with a transparent liquid substance. Histo-logical sections through the naked mole rat eyes showedthat almost the entire vitreous chamber was filled by theextensively folded retina, which appeared to be closelyassociated with the posterior of the lens. The mole ratretina was folded to the extent never seen in the mouse, asif it was too big for the size of the eyeball (Fig. 2H).Because this condition of the retina was common in theadult mole rat, but was never seen in the mouse, weconsider it a true difference between these species ratherthan a histological artifact. The general organization ofthe neural retina was similar to that of the mouse, which

is composed of nine layers, namely, the photoreceptorlayer, the external limiting membrane, the outer nuclearlayer, the outer plexiform layer, the inner nuclear layer,the inner plexiform layer, the ganglion cell layer, the opticnerve fiber layer, and the internal limiting membrane(Fig. 3H and I). All except two of the retinal layers werewell defined. The nerve fiber layer was absent in all adultmole rats examined and the ganglion cell layer appearedto be progressively reduced as the animals age (compareFig. 3H to I). This reduction in the number of ganglioncells was apparent from examination of histological sec-tions, and our observations have to be confirmed by gan-glion cell counts. Besides the intensely staining ganglioncell bodies, the paler-staining large frothy cells, identifiedas microglial cells, were seen in increased numbers in theganglion layer of the mole rat retina (Fig. 3I, arrowheads).The optic nerve contains cells arranged in an irregularpattern, which is different to the regular, more parallelarrangement seen in the mouse (Fig. 3D and E). Thus,while the retinal structure in the naked mole rat appearsto be essentially normal, some histological features ofreactive gliosis are evident.

One of the most prominent features of the mole rat eyeis the large size and abnormal shape of the ciliary body. Inthe adult mouse eye, the ciliary body is folded into pro-cesses (Fig. 4D) and covered by two layers of columnarepithelium, the outer layer of which is pigmented and theinner unpigmented (Fig. 3O). The border between theciliary body and the iris is clearly distinguishable. In themole rat eye, however, typical ciliary processes were notobserved, and there was no clear border between the cili-ary body and the iris (Fig. 3P and Q). Overall, the ciliarybody appeared flattened and elongated. In some speci-mens, extensive peripheral anterior synechias (abnormalattachment of the ciliary body to trabecular meshworkand to the peripheral posterior corneal surface) were ob-served (Figs. 2H and 3P and Q, arrows). Both the ciliarybody and iris of all specimens were highly pigmented. Theouter (pigmented) layer of the ciliary body shows slightfolding, while the inner layer remains unfolded. The lackof ciliary processes was confirmed by our SEM studies.The adult mouse ciliary body (Fig. 4D) has macroscopicfolds along its surface epithelium, whereas that of the5-year-old mole rat is flat (Fig. 4E). Intriguingly, we ob-served deep pores or holes on the surface of the mole ratciliary body, with an average width of 1.9 �m and lengthof 4.2 �m (Fig. 4F), and the average distance betweenthese pore-like structures being 16.1 �m. The presence ofthese pore-like structures was confirmed by TEM, whichshowed intercellular spaces about 8 �m in diameter (datanot shown).

The differences in the iris structure between the molerat and the mouse became apparent during the examina-tion of the freshly dissected eyes. Contrary to what isobserved in the mouse, the shape of the pupillary border isirregular and not clearly defined; moreover, brown pig-ment granules are often observed in the cornea and withinthe anterior chamber, suggesting that adult mole ratsexhibit iris degeneration and dispersion of pigment (Fig.1C and D). These observations were confirmed by histo-logical and ultrastructural analysis. In the mouse, the irisis composed of three layers, the anterior pigmented bor-der, the stroma, and the inner pigmented epithelium. Theiris muscle is clearly visible in the anteriormost tip of theiris (Fig. 3M, arrowhead). In some mole rats, the iris had

Fig. 2. Comparative histology of the mouse (A and F) and the nakedmole rat (B, C, D, E, G, and H) eyes. A: Neonate mouse has a well-developed lens (l), anterior chamber (ac), distinct ciliary body (cb) and iris(i), and an undifferentiated retina (r). B: Neonate mole rat has no anteriorchamber, greatly enlarged and irregularly shaped ciliary body, and retinawith clearly defined layers. C: Fourteen-day-old mole rat showing whatappears to be the developing iris (arrowheads). D: Twenty-one-day-oldmole rat has no anterior chamber and its lens is irregularly shaped at theposterior margin (arrowheads). E: Thirty-two-day-old mole rat has ante-rior chamber (ac) and abnormally shaped lens (arrowheads). F: Adultmouse eye with round lens, typical elongated iris, ciliary body withevident ciliary processes, and differentiated layers in the retina. G:Four-year-old naked mole rat eye, showing flat ciliary body withoutciliary processes, very large round lens with vacuole-like structures atthe posterior margin (arrowheads), iris undergoing thinning and depig-mentation, and pigment-filled trabecular meshwork (arrows). H: Twelve-year-old naked mole rat with extremely large irregularly shaped lens,ciliary body abnormally associated with sclera (arrow), and degenerateiris. Scale bars � 200 �m (A, D, E, G, H); 100 �m (B, C); 500 �m (F).

323EYE DEVELOPMENT IN NAKED MOLE RAT

Figure 3.

324 NIKITINA ET AL.

the characteristic elongate shape and well-defined irismuscle, while in other specimens we observed the de-crease in the thickness of the iris and degeneration of thepigmented epithelium (Fig. 3N, black arrowheads). Thepigment, presumably released from these degeneratingepithelial cells, was found in the anterior chamber angleand sometimes appeared to fill the trabecular meshwork(Fig. 2G, arrows). The degree of iris thinning variedgreatly among the mole rats examined and did not appearto be directly related to their age.

