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Note to the Reader Please refer to: Hogan D, Williams RW (1999) Analysis of the Retinas and Optic Nerves of Achiasmatic Belgian Sheepdogs. J Comp Neurol 352:367–380.

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Analysis of the Retinas and Optic Nerves of Achiasmatic Belgian Sheepdogs

Dale Hogan and Robert W. Williams
Department of Anatomy and Neurobiology School of Medicine University of Tennessee Memphis, Tennessee 38163


     An autosomal recessive mutation carried in a family of black Belgian sheepdogs eliminates the optic chiasm—all retinal ganglion cell axons extend directly into the ipsilateral optic tract. One key issue we are trying to resolve is whether the retina or the chiasm is the principal site of mutant gene action. In this study, we have examined retinas of mutants to discover any associated changes in retinal structure.
     Retinas of mutant animals are relatively normal. Inner and outer nuclear layers are qualitatively indistinguishable from those of normal dogs. The principal difference is that the area centralis of mutants is smaller and has a lower peak ganglion cell density than that of normal dogs (8,100 vs. 10,500/mm2, &NBSP;P < 0.05). This mutant phenotype is similar to that seen in retinas of Siamese cats and albino ferrets. Beyond area centralis, the central-to-peripheral gradient in ganglion cell density is normal in mutants. The size of the optic nerves, density of axons, and total number of axons do not differ between mutant and normal dogs.
     One of three mutant dogs had a small abnormal optic chiasm. Retrograde labeling of ganglion cells demonstrated that the residual crossed projection originated from cells in a widespread region in nasal retina and not solely from the peripheral nasal region, as might be expected of an anti-albino.
     Although our analysis does not rule out the retina as a site of mutant gene action, the modest differences between mutant and normal retinas suggest that the mutation either acts outside the retina or exerts a highly specific effect on ganglion cell trajectories alone.

     For the past 30 years, mutations at a single gene locus, the tyrosinase locus, have had a significant impact on our understanding of the development of central visual path ways and the formation of sensory maps (Lund, 1965; Guillery, 1986, 1991). Mutations of this gene are common in many animals, including rodents, rabbits, cats, ferrets, monkeys, and humans, and result in varying degrees of pigment deficits (Lerner et al., 1950; King and Witkop, 1976; Searle, 1989). Two examples are the albino mutation (the &NBSP;c allele), characterized by a complete lack of melanin pigmentation, and a temperature-sensitive variant (the &NBSP;ch&NBSP;allele), typified by the well-studied Siamese cat (Hubel and Wiesel, 1971). These mutations are of interest to neuroscientists because they also consistently reduce the number of retinal ganglion cells with uncrossed projections into the thalamus (Guillery, 1986). Mutations in which the neuronal connections of the visual system are altered provide excellent natural experiments and have been instrumental in the rapid progress made in understanding how the brain processes and represents sensory information.
     We recently identified and described another mutation that changes the projection pattern of retinal ganglion cells. This novel mutation eliminates the optic chiasm and results in a complete misrouting of optic axons into the ipsilateral optic tract (Williams et al., 1994). The achiasmatic mutation was found in a family of Belgian sheepdogs. This breed of dog, also known as the Groenendael, weighs 20–30 kg, has a long solid black coat, and normally pig mented eyes. The pattern of inheritance indicates that the achiasmatic mutation is carried as an autosomal recessive allele (Williams et al., 1991). All mutant dogs that we studied have a persistent horizontal nystagmus often associated with head tilt and head oscillation (Dell'Osso and Williams, 1994). This behavioral complex is remarkably similar to that seen in some human albinos and human achiasmatics (Apkarian et al., 1994).
     The achiasmatic mutation shares several features with tyrosinase mutations: both change the pattern of retinal projections and both are often associated with abnormal eye position or movements. However, the polarity of the projection defect is opposite: in achiasmatic animals the ipsilateral projection is increased, whereas in albinos the ipsilateral projection is decreased. In addition, the achiasmatic error is usually complete—the entire nasal projection is redirected into the ipsilateral tract. Even in the most severely affected albinos, there is always a residual population of ganglion cells with ipsilateral projections (Balkema and Drdger, 1990). Because the achiasmatic mutation misroutes the larger nasal retinal projection, this mutation is quantitatively much more severe than the partial misdi rection of the smaller temporal projection in albinos. By pushing the visual system so far out of kilter, the achiasmatic mutation promises to illuminate processes important for generating normal retinal connections and sensory maps.
     Despite the marked differences between albinos and achiasmatics, our initial experiments in the lateral geniculate nucleus have shown that the anatomical and functional consequences are surprisingly similar. The lateral genicu late of mutant dogs contains a complete functional map of the ipsilateral visual field in layer A that is contiguous with the normal map of contralateral visual fields in layer A1 (Williams et al., 1994). This is similar to the result found in Siamese cats, in which the part of A1 that receives the abnormally crossed projection contains an aberrant map of part of ipsilateral visual field. In both Siamese cats and achiasmatic dogs, the maps in the fused layers A and A1 are arranged in a mirror-image fashion.
     There are two candidate sites at which the achiasmatic gene might act during development to alter the pattern of retinal projections. The most obvious site is the developing optic chiasm. The mutation could change the physical or molecular characteristics of this region and redirect nasal axons into the wrong optic tract. The other candidate site is the retina. The mutation could change the cytochemical identity of nasal retinal ganglion cells, labeling all of them with an "ipsilateral only" marker that might compel them into the ipsilateral tract. The midline itself might be perfectly normal. If the mutant phenotype is generated by changes that are intrinsic to the retina or retinal ganglion cells, then one might expect correlated changes in retinal structure. The purpose of this study is to examine the retinas of mutant and normal dogs to determine whether there is morphological evidence that the mutation acts within the retina.





