Multiple pathologies in the outer segment layer in Crx-/- mice
A standard knock-out protocol was used to generate mice in which the homeodomain of Crx-/- was targeted and deleted. Generation of these Crx-/- mice has been reported elsewhere [34]. In this study, in order to characterize further the role of Crx in photoreceptor morphogenesis, the outer retinae from Crx-/- mice were examined using transmission electron microscopy. At postnatal day 21 (P21), when Crx+/+ photoreceptors exhibited robust outer segments (Figure 1A, os), Crx-/- retinas were without a recognizable outer segment layer (Figure 1B). Crx-/- photoreceptors had inner segments, demonstrating at least limited photoreceptor polarization in the Crx mutant, but the inner segments were extremely short (Figure 2). Furthermore, the majority of inner segments showed some morphological differentiation, having approximately as many mitochondria as the control (Figure 1 and 2). Occasionally, an inner segment undergoing lysis was noted, appearing swollen or with vacuoles and swollen mitochondria (data not shown).
Figure 1  Transmission electron microscopy of the outer retina at P21 in (A) Crx+/+ and (B) Crx-/- retinas. pe, pigmented epithelium. os, outer segments. is, inner segments. onl, outer nuclear layer with photoreceptor nuclei. Scale bar = 5 μm for A and B.
Figure 2  Transmission electron micrograph of the outer segment layer of Crx-/- retina at P21. Inner segments of Crx-/- photoreceptors exhibit numerous mitochondria (m indicated by arrow) as in Crx+/+ (Figure 1A). pe, pigmented epithelium. is, inner segments. onl, outer nuclear layer. Scale bar = 2 μm. Photoreceptor inner segments and outer segments are joined by a non-motile connecting cilium that exhibits a characteristic 9 + 0 arrangement of microtubule doublets when viewed in cross-section. At P21, in Crx-/- retinas, numerous cross sections of connecting cilia were noted (Figure 3A and 3B). Sporadically, connecting cilia contained other than the typical complement of microtubule doublets. For example, in Figure 3A, the connecting cilium labelled by arrowhead 1, shows 7 + 0 doublets. The majority exhibited the characteristic 9 + 0 doublets (arrowhead 2 and 3 in Figure 3A and Figure 3B). These observations indicate that in addition to inner segment formation, ciliogenesis is also largely intact in Crx-/- photoreceptors. Further, in Crx-/- retinas the retinal pigmented epithelium (PE) appeared normal, at least up to P21 (data not shown), the oldest age examined.
Figure 3  Transmission electron micrograph of Crx-/- retina at P21 (A and B), and scanning electron micrograph of Crx-/- at P10 (C) of outer segment layer. (A) Evidence of ciliogenesis in the photoreceptor layer of Crx-/- retina. Nonmotile connecting cilia were observed in cross section (arrowheads 1,2, and 3, for examples). Connecting cilium 1 (arrowhead 1) demonstrated seven microtuble doublets, while cilium 2 and cilium 3 exhibited nine. In A, a displaced cell nucleus (n) appearing pyknotic and abnormal deposition of matrix (mx) material of unknown identity were seen, along with large amounts of membranous vesicles (arrow) which filled the photoreceptor space and appeared to be released from inner segments. Scale bar = 3.7 μm. (B) Nonmotile connecting cilium in cross section, from a Crx-/- photoreceptor, demonstrating characteristic 9+0 radial array of microtubule doublets. Scale bar = 88 nm. (C) Scanning electron micrograph (SEM) of membranous vesicles (arrow shows one example) shed from inner segments of Crx-/- photoreceptors at P10. Figure shows inner segments viewed from the scleral side with the pigmented epithelium removed. Scale bar = 1 μm. In addition to the complete absence of outer segments, Crx-/- retinas exhibited three other notable pathologies in the outer segment layer. First, an abnormal deposition of matrix of unknown identity was noted (Figure 3A, mx). Second, sporadically displaced nuclei were found residing in the space abutting the PE. Occasionally, these nuclei appeared pyknotic (Figure 3A, n); but, more frequently exhibited the heterochromatin pattern typical of photoreceptors (data not shown), strongly suggesting that they belonged to ectopic photoreceptors. The third pathological entity noted in the outer segment layer were numerous small vesicles (Figure 3A arrow) 100 to 200 nm in diameter. They appeared to be emerging from the inner segments, as scanning electron microscopic images showed spherical structures budding from the inner segments (Figure 3C, arrow).