The trabecular meshwork of some adults is very exten-sive, spanning the whole length of the elongated ciliarybody. The trabecular beams of a 4-year-old specimen arepigmented and run almost parallel to each other, andlarge and well-developed aqueous channels are visible(Fig. 2G). In 8- to 12-year-old specimens, the size of thetrabecular meshwork decreases dramatically, the trabec-ular beams are compacted and highly pigmented, and thespaces between them seem to be severely reduced (Figs.2H and 3P). In some specimens, pigment-filled roundstructures were seen floating in the anterior chamber, ortrapped in the trabecular meshwork (Figs. 2G and 3N, redarrows). A TEM micrograph of a conglomerate of these

Fig. 3. Comparative histology of the mouse and mole rat cornea (A–C),optic nerve (D, E), retina (F–I), iris and ciliary body (J–Q), and lens (R, S). A:Adult mouse cornea with clearly defined epithelium (e), stroma (s), andendothelium (ce). B: Adult (12-year-old) mole rat cornea with obvious epi-thelium and stroma but no visible endothelium (arrowhead). C: Neonatemole rat cornea (c) is attached to the lens (l). D: Optic nerve of an adultmouse has a regular arrangement of nuclei. E: Optic nerve of an 8-year-oldmole rat exhibits an irregular arrangement of nuclei. F: Neonate mouseretina with undifferentiated photoreceptor layers. G: Neonate mole rat ret-ina. All 10 retinal layers are clearly identifiable at birth. H: Adult mouse retinawith characteristic retinal architecture. In G and H, note nerve fiber layer(arrowheads). I: Adult (12-year-old) mole rat retina, with no nerve fiber layer,and with ectopic cell bodies in the inner plexiform layer (white arrow) andpossible microglial cells in the retinal ganglion cell layer (arrowheads). J:Neonate mouse ciliary body and iris. K: Neonate mole rat ciliary body andiris. L: Juvenile (14-day-old) mole rat ciliary body and iris. M: Adult mouseiris with characteristic elongated shape and visible iris muscle (arrowhead).N: Four-year-old mole rat iris exhibiting thinning and depigmentation (ar-rowheads). Red arrows indicate pigment-filled particles in the anteriorchamber. O: Adult mouse ciliary body. Twelve-year-old (P) and 3.5-year-old(Q) mole rat ciliary body. Arrows indicate line of adhesion of iris to cornea.R: Neonate mole rat lens. Bow regions are boxed, and arrowheads point atthe nuclei found at abnormal positions in the center of the lens. S: Neonatemouse lens. Bow regions are boxed. Scale bars � 50 �m (D, K, M); 100 �m(R); 200 �m (S); 20 �m in all the rest.

Fig. 4. Scanning electron micrographs of mouse and naked mole ratcorneal endothelium (A–C) and ciliary body (D–F). A: Corneal endothe-lium of the adult mouse showing typical arrangement of hexagonal cellsin a cobblestone pattern. B: Juvenile (21-day-old) mole rat cornealendothelium. C: Five-year-old mole rat corneal endothelium. D: Mouse

ciliary body showing characteristic ciliary processes (arrow). E: Ciliarybody in a 5-year-old mole rat, showing an absence of ciliary processes.F: Higher magnification of the region boxed in E. Scale bars � 10 �m (A,D, E); 2 �m (B); 4 �m (C); 1 �m (F).

325EYE DEVELOPMENT IN NAKED MOLE RAT

Figure 5.

326 NIKITINA ET AL.

pigmented particles in a single cell, located within anintertrabecular space, is shown in Figure 5C. The finger-like projections on the surface of the plasma membrane ofthis cell, the shape of its nucleus, and the presence of alarge number of lysosomes in the cytoplasm suggest that itis a macrophage.

The most striking difference between the mouse and themole rat eye is the structure of the lens. The mouse lens ispositioned in the anterior hemisphere of the eyeball and isheld in place by the zonula fibers, which are also attachedto the ciliary body. It is typically round in shape, with acharacteristic pattern of nuclear distribution; a singlelayer of nucleated epithelial cells covers its anterior sur-face, and a group of nuclei belonging to the differentiatingfiber cells is found at the equator (the bow region; Fig. 2F).The interior and posterior edge of the mouse lens are freeof nuclei. In the adult mole rat, the lens appears to occupymost of the interior of the eyeball, and our observationsduring dissections and the absence of zonula fibers fromall histological sections suggests that the lens floats freelyinside the eyeball, rather than being attached to the cili-ary body. In the majority of the specimens studied, thelenses were round, but often exhibited various irregular-ities in shape at the posterior margin or at the equator(Fig. 2G and H, arrowheads). A prominent lens capsule,up to 23 �m thick in older specimens, was present. Thenuclei are found in a thin layer surrounding the entirelens. Our deduction is that it corresponds to the epitheliallayer of the mouse lens, but it appears to cover a muchmore significant proportion of the lens surface. The bowregion of fiber differentiation is often absent or very poorlydeveloped in adult mole rats, and nuclei can be altogetherabsent from the region of the lens epithelium closest tocornea (Fig. 2H). When TEM was used to examine epithe-lial cell morphology, we observed that the cells appear tobe very flattened and resemble squamous epithelium,rather than cuboidal epithelium that is found in adultmouse lens (Fig. 5E). The nuclei of these cells had veryflat, pancake-like appearance. Since we did not observe anobvious bow region in the adult mole rat lens, BrdU in-corporation assays were performed to establish whethernew fibers would still be formed. Our results (Fig. 7C andD) indicate that some of the cells located within the pos-terior third of the epithelium were dividing, and that themole rat epithelium was thus still mitotically active evenin 12-year-old mole rats. TEM of the lens equator in a4-year-old mole rat confirmed the presence of maturinglens fibers, which still contained some of the organellesand exhibited highly variable thickness and length (Fig.5B). Therefore, despite its abnormal architecture, the na-ked mole rat lens appears to maintain its growth and fiberdifferentiation activity throughout the life of the animal.

Postnatal Development of Naked Mole Rat EyeIn order to begin to define the sequence of the major

developmental events in the postnatal mole rat eye, weexamined the structure and histology of the naked molerats of different ages, ranging from neonate to 34 days old.Due to the peculiar social structure of the naked mole rats,breeding females are essential for the survival of the col-ony and consequently could not be sacrificed to obtainembryos; therefore, with the exception of one batch ofembryos at a very late developmental stage, the neonatewas the earliest developmental stage available for ourinvestigations.