Animals used

     Retinas and optic nerves were taken from six Belgian sheepdogs (BSD) and two normal beagles (Table 1). Three of the six sheepdogs from our colony were mutants. All three displayed marked congenital nystagmus and were used for mapping studies described elsewhere (Williams et al., 1994). The other three sheepdogs had normal pheno types and normal eye movements (DellOsso and Williams, 1994). However, since the achiasmatic mutation is autoso mal recessive, any of these three normal animals may have been heterozygous carriers of the mutant gene. For this reason, retinas and optic nerves from two wild-type normal beagles were also examined.



Table 1. Dogs Used in These Experiments


Dog Sex Breed/genotype Age at death



Retrograde tracer injection


      Horseradish peroxidase (HRP) was injected unilaterally into the dorsal lateral geniculate nucleus and optic tract of four animals. These animals were prepared as described elsewhere (Williams et al., 1994). In brief, dogs were sedated with ketamine hydrochloride (7 mg/kg) and xyla zine (0.4 mg/kg). Anesthesia was induced with sodium pentobarbital (15–20 mg/kg) and maintained by infusing sodium pentobarbital (3 mg/kg/hour). A craniotomy was made and the lateral geniculate located by electrophysiological means. HRP (30% solution of Sigma Type VI-A) was injected at several sites with a 10-µl syringe (for detailed methods, see Chalupa et al., 1984). In three of four cases we then recorded activity in the noninjected lateral geniculate nucleus for 12 to 24 hours. At the conclusion of the recording session, animals were killed with a lethal over dose of sodium pentobarbital (30–40 mg/kg) and perfused transcardially with either 2.5% gluteraldehyde/ 1% paraformaldehyde or 4% paraformaldehyde/0. 1% gluteraldehyde. In one case, the incision was closed and the animal was kept sedated for an additional 24 hours. The postinjection survival periods were not ideal for retrograde labeling in animals as large as these dogs. In only two cases did we successfully label a significant number of retinal ganglion cells.


Retinal wholemounts


      Retinas were dissected, and wholemounts were prepared and placed between two oversized coverslips in Gelvatol (Heimer and Taylor, 1974). Retinas from animals injected with HRP were processed by using the Hanker-Yates protocol (Hanker et al., 1977; Perry et al., 1983) and subsequently mounted in Gelvatol. Mounting in Gelvatol minimizes distortions and tears that commonly result from dehydrating retinas fixed to gelatinized slides. Repeated measurements show that this procedure introduces less than a 2% change in linear dimensions (Williams, 1991). A second advantage of Gelvatol is that a larger refractive index gradient is retained in the retina during processing, so that differential interference contrast (DIC) images of the retinal cell types have more contrast, making it possible to detect more easily unstained cells in the nuclear layers. Retinas were examined by using a video microscope equipped with DIC optics (Curcio et al., 1987). The size of stained and unstained retinal ganglion cells were measured by using a video-overlay camera system (Williams et al., 1993).


EM of optic nerves


      Optic nerves were prepared for examination with electronmicroscopy (Williams et al., 1983). In brief, cross sections of optic nerve approximately 1 mm thick were taken from the midorbital portion of the optic nerve. Tissue was postfixed with 2% osmium tetraoxide, stained en bloc overnight with 0.5% uranyl acetate, dehydrated, and embedded in Spurr's medium. Thin sections were stained with uranyl acetate and lead citrate. The cross section was photographed at both low power (80–lOOx nominal magnification) and high power (4,000–6,000x nominal magnifica tion) with an electron microscope. Actual magnifications were calculated by using a calibration grid. Low-power micrographs were used to calculate the cross-sectional area of the optic nerves. High-power micrographs were taken at 45–55 evenly distributed points across the nerve. Axons in each micrograph were counted, and the average density of axons was multiplied by the area of the nerve to estimate the total number of axons.




      As early as 4 weeks of age, mutant animals can be recognized by their abnormal visual behavior. By 8 to 10 weeks, the horizontal nystagmus is prominent. Mutants occasionally have other unusual behaviors. Several mutants in our colony, including two animals used in this study, circled almost incessantly in their cages when excited. Both also exhibited uncontrollable self-mutilating behavior that compelled us for ethical and veterinary reasons to use these animals in terminal experiments.

Figure 1. Vertical views of the chiasm areas of the brain.
Figure 1a Figure 1b Figure 1c
Figure 1. Ventral views of the brains of dogs showing chiasm area. A: Normal sheepdog (U3). B: Mutant sheepdog with no chiasm (M3). The upper little hole is the lamina terminalis, which is usually covered by the chiasm. C: Sheepdog (M2) with a partial chiasm. Scale bar = 5 mm.