In order to characterize further the morphogenesis of Crx-/- photoreceptors, the developing outer segment layer was viewed by scanning electron microscopy at P7, P14 and P21 (Figure 4). In Crx+/+ retinas, photoreceptor inner segments, connecting cilia, and the first rudimentary outer segment structures were noted at P7. In the Crx-/- retina, only an occassional connecting cilium was noted emerging from inner segments at this stage (Figure 4A and 4B). This observation was confirmed by comparison with transmission electron micrographs (Figure 5). These differences are the earliest noted differences between Crx+/+ and Crx-/- photoreceptors. At P14, elongating outer segments were noted on Crx+/+ photoreceptors, occasionally demonstrating a paddle-like structure at their apex (Figure 4C, os). In Crx-/- retinae, the vast majority of photoreceptors at this stage demonstrated connecting cilia without outer segments (Figure 4D, cc). Sporadically, Crx-/- photoreceptors would exhibit an irregular structure extending from a connecting cilium (Figure 4D, cc*) perhaps representing a malformed outer segment. Such structures were also observed at P21 (Figure 4F, cc*). These putative, abnormal outer segments were only rarely noted in Crx+/+ at P14, and never at P21 (Figure 4C and 4E, cc*). Further, in Crx-/- photoreceptors, unusually long connecting cilia were noted (Figure 4F, cc). Serial examination of Crx-/- photoreceptors at P7, P10, P14, and P21 by TEM, demonstrated a distinctive lack of any structure interpretable as orderly stacks of discs or forming discs. These data demonstrate a complete absence of normal outer segment formation in the Crx mutant mouse, and the arrest of development of the photoreceptor appendage at the elongation stage of outer segment formation.
Figure 4  Outer segment morphogenesis in Crx-/- photoreceptors. Scanning electron microscopy of developing photoreceptors viewed from the scleral side with the pigmented epithelium removed at P7, P14, and P21 for Crx+/+ (A, C, and E) and Crx-/- (B, D, F) littermates. In Crx+/+ retina, numerous connecting cilia (A, cc) were evident at P7 with rudimentary outer segments. After P7, in Crx+/+ outer segment elongation occurs. Initially, outer segments have a paddle-like structure (C, os) which is later shed (E, os). In Crx-/- photoreceptors, few connecting cilia were observed at P7 (B, cc). After P7, connecting cilia were more numerous and occasionally a malformed outer segment was noted extending from a connecting cilium (D and F, cc*). These were rarely observed in Crx+/+ and only at P14 (C, cc*). At P21, abnormally long connecting cilia are noted in Crx-/- (F, cc). Scale bars = 10 μm
Figure 5  Transmission electron micrographs of Crx-/- photoreceptors in the photoreceptor layer at P7. (A) Photoreceptor layer of Crx+/+ retina demonstrating nascent outer segment structures (arrow) emerging from photoreceptor connecting cilia (cc). (B) Crx-/- photoreceptors exhibited connecting cilia (cc) at this early stage, however, nascent outer segment structures were not observed. Scale Bar = 1 μm. Finally, the morphology of the malformed Crx-/- photoreceptors was compared to rhodopsin-/- and peripherin-/- photoreceptors. Rhodopsin and peripherin are two photoreceptor-specific genes whose expression is significantly downregulated in the Crx-/- retinae [10,34,35]. Loss of function mutations in each of these genes separately have been reported to result in a failure to elaborate outer segments [29,30]. Photoreceptors from these two mutant mice examined by SEM from the scleral side appeared highly similar to Crx-/- photoreceptors (compare Figure 4F to Figure 6A and 6B).
Figure 6  Scanning electron microscopy of peripherin-/- (A) and rhodopsin-/- (B) photoreceptors at P21, viewed from the scleral side with the pigmented epithelium removed. cc, connecting cilium. is, inner segment. Scale bar = 10 μm.