In newborn mice, the anterior chamber is fully formed,and the squamous endothelium is clearly visible on theinner surface of the cornea (Fig. 2A). In the newborn molerat eye, however, there was no space separating the cor-nea from the lens. In fact, in all three neonate specimensexamined, the cornea appeared to be attached to the an-terior surface of the lens and the ciliary body (Fig. 2B). Inorder to establish when the anterior chamber is firstformed, histological sections from juvenile mole rats ofvarious ages were examined. The presence of a spacebetween the cornea and the lens was noted in a 14-day-and three 32- to 34-day-old mole rats, while no such spacewas observed in a 21-day-old mole rat (Fig. 2C–E). There-fore, we deduced that the time of anterior chamber forma-tion is variable, and that the process is usually completedby the time mole rats are 32 days of age. It has beenproposed that the formation of the anterior chamber in themouse is concurrent with the corneal endothelium forma-tion (Kidson et al., 1999; Reneker et al., 2000). In anattempt to determine whether these processes also occurconcurrently in the mole rat, we examined semithin (0.5�m thick) resin sections of a late embryonic, 5-, 12-, 14-,and two 34-day-old mole rats eyes. Unfortunately, wewere not able to observe anterior chamber in any of thesesections because the lens was always found to havechanged its position considerably with respect to otherocular structures, sometimes being turned so that its an-terior epithelium was facing the retina. Thus, the spacebetween the lens and the cornea in these specimens ap-pears to be a resin-embedding artifact (except in the em-bryo, where the lens is not displaced and is still adherentto the cornea). Since we never observed such lens displace-ment when mouse eyes were processed in an identicalway, it appears that the mole rat lens is not firmly at-tached to the cornea even in those specimens that appar-ently lack anterior chamber. The typical flattened endo-thelial cells filled with transcellular vacuoles wereobserved in both the central and the peripheral regions ofthe cornea in the 34-day-old and the 14-day-old specimen.In the 12-day-old mole rat, the flattened endothelial celllayer was observed, but transcellular vacuoles were notfound. In the 5- day-old specimen, corneal endothelial cellswith transcellular vacuoles were seen in the peripheralregions of the cornea, but not in the center. No endotheliallayer was seen in the embryonic specimen, and the spacebetween the lens and the cornea contained stromal cells,which appeared to adhere to the lens. TEM and SEM wasused to confirm these observations. True corneal endothe-lial cells were seen in the peripheral regions of the TEMsections of a 5-day-old specimen, and in the cornea of a32-day-old specimen (data not shown). A corneal endothe-lial cell layer with a cell morphology closely resembling

Fig. 5. Transmission electron micrographs of various structures ofthe naked mole rat eye. A: Corneal endothelium of a 4-year-old mole rat,showing characteristic transcellular vacuoles (v). Insert: Semithin sectionthrough the central region of 4-year-old mole rat cornea. dm, Descem-et’s membrane; s, corneal stroma. B: Equatorial lens fibers (lf) in a4-year-old mole rat. lc, lens capsule. C: Pigment-filled cell in the angle ofthe eye of a 4-year-old mole rat. D: Anterior lens epithelium of a 5-day-old mole rat with rounded nuclei and cube-shaped cells. E: Posteriorlens epithelium of a 4-year-old mole rat, with flattened cells. Scalebars � 2 �m.

327EYE DEVELOPMENT IN NAKED MOLE RAT

Figure 6.

Figure 7.

328 NIKITINA ET AL.

that of an adult mole rat could also be seen in the scanningelectron micrograph of a 21-day-old mole rat cornea (Fig.4B). These findings suggest that, contrary to what is ob-served in the mouse, the formation of the anterior cham-ber in the naked mole rat occurs after the formation of thecorneal endothelium.

In the mouse, the ciliary body and iris morphogenesisstarts prenatally, at about E17 (Theiler, 1989), and con-tinues until P10, when both of these structures are mor-phologically mature (Monaghan et al., 1991; Smith et al.,2001). The ciliary body and iris primordia are distinguish-able by E18, and there is a clear boundary between thesestructures in the neonate mouse (Fig. 3J). Contrary towhat is observed in the mouse, there are no clearly dis-tinguishable ciliary body and iris primordia in the eye ofthe neonate naked mole rat, though a highly pigmentedstructure covered with a nonpigmented epithelial layer isclearly visible (Fig. 3K) and probably represents the com-mon primordium of the iris and ciliary body. This ciliarybody-iris complex of the mole rat is greatly enlarged, mak-ing up about 30% of the eye circumference, while in themouse the combined lengths of the ciliary body and theiris make up only 8% of the circumference (Table 2). Bythe time the naked mole rat is 14 days old, the ciliary bodyappears to have become distinct from the iris (Fig. 3L).Interestingly, the combined length of the iris and theciliary body at that age is approximately equal to thelength of the neonate ciliary-body-like complex (Table 2),suggesting that in the mole rat, part of this initial struc-ture differentiates to form the iris.

Ciliary fold morphogenesis in the mouse begins aroundE18, and the elongated adult-like ciliary processes areevident by P4 (Theiler, 1989; Smith et al., 2001). In theneonate mole rat, there is no evidence of the ciliary foldformation: only the outer (pigmented) layer shows somefolding, while the inner (nonpigmented) layer remains flatand accommodates the folds of the pigmented layer by thedecrease in the height of the cells lying directly above thefolds (compare Fig. 3J and K). True folding of the ciliaryepithelium, similar to that of the neonate mouse, is ob-served in the 14-day-old mole rat (Fig. 3L). Our BrdU-labeling experiments show large numbers of proliferatingcells in the inner (pigmented), but not in the outer (non-pigmented) epithelial layer of the ciliary body in the 14-day-old mole rat (Fig. 7A and B, arrowheads), suggestingthat increased levels of cellular proliferation in the innerepithelial layer can be responsible for the ciliary folding.However, as the animal grows, the ciliary processes ap-pear to become flattened out. The absence of the typicalfolding of the ciliary body epithelium into processes in the21-day-old mole rat was confirmed by scanning electronmicroscopy. The surface of the 21-day-old mole rat ciliarybody is flat and very similar to what is observed in the5-year-old mole rat (data not shown).

In the mouse, formation of the trabecular meshwork is apostnatal event, which starts around P6 and reaches itsfull structural and functional maturity by P21–P42(Smith et al., 2001). The mole rat trabecular meshworkappears to form at about the same time as the anteriorchamber, when the ciliary body separates from the cornea.Well-defined trabecular beams and aqueous channelswere visible in the eye angle of a 32-day-old mole rat (Fig.2E), but in younger specimens the state of differentiationof this structure cannot be easily observed due to theadherence of the iris and ciliary body to the cornea andconsequent absence of the iridocorneal angle. The trabec-ular meshwork in all juvenile mole rats examined re-mained unblocked, and no signs of pigment dispersion arevisible, suggesting that this phenotype develops slowly asthe animal ages.

While the anterior chamber development is apparentlydelayed in the mole rat when compared to the mouse, thereverse is true for the retina. The retina of the neonatemouse is not yet fully differentiated, and only RPE, outer

Fig. 6. TUNEL assay showing cell death in tissues of the naked molerat eye. A: Neonate mole rat eye. B: Juvenile (21-day-old) mole rat eyeshowing a number of TUNEL-positive cells at the posterior margin of thelens (arrowheads).