     Two of the three mutants used in this study had no crossed retinofugal projection. Their optic nerves failed to approach the midline (Fig. 1B) and were separated by 3–5 mm as they entered the thalamus. The other mutant with congenital nystagmus (M2) had a small and abnormal optic chiasm (Fig. 1C). With the exception of the optic chiasm, we did not note any other obvious midline abnormalities in mutants. The corpus callosum and the anterior and posterior commissures were intact. The hypothalamus and third ventricle also appeared normal. The three phenotypically normal sheepdogs, one or more of which may have been heterozygous carriers of the mutation, had normal optic chiasms (Fig. 1A). In the normal dog, 80–82% of the axons of retinal ganglion cells cross in the chiasm (Stone, 1983).


Optic nerves


      Despite their failure to decussate at the chiasm, the size of the optic nerves and the density of axons were similar in normal and mutant dogs (Table 2). The appearance of the axons within the nerves was also normal in the mutants (Fig. 2). Six optic nerves taken from three normal dogs gave counts that range from 190,200 to 256,500 (average 211,500±11,000; see Table 3). Counts from three mutants yielded estimates that ranged from 176,000 to 239,000 (average 215,000±14,900). These averages were not significantly different (t test). For comparison, three optic nerves from two beagles were counted and averaged (192,200±15,800). This number was not significantly different from either group of sheepdogs.


Anatomy of the retina


      In general, the retinas of mutant dogs are remarkably normal. Retinas from mutants are slightly smaller than those from normal dogs (515 mm2vs. 580 mm2; &NBSP;P < 0.05: Table 3). Two of the mutants were killed before maturity (Table 1), and the smaller retinal areas may be due only to age differences. However, mutant M3 had reached maturity and this animal's retinas were also smaller than those of any of the normal sheepdogs. The proportion of retina in the area nasal to a vertical line drawn through the area centralis was calculated. The mutants we studied had a slightly higher percentage of their retinal area located in nasal retina, 81 vs. 78%. The distance from the area centralis to the optic disk of mutant and normal animals was identical.
     The photoreceptor mosaic of mutant and normal animals is similar in density and distribution (Fig. 3A,B). The density of cones in the peripheral retina (5–10 mm from area centralis) is about 13,400/mm2 in both mutant and normal dogs. The density of rods in the same areas is S03,000/mm2 for mutant M1 and 493,000/mm2 for the normal dog U2. The outer nuclear layer of mutants and normal dogs are similar: both are 8–9 cell nuclei thick (Fig. 3C,D). The inner nuclear layer of mutants and normal dogs are also similar: both are 3 cell nuclei thick. In addition, the thickness of both the inner and outer plexiform layers are the same in mutant and normal dogs.


TABLE 2. Optic Nerve Data


Number of
(mean ± S.E.M.)
Density of
in nerve
Normal BSDs



Mutant BSDs
















TABLE 3. Retinal Data


  Eye Retinal
AC to OD
Peak RGC
Normal BSDs


Mutant BSDS


















*Significantly different from normal BSDs (P < 0.05).



Retinal ganglion cells


Distribution of ganglion cells. Collectively, the total cell population in the ganglion cell layer, including glial cells and displaced amacrine cells, shows a normal central-to-peripheral gradient in the mutant retina (Fig. 4A). The ganglion cell layer contains about 900,000 cells. This fignre, combined with estimates of the number of retinal ganglion cells from optic nerve counts, yields a ratio of 3.75:1 of all cells in the ganglion cell layer to retinal ganglion cells. This is within the range of 3:1 to 4:1 reported for dogs (Arey and Gore, 1942). The distribution of retinal ganglion cells in the mutant dog retina is also similar to that of normal dogs (Fig. 4B; Krinke et al., 1981; Peichl, 1992b). The number of ganglion cells estimated from the isodensity profile of mutant M2 is 110,000 cells. This fignre is 54% lower than the axon count from the optic nerve of the same animal (see Discussion).
     The retinas of different breeds of dogs have been shown to be characterized by either a prominent horizontal streak, identified by a threefold increase in the density of ganglion cells along the horizontal axis of the eye, or by a moderate horizontal steak characterized by only a twofold increase in ganglion cell density (Fig. 5; Peichl, 1992b). The isodensity profile of ganglion cells in both our normal and mutant sheepdogs is typical of dogs with a moderate horizontal streak (Figs. 4, 5). In contrast, the retinas of a beagle we examined had a prominent horizontal streak (Fig. 5).
     In nasal retina, the number of alpha ganglion cells is similar in both mutant and normal dogs. In a typical sample area 10 mm nasal and 5 mm superior to the area centralis (Fig. 6), the proportion of alpha ganglion cells to all ganglion cells is 3.3% in the normal dog and 3.2% in the mutant. An unusual feature that has been reported in the dog retina is the absence of alpha ganglion cells in the peripheral temporal retina (Peichl et al., 1987; Peichl, 1992b). We also failed to find alpha cells in this area of temporal retina in either normal or mutant dogs (Fig. 7).