Fig. 7. Cellular proliferation in various tissues of the naked mole rateye. A: Fourteen-day-old mole rat showing BrdU-positive cells (green) inthe lens (arrows), ciliary body (arrowheads), and cornea. The yellowsignal is due to autofluorescence of red blood cells. B: Phase-contrastimage of the section shown in A. C: Phase-contrast image of a BrdU-labeled section through the eye of a 12-year-old mole rat. D: Fluorescentimage of a region boxed in C, showing a BrdU-positive nucleus (green)in the lens epithelium (arrow). The nuclei are stained blue with DAPI stain.

TABLE 2. Comparative measurements of the eye structures

Specimen

Eyediameter

(mm)

Lensdiameter

(mm)

Lensflattening

ratio

Cornealthickness

(�m)

Irislength(mm)

Ciliary bodylength (mm)

Lens/eyeproportion

Mole-rat neonate(n � 3) 0.91 0.45 1.4 36 none 0.43 49%

Mouse neonate(n � 3) 2.0 1.1 1.21 86 0.12 0.13 55%

14-day old mole-rat(n � 1) 1.0 0.50 1.24 44 0.23 0.19 50%

21-day old mole-rat(n � 2) 1.1 0.48 1.4 24 0.24 0.29 44%

32-day old mole-rat(n � 1) 1.1 0.50 1.6 28 0.25 0.27 45%

Adult mouse(n � 4) 2.7 1.84 1.07 111 0.62 0.21 68%

Adult mole-rat(n � 9) 1.2 0.84 1.2 52 0.40 0.29 70%

329EYE DEVELOPMENT IN NAKED MOLE RAT

neuroblastic layer, inner plexiform layer, ganglion celllayer, and forming nerve fiber layer can be clearly distin-guished (Fig. 3F). In the neonate mole rat, on the otherhand, all 10 retinal layers characteristic of the adult arefully formed (Fig. 3G). Traces of nerve fiber layer, whichare not distinguishable in the adult mole rat, can be seenin the neonate mole rat retina (Fig. 3G, arrowhead).

The neonate mole rat lens is oval-shaped, which is sim-ilar to what is observed in the neonate mouse (Table 2,Fig. 3R and S). However, as the mole rat grows, the shapeof the lens becomes more irregular, with an abnormallydefined, collapsing posterior margin observed in 21- and32-day-old mole rat lenses (Fig. 2D and E). Although theshape of the lens and the degree of collapse vary greatlyamong specimens, it was consistently observed in the ju-venile mole rat specimens (n � 5). High-magnificationexamination of these abnormal lenses revealed that theindividual fibers at the posterior margin were bent ormisshapen and were not arranged in the regular fashioncharacteristic of the mouse lenses. We also noticed signif-icant differences in the nuclei distribution in the lenses ofthese two species. In the mouse, a layer of nucleatedepithelial cells covers the anterior surface of the lens, andthere are accumulations of nuclei at the bow region, lo-cated at the equator of the lens (Fig. 3S, boxes). In theneonate mole rat, the epithelial cell layer appears to ex-tend over a significantly larger area of the lens. The regionof fiber differentiation (the bow region), identified by theelongated shape and the clustered arrangement of theirnuclei, is shifted toward the posterior pole of the lens (Fig.3R, boxed regions). Moreover, in the mole rat, nucleatedcells are also found throughout the center and the poste-rior of the lens (Fig. 3R, arrowheads), while in the mousethese areas contain only mature fibers and are thereforenuclei-free (Fig. 3S). Interestingly, in juvenile mole rats,the center of the lens gradually becomes nuclei-free, sug-gesting that the fiber maturation process is delayed in thisanimal compared to the mouse. The lens epithelium inboth the neonate mouse and the mole rat is a simplecuboidal epithelium. However, in the mole rat, the mor-phology of the lens epithelial cells changes from regularcuboidal, with round nuclei in a 5-day-old specimen (Fig.5D), to pseudostratified cuboidal in a 32-day-old (data notshown), until the cells become completely flattened andresemble squamous epithelium in the adult (Fig. 5E).

Molecular Basis of Abnormalities in Mole RatLens

To begin to investigate the molecular basis of the struc-tural abnormalities in the naked mole rat lenses, we car-ried out a series of experiments designed to establishwhether these abnormalities are due to changes in celldeath, proliferation, or differentiation.

Using the TUNEL reaction to identify apoptotic cells,we found no evidence of cells undergoing programmed celldeath in the lens in any of the neonate eye sections exam-ined (Fig. 6A). However, in a 21-day-old mole rat, a num-ber of nuclei at the posterior margin of the lens wereTUNEL-positive (Fig. 6B, arrowheads). The staining wasconsiderably fainter than in our DNase-treated positivecontrols (data not shown). The nuclei of differentiatinglens fibers undergo DNA fragmentation and therefore canbe labeled by TUNEL technique under certain experimen-tal conditions (Bassnett and Mataic, 1997; Ishizaki et al.,1998; Wride and Sanders, 1998), though the staining is

not as intense as that of apoptotic cells. Moreover, darkerstaining apoptotic cells were observed in the ganglion celllayer in the neonates and in the inner nuclear layer of the21-day-old mole rats (data not shown). We conclude thatincreased levels of apoptosis are not observed in the nakedmole rat eye.

We next carried out labeling studies (phospho-histoneH3 and BrdU incorporation) to obtain a picture of thezones of cell proliferation in the lens. In particular, wewere interested in establishing whether the nucleatedcells located at the posterior margin of the lens wereproliferative cells. Eyes from three juvenile mole rats wereexamined (two 32-day-old mole rats and one 14-day-oldmole rat). In all of the mole rats examined, we foundevidence of dividing cells in the region of the epitheliumlocated just posteriorly to the lens equator (Fig. 3A). Noneof the nuclei in the center or posterior border of the lenswere BrdU-positive. These results suggest that the overallpattern of proliferation is similar in the mole rat and inthe mouse, but the position of the proliferative compart-ment (equatorial or bow region) in the mole rat is shiftedtoward the posterior end of the lens. These results led usto the next question of whether the presence of nuclei inthe center of the mole rat lens, as well as the abnormalshape of the lens fibers, can be attributed to a defect inlens fiber differentiation.

�-crystallin synthesis in mammals is the essential partof the lens fiber differentiation process. In the mouse, thesynthesis of �-crystallins commences at about 14 day ofembryonic development and continues until the mousereaches reproductive age (about P40), after which it grad-ually declines (Goring et al., 1992). The proteins persist inthe lens fibers throughout the life of the animal, and theirpresence in the lens at appropriate concentrations is es-sential for maintaining its transparency. In order to in-vestigate further the process of lens differentiation in thenaked mole rats, we used SDS-PAGE and Western blot-ting to analyze the 2-day-old and the 6-year-old mole ratlens protein extracts for the presence of �-crystallins. Theadult (6-month-old) and 2-day-old mouse lens extractswere used as positive controls. A protein band of theexpected molecular weight of 21–22 kDa, corresponding toall six �-crystallin proteins (Siezen et al., 1988), was ob-served in the immunoblot of the mouse lens extracts (Fig.8B, lanes 7 and 8). Our results show that at least some ofthe �-crystallins are present in the soluble protein fractionfrom the lenses of a 2-day-old mole rat (Fig. 8B, lane 4),but we were unable to detect crystallins in the lens of a6-year-old mole rat (Fig. 8B, lanes 1 and 2) despite re-peated analysis. Moreover, the diffuse appearance of the6-year-old mole rat protein bands on the gel is an indica-tion of increased protein degradation.

The distribution pattern of �-crystallins within thelenses of mole rats of different ages was analyzed byimmunocytochemistry (ICC), and the results are shown inFigure 9. Sections of adult and neonate mouse eyes wereused as positive controls. In the mouse, �-crystallins areexpressed throughout the lens, with the exception of theepithelium and the equatorial region (which includes fi-bers that have just begun differentiating; Fig. 9F and G).The pattern of �-crystallin expression in the mole rat wasfound to be different to that of the mouse. In the neonatemole rat, most of the lens fibers stain positive for �-crys-tallins, leaving the epithelium and a thin circle of two tothree fiber layers extending around the exterior of the lens

330 NIKITINA ET AL.

(presumably corresponding to the new differentiating fi-bers) signal-free (Fig. 9B). In the 21-day-old mole rat, this�-crystallin-free zone was extended to 7–10 layers (Fig.9C), suggesting that fibers formed, after the mole ratwas born, do not express this protein. Only the center ofthe lens of a 3-year-old mole rat stained positive for�-crystallin (Fig. 9D), while no staining was observed inthe lens of the 12-year-old adult (Fig. 9E). We concludethat the synthesis of �-crystallins in the mole rat lens isterminated around the time of birth, and that the�-crystallin produced earlier undergoes degradation asthe animal ages. This process could be responsible forthe irregular shape and fiber arrangement observed inthe mole rat lenses.

DISCUSSIONBehavioral observations on the naked mole rat suggest

that this animal relies almost solely on olfactory and tac-tile cues to navigate and when interacting with conspecif-ics. There is no evidence that they rely on visual informa-tion for any aspect of their daily lives (Narins et al., 1997).Our histological examination of the naked mole rat eyelends support to these observations. The small eye size ofthese animals probably results in their having a veryrestricted visual field. In addition, the irregular shape ofthe lens, and the presence of cellular nuclei along thevisual axis, might suggest that light scattering preventsclear images from forming on the retina. The absence ofthe zonula fibers and the reduced ciliary muscle of mostmole rat specimens suggest that light focusing on theretina does not occur. However, the presence of rudimen-tary iris muscle indicates that the mole rats are able, atleast to a degree, to regulate the amount of light thatenters the eye. Thus, we conclude that the visual abilitiesof the naked mole rats are limited to judging the intensityof the surrounding light (i.e., being able to distinguishbetween night and day, or between being inside the bur-row or outside it) and, possibly, to seeing the shadows castby large moving objects, without being able to see theirdetails.

The above findings lead us to the question of why, afterat least 25 million years of subterranean evolution (Ben-nett and Faulkes, 2000), the naked mole rats still retainall of their ocular structures and apparently a degree ofvisual ability? The conservation of the eye architecture ofthis species is especially surprising when compared to theregressed ocular phenotypes seen in other dark-adaptedvertebrates. For instance, the eyes of the blind cavefishAstyanax mexicanus do not develop a cornea, an iris, sec-ondary lens fibers, or differentiated retina (Yamamoto andJeffery, 2000; Jeffery, 2001). The cornea, iris and ciliarybody epithelia, differentiated retinal layers, as well as thevitreous and aqueous chambers are altogether absentfrom the eyes of the marsupial mole Notoryctes typhlops(Sweet, 1906) and the insectivorous moles Scalops aquati-cus and Eremitalpa granti (Slonaker, 1902; Gubbay,1956). These extremely reduced eyes are thought to haveevolved in response to the evolutionary pressure to de-crease the metabolic expenditure, associated with the for-mation and maintenance of the organ that is no longerused (Nevo, 1998). However, the naked mole rats, as wellas a number of other fossorial mammals, retain much ofthe normal ocular architecture and, in particular, an ap-parently normal retina. This suggests that retaining thecapacity for light-dark discrimination is important for thesurvival of these animals. The soil-removal activity of thenaked mole rats results in their direct exposure to sun-light, as the animals kick soil out of an open mound. Theopen mound poses a further threat of exposure toaboveground predators (Sherman et al., 1991). An abilityto detect light and dark and sudden transitions associatedwith the arrival of a predator at well-lit burrow entrancemay confer a survival advantage and hence be maintainedby natural selection.

It is interesting to notice that the degree of eye reduc-tion in various burrowing species correlates very well withthe method they use for soil digging. Thus, the eyes of theanimals that use their head to push the excavated soil(Spalax), or to force their body forward through the soil

Fig. 8. SDS-PAGE and Western blot analysis of �-crystallin expres-sion in the naked mole rat. A: SDS-PAGE of proteins from mouse andmole rat eyes. Proteins from a 6-year-old mole rat retina (lane 1); soluble(lane 2) and insoluble (lane 3) proteins from a 6-year-old mole rat lens;soluble (lane 4) and insoluble (lane 5) proteins from a 2-day-old mole ratlens; molecular weight markers: ovalbumin and carbonic anhydrase(lane 6); soluble proteins from the 2-day-old mouse lens (lane 7); solubleproteins from the adult mouse lens (lane 8). B: Western blot of the abovegel, showing �-crystallin expression.

331EYE DEVELOPMENT IN NAKED MOLE RAT

Figure 9.

Figure 10.