Figure 2. Electron photomicrographs of normal and mutant dog's optic nerves.
Figure 2

The area centralis of mutant and normal dogs. The most striking difference between the retinas of normal and mutant dogs is in the area centralis, the carnivore equivalent of the human macula. The area centralis is smaller and the peak ganglion cell density is lower in mutants than in normal dogs (Fig. 8). The peak ganglion cell density in retinas of three normal dogs averaged 10,500±800/mm2 (range 8,900–12,300; Table 3). In contrast, the peak ganglion cell density of mutants averaged 8,100±300/mm2 (range 7,300–8,700). This fignre is significantly lower than that of the normal sheepdogs (P < 0.05). The peak gan glion cell density of the normal beagle retina with a prominent horizontal streak was 13,500±500/mm2. These fignres are within the range of average peak ganglion cell densities reported in retinas of dogs with a moderate visual streak (range 6,400—9,800/mm2), as well as dogs and wolves with a pronounced horizontal streak (10,300— 14,400 cells/mm2; Peichl et al., 1987; Peichl, 1992b).
Retrograde tracing of the retinogeniculate pathway. HRP was injected unilaterally into the lateral geniculate nucleus of two mutant sheepdogs (M2 and M3), one normal sheepdog (U2), and one normal beagle (C2; Table 1). Although these cases yielded incomplete filling of retinal ganglion cells, cases M2 and C2 yielded sufficient labeling to identify unequivocally ganglion cells in large areas of the retina. In case M2, the sheepdog with a small residual chiasm, HRP tracing showed that the residual crossed projection originates primarily from ganglion cells in para central and peripheral nasal retina, although a few cells also appeared to originate in temporal retina (Fig. 9). The contralateral and ipsilateral projections did not originate in precisely the same part of each retina; the large population of nasal cells with ipsilateral projections was located closer to the area centralis of the ipsilateral retina than the labeled cells in the contralateral retina.
     The sizes of HRP-labeled cells were measured to determine if there was en masse respecification of nasal retinal ganglion cells that could be detected as changes in the distribution of ganglion cell size, particularly in the nasal hemiretina. In this respect, the mutant (M2) with the partial chiasm was particularly informative because we were able to compare the sizes of both aberrantly and normally projecting nasal ganglion cells within a single mutant animal as well as with nasal ganglion cells in the normal dog (C1). Cell body areas of HRP-labeled retinal ganglion cells of this mutant and that of the beagle were similar (Fig. 10). Cell areas are similar in the normal projections of the mutant (i.e., ipsilateral temporal: 500 in M2 vs. 460 µm2 in C1, and contralateral nasal: 500 vs. 500 µm2). The area of cells in the large aberrant projection of the mutant (ipsilateral nasal; 490 µm2) is similar to the areas of cells with normal decussation patterns.





      We have quantified several aspects of retinal structure in a novel mutation that is carried in dogs. In this mutation, all optic nerve fibers extend into the ipsilateral optic tract. Our intent was to determine whether there is evidence that the retina is a primary site of mutant gene action. The principal difference we observed between retinas of mutants and normal dogs is the lower density of retinal ganglion cells in area centralis.


Figure 3. Photomicrographs of photoreceptor layers from retinas of normal and achiasmatic sheepdogs.
Figure 3



General observations on the canine retina


      The extreme polymorphism among different breeds of dogs has led investigators to bypass this species as a model for vision research. Peichl exploited this variation and compared the topography of retinal ganglion cells in several breeds of domestic dog and in the wolf (Peichl, 1992a,b). Our work has largely confirmed key aspects of his work. We have also identified dog retinas with either a prominent or a moderate horizontal streak, have verified Peichi's estimates of peak ganglion cell density, and have noted the lack of alpha cells in far peripheral temporal retina.
     Our optic nerve counts in eight normal and mutant dogs (Table 2) range from a low of 160,000 to a high of 260,000 with an average of 210,000. By using another method, the isodensity contour method, Peichl (1992b) estimated that the retinas of two dogs in his study contained 110,000 and 121,000 ganglion cells. These estimates are 45% lower than our counts of fibers in the optic nerve. Peichl's estimates are also lower than those of Arey's group (Arey et al., 1942; Arey and Gore, 1942). These studies, based on light microscopic analysis of the optic nerve (a technique that consistently underestimates the number of axons), estimated 138,000–165,000 axons in the optic nerves of dogs of various breeds. Retinal analysis in these studies yielded estimates of 153,000–192,000 ganglion cells. These figures overlap the lower end of the range of values we obtained.
     To help resolve this problem we estimated ganglion cell number with the isodensity contour method (case M2; see Fig. 4B) and obtained a count of 110,000 ganglion cells. This value is similar to the estimates of Peichl from using the same method, but it is 54% lower than our estimates of axons in the optic nerve of the same animal. Estimates of axons in each optic nerve provide an accurate estimate of the total number of ganglion cells in the retina in both the cat (Chalupa et al., 1984) and the mouse (Rice et al., 1994). Therefore, we believe that this difference is most likely due to systematic undercounting of ganglion cells in the whole mounts. Regardless, our results unequivocally show that the achiasmatic phenotype is not due to the loss of ganglion cells in the nasal hemiretina.

Figure 4. Isodensity profiles of mutant sheepdogs.
Figure 4


Figure 5. Density of ganglion cells perpendicular to the horizontal streak of mutant and normal sheepdogs.
Figure 5

      The finding that there are no alpha ganglion cells in the far temporal retina of dogs is particularly intrigning be cause it is the only known example of this phenomena in mammalian retina (Peichl et al., 1987). Peichl speculated that this may be a result of the relatively large snout that results in a deprivation-like effect in the far temporal retina. Whether the lack of alpha cells in a portion of the temporal retina is a deprivation induced effect or is an intrinsic attribute of canine retina is unknown. Whatever the cause, this unusual distribution is unaffected by the achiasmatic mutation.


Effects of the achiasmatic mutation on the retina


Deficits in the area centralis. The primary defect found in the retinas of mutant dogs is a reduction in the size of the area centralis and a reduction in the peak ganglion cell density. This deficit is similar to those observed in the retinas of tyrosinase mutations of other species. In the retinas of Siamese cats, albino cats, and albino ferrets, the area centralis is smaller and has a lower peak ganglion cell density when compared with normally pigmented animals (Stone et al., 1978; Cucchiaro and Guillery, 1984; Leven thal and Creel, 1985; Morgan et al., 1987). In albino primates the fovea is also undeveloped. Both human and green monkey albinos lack a fovea (Fulton et al., 1978; Guillery et al., 1984). Like the mutant dogs, the gradient of ganglion cells in the periphery of the retinas of albinos is normal.
      Guillery et al. (1984) speculated that the albino mutation may act directly to modify the structure of the area centralis. The fact that achiasmatic dogs have similar structural deficits suggests that the defects in area centralis is not caused directly by the albino mutation itself but is a secondary effect produced by decussation errors. Albinos of many species and achiasinatic humans and dogs exhibit abnormal eye movements or position which could prevent the affected animal from maintaining a stable image on the retina. It is possible that the deficits in the central retinas of these two different mutations is secondary to these abnormalities.

Figure 6. Distribution of ganglion cells in one square millimeter of a normal and mutant sheepdog.
Figure 6

Other retinal differences. Although the major effect in mutant retina was seen in area centralis, several other differences were noted, namely, retinal area and the ratio of nasal and temporal retinal areas. Given the small sample size and age differences between groups, we do not believe that these differences have much biological significance.


Visual system mutations and decussation patterns


Site of gene action in misrouting of the retinal projection. An aim of this study was to determine whether the retina is a site of mutant gene action. If we had found highly unusual structure in the nasal hemiretina, this would have provided strong evidence of a retinal site of gene action that might account for the failure of nasal ganglion cells to cross at the optic chiasm. We did not find any unusual cellular properties in either nasal or temporal retina. However, our study and a review of previous work do provide some clues regarding possible sites of gene action in this mutation.
      Studies have shown that the ratio of axons that project ipsilaterally or contralaterally changes during develop ment. In most species, the first axons to reach the optic chiasm project ipsilaterally (Godement et al., 1990; Reese et al., 1992; Baker and Reese, 1993). At later stages, more axons are directed contralaterally. Nearly all of the last axons to arrive at the chiasm cross. This has led several researchers to propose that a signal that is expressed early in retinal development favors ipsilateral projections and that this signal weakens over time (Walsh et al., 1983; Reese et al., 1991, 1992; Reese and Colello, 1992; Baker and Reese, 1993).
      Studies in mice and cats have shown that axons from nasal and temporal retinas are intermingled in the optic nerve throughout development. As the fibers approach the chiasm, they sort and either cross or do not cross with a surprising degree of accuracy (Godement et al., 1990; Jeffery, 1990; Sretavan, 1990; Chalupa and Lia, 1991). These data implicate a factor near the chiasm that the axons respond to differentially. Temporal axons frequently grow right up to the midline before turning into the ipsilateral optic tract. This suggests that there is a repulsive interaction specific to a subpopulation of temporal ganglion cell growth cones (Bonhoeffer and Huf, 1985; Godeinent et al., 1990).
      Given this background, what types of developmental changes could account for the complete elimination of a crossed retinal projection? One possibility, suggested by the mostly ipsilateral projection of the earliest arriving axons, is that the achiasmatic mutation results from an accelera tion in the time of genesis of retinal ganglion cells. How ever because the achiasmatic phenotype is so extreme and because retinal ganglion cells in carnivores are born over a 2-week period (Walsh and Polley, 1985), it is difficult to imagine that a disruption in timing alone that would be sufficient to cause this massive misrouting.

Figure 7. Photomicrographs from the retinal ganglion cell layer of beagle and mutant dogs.
Figure 7

      A second possibility could be a mispecification of retinal ganglion cells in the achiasmatic mutants. Chan et al. (1993) postulated that the albino mutation acts within the retina to change a putative retinal gradient that causes most early cells to remaln ipsilateral, resulting in more crossed axons, as well as a shift in the decussation line toward the ventral temporal periphery. The data from the mutant dog with a partial chiasm argnes against a change in retinal gradients analogous (but opposite) to that of the albino because this animal did not exhibit a simple shift in the line of decussation into the nasal periphery. In addition, our previous study in the lateral geniculate nucleus of mutant dogs strongly suggest that retinal positional specificity is not affected by this mutation because axons are correctly targeted within the lateral geniculate nucleus: nasal axons terminate in layer A and temporal axons in layer Al with appropriate retinotopy despite the decussa tion error (Williams et al., 1994).