332 NIKITINA ET AL.

loosened by their forelimbs (Notoryctes, insectivorousmoles), are much more reduced than those of the animalsthat use both their incisors and forelimbs to scrape off thesubstrate and push the loosened soil backward with theirlimbs (Bathyergidae, Rhyzomyidae) (Vaughan, 1978;Nevo, 1979; Webb et al., 1979; Bennett and Faulkes,2000). The forelimb diggers appear to be more at risk ofeye damage and infection than the teeth diggers. It ispossible that one of the major selective forces favoring thereduction in the eye structures in subterranean mammalsis the need to protect this soft and sensitive organ from theabrasive effects of soil.

Studies to date on chick (Hay and Revel, 1969) andmouse eyes (Kidson et al., 1999) suggest that the forma-tion of the anterior chamber is coupled to the morphogen-esis of the corneal endothelium. Thus, the anterior cham-ber forms as the neural crest cells, closest to the lens,undergo mesenchymal-epithelial transformation and be-come the tightly packed corneal endothelial layer, whichseparates the extracellular matrix of the corneal stromafrom the surfaces of the lens and iris (Kidson et al., 1999;Reneker et al., 2000). Interestingly, however, in the nakedmole rats, the anterior chamber appears to form signifi-cantly later than the corneal endothelium. Thus, it seemsthat the formation of the corneal endothelial layer is in-sufficient for the establishment of the proper anteriorchamber architecture.

The anterior chamber of the adult mouse is filled withaqueous fluid, which is produced by the ciliary body. How-ever, when the anterior chamber is first established (E15in the mouse), the ciliary body has not yet formed (Pei andRhodin, 1970; Theiler, 1989). Therefore, it is currently notclear what the origin of the fluid that fills the anteriorchamber is when it first appears. Perhaps the reason forthe delayed formation of the anterior chamber in the na-ked mole rats is the fact that there is nothing to fill theanterior chamber until the ciliary body is mature enoughto synthesize the aqueous humor. The molecular mecha-nisms responsible for this phenotype are still to be eluci-dated. The close similarity of the mole rat anterior cham-ber architecture (adherence of the base of the ciliary body/iris to the cornea, low amplitude of the anterior chamber,poorly developed iridocorneal angle) to the phenotype ofthe Lmx1b�/� mouse mutants (Pressman et al., 2000)suggests that altered Lmx1b expression could play a role.Interestingly, the lack of or decrease in the size of theanterior chamber occurs among other fossorial mammalswith reduced eyes (Rhizomyidae) (Cei, 1946a), suggestingthat a similar change in the eye development could be

responsible for the convergent evolution in these subter-ranean mammals.

Both the iris and the ciliary body of the naked mole ratsare highly pigmented, and the ciliary body is very largerelative to the size of the eye. The extreme pigmentationand enlargement of these anterior chamber structuresappears to be very common in the mammals adapted to afossorial lifestyle. This phenomenon is most noted inSpalax ehrenbergi and Notoryctes typhlops, where the pu-pil is completely obliterated by a mass of pigmented tissue(Sweet, 1906; Cei, 1946a; Quilliam, 1966; Sanyal et al.,1990). The presence of enlarged ciliary body could be ofadaptive value to these animals, as it probably protectsthe retina from sudden exposure to bright light when theanimal emerges from its burrow during the day (Sanyal etal., 1990). Alternatively, the increase in the relative ciliarybody size might not in itself confer any evolutionary ad-vantage, but result from the selective pressure to decreasethe size of the retina. As have been pointed out previously,neuronal tissue uses larger amounts of energy than mostother tissues, and therefore maintenance of excess neu-rons is an evolutionary luxury that is strongly selectedagainst (Nevo, 1998).

In the mouse, the iris and ciliary body epithelia areformed from the tip of the optic cup, while the iris stromaand the ciliary muscle are derived from the cephalic neu-ral crest (Beebe, 1986). A part of the anterior optic cup isspecified as the ciliary epithelium at around E12 by yetunidentified signals from the lens epithelium (Genis-Galvez, 1966; Stroeva, 1967; Beebe, 1986; Thut et al.,2001). The morphologically distinct ciliary body is firstformed in the mouse around E16.5–17, and the formationof the ciliary processes and its functional maturation iscompleted postnatally (Theiler, 1989). In the neonate molerat, the size of the ciliary body and iris, relative to the eyecircumference, is three times greater than in the newbornmouse (Table 2). This suggests that, in the naked mole rat,a greater proportion of the anterior optic cup is instructedto adopt the ciliary body/iris fate during embryogenesis. Itis interesting that the area of the lens epithelium, theproposed source of the ciliary body-specifying signal, isalso increased in the naked mole rat. Possibly, this ex-tended area of lens epithelium is responsible for the es-tablishment of the larger ciliary body.

As in the mouse, the ciliary body and iris of the nakedmole rats are not mature at birth. In fact, there is nodistinguishable border between the ciliary body and theiris in the neonate mole rat. Our measurements (Table 2),however, suggest that the iris primordium is present inthe neonate mole rats, because the length of the ciliarybody, as measured on the juvenile and adult mole rats, is0.4–0.7 times that of the initial ciliary body-like structureof the neonates (Table 2). This piece of data suggests thatthe iris specification in the mole rat also occurs during theembryonic life, similar to the mouse, even if the morphol-ogy of this structure is initially quite different. As in themouse, most of the growth of the iris in the mole ratsoccurs postnatally. We observed thinning of the irisstroma and loss of the iris pigment in many old mole ratspecimens. This interesting phenotype could be a conse-quence of the elevated levels of melanogenesis in the iris.The increased eye pigmentation in the naked mole ratssuggests that the melanocytes of the anterior surface ofthe iris, which are not active in the adult mouse, are stillproducing pigment in the adult mole rats. These greatly

Fig. 9. Expression of �-crystallins in the naked mole rat (A–E) and inthe mouse (F and G). Neonate mole rat lens: (A) negative control (noprimary antibody added) and (B) expression of �-crystallins (brown). C:Juvenile (21-day-old) mole rat. D: Three-year-old mole rat. Note absenceof �-crystallin expression. E: Twelve-year-old mole rat. Neonate mouse(F) and adult mouse (G) showing a typical expression of �-crystallins.

Fig. 10. Model for the development of lens abnormalities in thenaked mole rat. Blue, lens epithelium; yellow, proliferative region of theepithelium; purple, primary lens fibers; green, secondary lens fibers. Toprow: Specification of the bow region and development of the secondaryfibers in the mouse. Bottom row: A shift in the position of the bow regiontoward the posterior pole of the lens results in the malformation of thesecondary fibers in the naked mole rat.