Figure 8. Density of ganglion cells in the area centralis of mutant and normal dogs.
Figure 8

      A third possibility is that the optic nerve itself grows in an aberrant direction. Guillery and Walsh (1987a,b) reported the early appearance of a small bundle of optic fibers in ferrets that do not enter the chiasm. These fibers, collec tively called the ipsilateral optic bundles, grow laterally and ventrally around the hypothalamus and never approach midline. Even though these are the first fibers to leave the optic stalk and enter the brain, the later arriving fibers do not normally follow the ipsilateral optic bundles but enter the chiasm. However, in the achiasmatic dogs, all axons may follow the pathway taken by the ipsilateral optic bundles. This hypothesis is attractive because it would explain why, in most mutants, the optic nerves do not approach midline. If the defect were at the midline itself, we would expect that the nerves would grow up to, but not cross, the chiasm.

Figure 9. Drawings of ganglion cells in the retinas of mutant M2.
Figure 9

Is the achiasmatic mutant an "anti-albino"? Despite the obvious reversed polarity of the decussation defect in the achiasmatic dog, this mutation is not simply an anti- albino. In albinism, the line of decussation, although not as sharp as that in normal animals, is shifted toward the temporal edge of the retina. In addition, there is always a residual uncrossed projection. In the achiasmatic dog, there is usually no residual crossed projection. However, as case M2 illustrates, the achiasmatic mutation can occasionally be incomplete. If this mutation were an anti-albino, the expected effect of a partial decussation would be a line of decussation that is shifted to the nasal side of the retina, perhaps to the other side of the optic disk. In fact, the residual crossed projection originates throughout nasal retina, not solely in far peripheral nasal retina, as would be expected of an anti-albino.

Figure 10. Comparison of cell body areas between achiasmatic BSD and normal beagles.
Figure 10

      Although the albino mutation usually causes a shift in the line of decussation toward the temporal periphery, there is a report in the literature of a single individual, an albino ferret, with a decussation pattern analogous to that of the dog with a partial chiasm (Morgan et al., 1987). In this anomalous albino, the ipsilateral component arose from the nasal retina (see Fig. 9; Morgan et al., 1987). As in our mutant dog M2, this particular ferret did not have a shift in the decussation line, but the areas of the highest densities of labeled cells were found in approximately the same area of both nasal retinas.
A comparison with the acallosal mutation. The acallo sal mutation is another mutation in which axons fall to cross midline. In four strains of mice (BALB/cWahl, BALB/cWah2, J/Ln, and 129/Re), this callosal abnormality is highly variable and appears to be a polygenic trait involving two or three loci (Livy and Wahlsten, 1991). In contrast, the pattern of inheritance of the achiasmatic mutation is most consistent with an autosomal recessive mutation at a single locus. The elegant studies of Silver et al. (1982) and Silver and Ogawa (1983) showed that the acallosal phenotype is a mutation that directly affects midline. Callosal axons cannot cross because the substrate that they normally grow along, the glial sling, fails to form. Comparable developmental studies of achiasmatic dogs could may reveal whether or not there is an analogous defect in the floor of the hypothalamus that does not support the crossing of nasal axons.
      In acallosal mice and humans, cortical efferents, both "callosal" and ipsilateral corticocortical, accumulate to form a swirling ipsilateral pathway or neuroma known as "Probst's longitudinal bundle" (King, 1936; Silver et al., 1982). By analogy, one might expect retinal axons in achiasinatic mutants to form a similar tangle in the area where the optic chiasm should form. However, this does not happen—axons extend straight back into the optic tract.
      Some axons are able to escape Probst's bundle and form appropriate connections to both ipsilateral and contralat eral hemispheres (Ozaki and Shimada, 1988; Ozaki et al., 1989). The fact that these connections are topographically appropriate is analogous to the condition in achiasmatic mutants: optic axons terminate in the ipsilateral lateral geniculate nucleus with appropriate retinotopy despite their failure to cross midline (Williams et al., 1994). This suggests that both mutations specifically perturb crossing at the midline but do not perturb target recognition. The fact that the optic chiasm is normal in acallosal mice and that the corpus callosum is apparently normal in achias matic dogs emphasizes that multiple loci control the devel opment of midline structures.
Specificity of mutation. This study of the effects of the achiasmatic mutation on the retina reinforces the results of our physiological and anatomical studies of the lateral geniculate nucleus of achiasmatic sheepdogs. It is surprising how normal the visual system of these animals is considering over 80% of the retinal ganglion cells are misrouted. This massive rerouting might be expected to disrupt severely the normal architecture in the brains and retinas of affected animals. The relatively minor perturbation of retinal structure also reinforces the remarkable selectivity of action of this achiasmatic mutation.