333EYE DEVELOPMENT IN NAKED MOLE RAT

increased levels of pigment synthesis could lead to themelanocytes becoming filled up with pigment, burstingand releasing their contents into the anterior chamber.This is very similar to some of the phenotypic features ofthe mouse models of pigment-dispersion glaucoma (Johnet al., 1998; Anderson et al., 2002). Genetic dissection ofthe mechanisms regulating the pigment synthesis in thenaked mole rats could provide additional insights into theetiology of human pigmentary glaucoma.

The ciliary body of all adult and most juvenile mole ratspecimens is flat and elongated in shape, and the ciliaryprocesses are absent. This lack of the ciliary processes isanother feature that mole rats share with other subterra-nean mammals [e.g., the common mole (Quilliam, 1966)],but not with any other vertebrate models exhibiting nor-mal visual acuity, i.e., chick, mouse, frog Rana (Beebe,1986). It is currently not clear whether this lack of theciliary processes affects the functioning of the ciliary bodyin a way that is somehow advantageous to these animals,or whether it is simply a developmental consequence ofthe altered signaling within the eye, leading to the in-creased ciliary body size. The ciliary fold morphogenesis inchick appears to be dependent on the intraocular pressure(Bard and Ross, 1982). Our observations during dissec-tions of the mole rat eyes suggest that its intraocularpressure is reduced, which could be another factor contrib-uting to the absence of the ciliary processes.

The mouse trabecular meshwork is derived from agroup of mesenchymal cells situated in the angle betweenthe base of the iris and the cornea. These cells undergodifferentiation to form trabecular beams separated bychannels, which serve to allow the exit of the aqueous fluidfrom the anterior chamber. The process of trabecularbeam differentiation commences around P10 in themouse, and the mature meshwork is established by P21(Smith et al., 2001). In the mole rat, the formation of thetrabecular meshwork cannot be assessed at early postna-tal stages due to the absence of the anterior chamber andthe consequent adherence of the iris and ciliary body tothe inside of the cornea, obliterating the iridocorneal an-gle. It appears, however, that in the 5-day-old specimen,no identifiable trabecular beams and channels are yetevident, making the state of differentiation of that speci-men comparable to P6–P10 mouse. At 30–34 days of age,the trabecular meshwork (TM) of the mole rat is extensiveand well-differentiated and comparable to the mouse TMat P21–30. Moreover, no TUNEL-positive cells were no-ticed in the TM of the 21-day-old specimen, suggestingthat, similar to the mouse but contrary to what has beenreported in the rats, the TM development in the mole ratsdoes not involve apoptosis (Smith et al., 2001). A veryunusual feature of the naked mole rat eye, often observedin mature specimens, is a closure of the eye angle anddegeneration of the trabecular meshwork. These changescould be related to the unusual longevity of the mole rats(Buffenstein and Jarvis, 2002), but the extensive tissueloss from the trabecular meshwork observed in these an-imals is not a feature of the normal aging process even inhumans (Oates and Belcher, 1994). It is possible thatincreased macrophage activity associated with the pig-ment dispersion results in the age-related deterioration ofthe trabecular meshwork.

The structure and retinal layer organization in the molerat eyes are essentially normal, except that, in the adults,no nerve fiber layer and an apparent decrease in the

density of the retinal ganglion layer are observed. Retinalganglion cells appear to be the most susceptible to apopto-sis as the result of increased intraocular pressure, as seenin glaucomas (John et al., 1998). It is possible that, in themole rats, the impaired circulation of the aqueous fluiddue to the blockage and degeneration of trabecular mesh-work causes the loss of retinal ganglion cells. However,the reduction in the numbers of ganglion cell layers hasbeen observed in other completely or partially subterra-nean mammals (Herbin et al., 1994) and is not accompa-nied by the pigment-dispersion phenotype seen in thenaked mole rats. Other factors, perhaps related to de-creased exposure to light in subterranean habitats, couldtherefore play a role. In order to investigate the age-related changes in retinal architecture, ganglion cellcounts and extensive quantitative studies of apoptosis inthe retina of mole rats of different ages will need to beperformed.

Cellular Organization of Mole Rat Lens IsAbnormal

Our histological studies demonstrated that the nucleardistribution in the naked mole rat lens differs significantlyfrom that of the mouse lens. In the neonate and juvenilemole rats, the nuclei are found in a thin layer coveringabout two-thirds of the anterior surface of the lens (pre-sumably corresponding to the nuclei of the lens epitheliumin the mouse), as well as within the posterior hemisphereof the lens, which in the mouse is occupied only by non-nucleated lens fiber cells. In order to ascertain whetherthis peculiar nuclear distribution is the result of the fail-ure of the prospective lens fiber cells to exit cell cycle, weassayed the levels of cellular proliferation using BrdUincorporation. Because continuous proliferation of lensfiber cells resulting from failure to exit cell cycle activatesan apoptotic response (Morgenbesser et al., 1994; Lahoz etal., 1999), we performed TUNEL assays to determine thelevels of programmed cell death in the mole rat lens. Wedid not find any BrdU-positive or TUNEL-positive cellswithin the fiber cell compartment. This suggest that theabnormal presence of the nucleated cells within the centerof the lens is due to the delayed nuclei degradation in thematuring fibers, rather then to the continuous cell prolif-eration and failure to exit the cell cycle. The only prolif-erating cells that were found in the mole rat lenses werelocalized at the equator. This suggests that the nucleibelong to the epithelial cells of the lens proliferative re-gion, which appears to be displaced posteriorly in the molerat compared to the mouse.

In the mouse, the lens polarity is established early inthe development, around E12, when the posterior cells ofthe lens vesicle are instructed to elongate and form lensfibers, while the anterior cells remain epithelial. The re-gion of high cell proliferation is established just anterior ofthe equator, and the cells, which are produced therethroughout the life of the animal, move posteriorly andundergo differentiation to form the secondary lens fibers(Pei and Rhodin, 1970; Theiler, 1989; Graw, 1996). Classictransplantation experiments have demonstrated that mo-lecular signals originating from the retina and present inthe vitreous humor of the eye are responsible for theestablishment of the lens polarity (Coulombre and Cou-lombre, 1964; Yamamoto, 1976). More recent tissue cul-ture-based studies identified that a high concentration ofa group of diffusible signaling molecules—fibroblast

334 NIKITINA ET AL.

growth factors (FGFs)—is able to cause lens fiber differ-entiation, and a lower concentration can initiate prolifer-ation of lens epithelial cells (McAvoy and Chamberlain,1989). The existence of the FGF concentration gradient invivo, compatible with its role as the inducer of cell prolif-eration and fiber differentiation in the lens epithelium,was demonstrated within the ocular media. Moreover,transgenic mice-based studies indicate that increase inthe FGF concentration within the eye leads to differenti-ation of lens fiber cells in the anterior region normallyoccupied by the lens epithelium (Chamberlain and Mc-Avoy, 1997; Lang, 1999). Since the role of FGFs in lensmorphogenesis has been documented in a range of mam-malian species (human, rat, mouse, cow) (Chamberlainand McAvoy, 1997), it is likely that a similar mechanismoperates in the mole rats. We suggest that the displace-ment of the proliferative region toward a more posteriorlocation in the naked mole rats could be caused by thedecreased FGF concentration in the vitreous humor. Fur-ther investigations are necessary in order to ascertainwhat role FGFs and their receptors play in the establish-ment of the lens polarity in this animal.