This work was supported by NRSA grant EY06560 to D. H. and NIH grant EY-6627 to R. W. W.



Apkarian, P., L. Bour, and PG. Barth (1994) A unique achiasmatic anomaly detected in non-alhinos with misrouted retinal—fugal projections. Eur. J. Neurosci. 6: 501—507.

Arey, LB., S.R. Bruesch, and S. Castanares (1942) The relation between eyehall size and the numher of optic nerve fibers in the dog. J. Comp. Neurol. 76: 417—422.

Arey, L.B., and M. Gore (1942) The numerical relation hetween the ganglion cells of the retina and the fibers in the optic nerve of the dog. J. Comp. Neurol. 77: 609—617.

Baker, G.E., and B.E. Reese (19931 Chiasmatic course of temporal retinal axons in the developing ferret. J. Comp. Neurol. 330: 95—104.

Balkema, G.W., and U.C. Drager (19901 Origins of uncrossed retinofugal projections in normal and hypopigmented mice. Vis. Neurosci. 4: 595—604.

Bonhoeffer, F., and J. Huf (1985) Position-dependent properties of retinal axons and their growth cones. Nature 351: 405—410.

Chalupa, L.M., and B. Lia (1991) The nasotemporal division of the retinal ganglion cells with crossed and uncrossed projections in the fetal rhesus monkey. J. Neurosci. 11: 191—202.

Chalupa, L.M., R.W. Williams, and Z. Henderson (1984) Binoclular interac tion in the fetal cat regulates the size of the ganglion cell population. Neuroscience 12: 1139—1146.

Chan, SO., G.E. Baker, and EW. Guillery (1993) Differential action of the albino mutation on two components of the rat's uncrossed retinofugal pathway. J. Comp. Neurol. 336: 362—377.

Cucchiaro,J., and R.W. Guillery (1984) The development of the retinogenicu late pathways in the normal and alhino ferret. Proc. R. Soc. Lond. [Biol.] 223: 141—164.

Curcio, C., 0. Packer, and RE. Kalina (1987) A wholemount method for sequential analysis of photoreceptor and ganglion cell topography in a single retina. Via. Res. 27: 9—15.

Dell'Osso, L.F., and R.W. Williams (1994) Ocular motor ahnormalities in achiasmatic Begian sheepdogs: Unyoked eye movements in a mammal. Vis. Res. in press.

Fulton, A.B., D.M. Alhert, and J.L. Craft (1978) Human alhinism: Light and electron microscopy study. Arch. Opthalmol. 96: 305—310.

Godement, P., J. Salaun, and CA. Mason (1990) Retinal axon pathfinding in the optic chiasm: divergence of crossed and uncrossed fibers. Neuron 5: 173—186.

Guillery, R.W. (1986) Neural ahnormalities of albinos. Trends Neurosci. 9: 364—367.

Guillery, R.W. (1991) Rules that govern the development of the pathways from the eye to the optic tract in mammals. In D.M.-R. Lam and C.J. Shatz (eds.): Development of the Visual System, Vol.3. Cambridge, MA: MIT, pp. 153—171.

Guillery, R.W., T.L. Hickey, J.H. Kaas, D.J. Fellemam, E.J. Dehruyn, and D.L. Sparks (1984) Ahnormal central visual pathways in the brain of an albino green monkey (Cercopithecus aethiops). J. Comp. Neurol. 226: 165—183.

Guillery, R.W., and C. Walsh (1987a) Changing glial organization relates to changing fiher order in the developing optic nerve of ferrets. J. Comp. Neurol. 265: 203—217.

Guillery, EW., and C. Walsh (1987b) Early uncrossed component of the developing optic nerve with a short extracerebral course: A light and electron microscopic study of fetal ferrets. J. Comp. Neurol. 265: 218—223.

Hanker, J.S., P.R. Yates, C.B. Metz, and A. Rustioni (1977) A new specific, sensitive and non-carcinogenic reagent for the demonstration of horse radish peroxidase. Histochem. J. 9: 789—792.

Heimer, G.V., and CED. Taylor (1974) Improved mountant for immunofiu orescence preparations. J. Clin. Pathol. 27: 254—256.

Hubel, D.H., and TN. Wiesel 11971) Aberrant visual projections in the Siamese cat. J. Physiol. 218: 33—62.

Jeffery, G. (1990) Distribution of uncrossed and crossed retinofugal axons in the cat optic nerve and their relationship to patterns of fasciculation. Via. Neurosci. 5: 99—104.

King, L.S. (1936) Hereditary defects of the corpus allosum in the mouse, Mus musculus. J. Comp. Neurol. 64: 337—363.

King, R.A., and Cd. Witkop (1976) Hairbulb tyrosinase activity in oculocutaneous albinism. Nature 263: 69—71.

Krinke, A., K. Schnider, E. Lundheck, and G. Krinke (1981) Ganglionic cell distribution in the central area of the hoagie dog retina. Zbl. Vet. Med. C. Anat. Histol. Embryol. 10: 26—35.

Lerner, A.B., T.B. Fitzpatrick, E. Calkins, and S.W.H. Summerson (1950) Mammalian tyrosinase: The relationship of copper to enzyme activity. J. Biol. Chem. 187: 793—802.

Leventhal, A.G., and D.J. Creel (1985) Retinal projections and functional architecture of cortical areas 17 and 18 in the tyrosinase-negative albino cat. J. Neurosci. 3: 795—807.

Livy, D.J., and D. Wahlsten (1991) Tests of genetic allelism between four inhred mouse strains with absent corpus callosum. J. Hered. 82: 459—464.

Lund, RD. (1965) Uncrossed visual pathways of hooded and alhino rats. Science 149: 1506—1507.

Morgan, J.E., Z. Henderson, and I.D. Thompson (1987) Retinal decussaton patterns in pigmented and aihino ferrets. Neuroscience 20: 519—535.

Ozaki, H.S., and M. Shimada (1988) The fihers which course within the Probst's longitudinal bundle seen in the hrain of a congenitally acallosal mouse: A study with the horseradish peroxidase method. Brain Res. 441: 5—14.

Ozaki, H.S., K. Iwahashi, and M. Shimada (1989) Ipsilateral corticocortical projections of fibers which course within Probst's longitudinal hundle seen in the hrains of mice with congenital absence of the corpus callosum: A study with the horseradish peroxidase method. Brain Res. 493: 66—73.

Peichl, L. (1992a) Morphological types of ganglion cells in the dog and wolf retina. J. Comp. Neurol. 324: 590—602.

Peichl, L. (1992b) Topography of ganglion cells in the dog and wolf retina. J. Comp. Neurol. 324: 603—620.

Peichl, L., H. Ott, and B.B. Boycott (1987) Alpha ganglion cells in mamma lian retinae. Proc. R. Soc. Lond. B 231: 169—197.

Perry, V.H., Z. Henderson, and R. Linden (1983) Postnatal changes in retinal ganglion cell and optic nerve populations in the pigmented rat. J. Comp. Neurol. 219: 356—368.

Reese, BE., and R.J. Colello (1992) Neurogenesis in the retinal ganglion cell layer of the rat. Neuroscience 46: 419—429.

Reese, B.E., R.W. Guillery, and C. Mallarino (1992) Time of ganglion cell genesis in relation to the chiasmatic pathway choice of retinofugal axons. J. Comp. Neurol. 324: 336—342.

Reese, B.E., EW. Guillery, CA. Marzi, and G. Tassinari 119911 Position of axons in the cat's optic tract in relation to their retinal origin and chiasmatic pathway. J. Comp. Neurol. 306: 539—553.

Rice, D.S., EW. Williams, and D. Goldowitz (1994) Genetic control of retinal projections in inbred strains of albino mice. J. Comp. Neurol., (in press).

Searle, A.G. (1989) Comparative genetics of albinism. Ophtal. Paed. Genet. 11: 159—164.

Silver, J., SE. Lorens, D. Wahlsten, and J. Coughlin (1982) Axonal guidance during development of the great cerebral commissures: Descriptive and experimental studies, in vivo, on the role of preformed glial pathways. J. Comp. Neurol. 210: 10—29.

Silver, J., and M. Ogawa (1983) Postnatally induced formation of the corpus callosum in acallosal mice on glia-coated cellulose bridges. Science 220: 1067—1069.

Sretavan, D.W. 119901 Specific routing of retinal ganglion cell axons at the mammalian optic chiasm during emhryonic development. J. Neurosci. 10: 1995—2007.

Stone, J. (1983) Parallel Processing in the Visual System: The Classification of Retinal Ganglion Cells and Its Impact on the Neurobiology of Vision. New York: Plenum.

Stone, J., M.H. Rowe, and J.E. Campion (1978) Retinal abnormalities in the Siamese cat. J. Comp. Neurol. 180: 773—782.

Walsh, C., and E.H. Polley (1985) The topography of ganglion cell production tn the cat's retina. J. Neurosci. 5: 741—750.

Walsh, C., E.H. Polley, T.L. Hickey, and EW. Guillery (1983) Generation of cat retinal ganglion cells in relation to central pathways. Nature 302: 611—614.

Williams, R.W. (1991) The human retina has a cone-enriched rim. Vis. Neurosci. 6: 403—406.

Williams, R.W., M.J. Bastiani, and L.M. Chalupa (1983) Loss of axons in the cat optic tract following prenatal unilateral enuclearion: an electron microscopic analysis. J. Neurosci. 3:133—144.

Williams, R.W., C. Cavada, and F. Reinoso-Suarez (1993) Rapid evolution of the visual system: A cellular assay of the retina and dorsal lateral geniculate nucleus of the Spanish wildcat and the domestic cat. J. Neurosci. 13: 208—228.

Williams, R.W., P.E. Garraghty, and D. Goldowitz (1991) A new visual system mutation: Achiasmatic dogs with congenital nystagmus. Soc. Neurosci. Abstr. 17: 187.

Williams, R.W., D. Hogan, and P.E. Garraghty (1994) Target recogntion and visual maps in the thalamus of achiasmatic dogs. Nature 367: 637—639.




Accepted August 8, 1994.


Neurogenetics at University of Tennessee Health Science Center

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