The unusual persistence of nuclei in the central fibers ofthe mole rat lens led us to ask whether other aspects oflens fiber differentiation are abnormal. We therefore usedICC and Western blots to investigate the expression of thelens-specific proteins �-crystallins, which are synthesizedonly in lens fiber cells and thus can be used as a marker oflens differentiation. In our Western blot experiments, wedetected �-crystallins in the lenses of 2-day-old mole ratpups, but not in the lens of the adult (6-year-old) mole rat.This suggests that either the synthesis of this protein isturned off in the adult lens, or the protein undergoesdegradation. This result was confirmed by our ICC exper-iments, which showed that at birth, �-crystallins wereexpressed throughout the entire fiber compartment of thelens, with the exception of a few lateral fibers, whichappear to be still undergoing the differentiation process.The same pattern of the �-crystallin staining is observedin the mouse eyes. However, as the mole rats matured, thearea of the �-crystallin-free fibers was expanded. More-over, only a very weak signal was detected in the center ofthe 3-year-old mole rat lens, and no �-crystallin-positivefibers were observed in a 12-year-old mole rat. It thereforeappears that the synthesis of �-crystallins is downregu-lated soon after birth, and also that the protein that wasalready synthesized becomes degraded as the animals age.Since we did not investigate the levels of �-crystallinmRNA, we could not establish whether the turning-off of�-crystallin synthesis is due to a translational or a tran-scriptional downregulation event. Six individual �-crystal-lin proteins are synthesized in the mouse and rat lenses(van Leen et al., 1987; Siezen et al., 1988; Goring et al.,1992), and promoter and knockout studies indicate thatall �-crystallins are under common transcriptional controlby a number of factors, including c-Maf (Kawauchi et al.,1999; Kim et al., 1999; Ring et al., 2000) and Sox1(Nishiguchi et al., 1998). Therefore, it is possible that amutation in one of these regulators can result in theobserved absence of �-crystallin synthesis.

The expression pattern of the �-crystallins in the nakedmole rat is unusual even when compared to that of otherblind subterranean mammals. For example, the commonmole, Talpa europaea, despite having a small poorly dif-ferentiated lens, consisting of what appears to be nucle-

ated primary fibers, shows a typical lens expression of �-,�-, and �-crystallins (Quax-Jeuken et al., 1985). The un-differentiated lens of the blind mole rat, Spalax ehren-bergi, does not express either �- or �-crystallins, but doesexhibit �-crystallin expression (Quax-Jeuken et al., 1985;Hough et al., 2002). In this animal, the lens developmentappears to be arrested earlier than in the mole, so that nofiber differentiation (and thus no synthesis of fiber-specificproteins) takes place. It is rather difficult to speculate whythere is a switching-off of �-crystallin expression in thenaked mole rat, unless we assume that formation of a lensthat allows at least some of the light through gives anevolutionary advantage to the naked mole rat. �-crystal-lins differ from all other crystallin types by having arelatively high phase-separation temperature, and thus atendency to precipitate when the body temperature isslightly lowered, as observed in cold cataract formation inneonate rats and mice (Siezen et al., 1988). The nakedmole rats, unlike any other mammals, are poikilothermic,i.e., have a variable body temperature (Buffenstein andYahav, 1992). It is possible, therefore, that switching offthe expression of �-crystallins was an evolutionary solu-tion to producing a lens that would not become completelyopaque when the body temperature of the mole rat fluc-tuates. Sequencing mole rat �-crystallin genes to deter-mine the cryostability of the proteins they encode, andinvestigating �-crystallin expression in warm-bloodedmembers of the family Bathyergidae, could help clarifythis matter.

As can be clearly seen on histological and even on ICCsections, the lens fiber arrangement in the naked mole ratlenses, especially at the posterior margin in 15- to 30-day-old juveniles, is abnormal. The newly differentiated lensfibers, which do not express �-crystallins, do not formconcentric circles around the embryonic nucleus, as in themouse, but are predominantly located in the posteriorhemisphere of the lens (Fig. 9B–D). We think that such anirregular arrangement of lens fibers could result fromdisplacement of the equatorial region. A consequence ofthis displacement is that the elongating secondary lensfibers are forced to grow into the posterior lens hemi-sphere, rather than to elongate equally in both the ante-rior and the posterior directions. The embryonic lens nu-cleus is progressively pushed toward the lens epithelium,rather then remaining in the center of the lens, with theresult that the lens fibers become organized into the ir-regular bent arrangement observed in the naked mole rat(Fig. 10).

In summary, we conclude that the naked mole ratsprovide a potentially very interesting model for investiga-tions into the developmental and genetic basis of evolu-tionary change and eye disease. However, the usefulnessof this animal as the model for human eye disease islimited by their slow reproduction rate and the fact thatthe embryos are not easily available.

ACKNOWLEDGMENTSWe thank Melaney Peterson for assistance with histo-

logical sectioning and TEM, Toni Wiggins for technicalsupport, Liz van der Merwe for the SEM micrograph of theadult mouse ciliary body and help with TEM sectioning,and Barbara Young for TEM sectioning. We also thank Dr.Linlin Ding (Horwitz Laboratory, Jules Stein Eye Insti-tute, University of California, Los Angeles, CA) for pro-viding us with anti-�-crystallin antibodies, Thandi

335EYE DEVELOPMENT IN NAKED MOLE RAT

Mgwebi for allowing us to use her SEM micrograph of theadult mouse corneal endothelium, as well as Dr. DirkLang for helpful discussions. This work was supported bygrants from the Medical Research Council of South Africa(to S.H.K.) and National Research Foundation (to M.J.O.)and by bursaries from the Medical Research Council ofSouth Africa and the University of Cape Town (to N.V.N.).

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