CEP290 myosin-tail homology domain is essential for protein confinement between inner and outer segments in photoreceptors

Mutations in CEP290 cause various ciliopathies involving retinal degeneration. CEP290 proteins localize to the ciliary transition zone and are thought to act as a gatekeeper that controls ciliary protein trafficking. However, precise roles of CEP290 in photoreceptors and pathomechanisms of retinal degeneration in CEP290-associated ciliopathies are not sufficiently understood. Using Cep290 conditional mutant mice, in which the C-terminal myosin-tail homology domain is disrupted after the connecting cilium is assembled, we show that CEP290, more specifically the myosin-tail homology domain of CEP290, is essential for protein confinement between the inner and the outer segments. Inner segment plasma membrane proteins including STX3, SNAP25, and IMPG2 rapidly accumulate in the outer segment upon disruption of the myosin-tail homology domain. In contrast, localization of endomembrane proteins is not altered. Trafficking and confinement of most outer segment-resident proteins appear to be unaffected or only minimally affected in this mouse model. One notable exception is RHO, which exhibits severe mislocalization to inner segments from the initial stage of degeneration. Similar mislocalization phenotypes were observed in rd16 mice. These results suggest that failure of protein confinement at the connecting cilium and consequent accumulation of inner segment membrane proteins in the outer segment combined with insufficient RHO delivery is part of the disease mechanisms that cause retinal degeneration in CEP290-associated ciliopathies. Our study provides insights into the pathomechanisms of retinal degenerations associated with compromised ciliary gates.


INTRODUCTION
Photoreceptor cells in the retina are highly compartmentalized neurons. While proteins involved in the phototransduction cascade are confined to a compartment called the outer segment, ones responsible for energy metabolism and protein/lipid synthesis are confined to another compartment, the inner segment. Proteins that constitute the outer segment are synthesized in the inner segment and transported to the outer segment through a narrow channel, called the connecting cilium. The outer segment is a primary cilium-related compartment and the connecting cilium is equivalent to the transition zone in primary cilia. Since connecting cilia are the only conduit between the inner and the outer segments, they are situated at a critical place to control protein movement between the two compartments and maintain compartment-specific protein localization.
Although outer segments share many features with primary cilia, they have several unique features that are not observed in primary cilia (reviewed in [1]). For example, outer segments are much larger than primary cilia, accounting for 25-60% of the total cell volume depending on cell types (rod vs. cone) and species. Outer segments are also filled with membranous structures called discs. The large size and high membrane content of the outer segment provides a space to accommodate a large amount of transmembrane and lipidated proteins that constitute the phototransduction cascade. In addition, outer segments are constantly and rapidly regenerated: old discs are shed from the distal end of the outer segment and new discs are formed at the base [2]. Constant and rapid regeneration of a large organelle necessitates a high volume of lipid and protein transport through the connecting cilium. At the same time, proteins that are not authorized to pass the connecting cilium (in either direction) should be kept in their designated compartments. Several proteins at the connecting cilium are thought to organize a gate to control protein movement between the inner and the outer segments [3].
CEP290 is one of such proteins. In model organisms Chlamydomonas reinhardtii and Caenorhabditis elegans, CEP290 is found at the ciliary transition zone and required to build Y-shaped linkers that extend from the axonemal microtubules to the ciliary membrane [4][5][6]. Although cilia/flagella still form in the absence of CEP290 in these organisms, ciliary protein compositions are altered [4,6]. In mammalian primary cilia, CEP290 is localized to the transition zone [7][8][9], and loss of CEP290 reduces ARL13B and ADCY3 levels within cilia while increasing the rate of ciliary entry of SMO in fibroblasts [10]. These studies establish the current model for CEP290 function: a ciliary gatekeeper that regulates protein trafficking in and out of the ciliary compartment at the transition zone [4][5][6][7][10][11][12][13]. In photoreceptors, CEP290 is localized to the connecting cilium [14,15] and expected to control protein movement between the inner and the outer segments.
Mutations in human CEP290 cause various ciliopathies ranging from isolated retinal dystrophy (e.g. Leber congenital amaurosis (LCA)) to syndromic diseases such as neonatal lethal Meckel-Gruber Syndrome (MKS) with multi-organ malformations [16][17][18][19][20][21][22]. Despite considerable variations in phenotypic severity, retinopathy is present in almost all cases regardless of the involvement of other organs. This suggests that photoreceptors are particularly susceptible to deficiencies in CEP290 function. Based on the ciliary gatekeeper model, anticipated functions of CEP290 in photoreceptors include i) permitting or facilitating entry of outer segment-bound proteins into the outer segment, ii) blocking unauthorized entry of inner segment proteins into the outer segment, and iii) preventing diffusion of outer segment proteins into the inner segment. In line with the first function listed above, Cep290 mutant mice fail to develop outer segments. The connecting cilium and the outer segment are entirely absent in Cep290 null mice [15]. Partial loss of CEP290 function in rd16 mice, which have an in-frame deletion of exons 36-40 (based on Reference Sequence transcript NM_146009), allows formation of membrane-bound connecting cilia, but outer segments are rudimentary and severely malformed [14,15,23]. These studies show that CEP290 is essential for outer segment biogenesis and, either directly or indirectly, required for the trafficking of outer segment proteins.
However, precise roles of CEP290 in photoreceptors and disease mechanisms that induce retinal degeneration in CEP290-associated ciliopathies are not sufficiently understood. This is partly because of the critical requirement of CEP290 for the outer segment biogenesis in mouse models. As described above, Cep290 mutant mice have no or only rudimentary outer segments [14,15,23]. While these phenotypes demonstrate the requirement of CEP290 for the outer segment biogenesis, complete lack or severe malformation of the outer segment precludes further investigation of CEP290's role in protein trafficking and confinement between the inner and the outer segments. For instance, although CEP290 is likely required for the trafficking of at least certain outer segment proteins, specifically which proteins require CEP290 is unclear. Requirement of CEP290 for protein confinement between the inner and the outer segments has not been demonstrated either. Contrary to the findings in mouse models, Cep290 (or the C-terminal half of Cep290) does not appear to be essential for outer segment biogenesis in zebrafish [24]. In cep290 fh297/fh297 mutants, which have a nonsense mutation (p.Gln1217X) near the middle of the reading frame, retinas develop normally during embryogenesis. In addition, retinal degeneration is slow and limited to cones in this model. Interestingly, although RHO mislocalization is detected at 6 months post fertilization, degeneration of rods is not observed. Apart from RHO mislocalization, obvious signs of disrupted ciliary trafficking (e.g. accumulation of vesicular materials near the ciliary base in electron microscopy) are also not observed in this model. Therefore, precise roles of CEP290 including its gating functions remain to be determined in photoreceptors.
In this work, we sought to test the current model of CEP290 function as a ciliary gatekeeper in photoreceptors and advance our understanding of the pathomechanisms underlying CEP290associated retinopathies. We avoided the aforementioned limitations of the CEP290 mouse models with constitutive mutations by using a model with a conditional allele and disrupting CEP290 functions after the connecting cilium assembly. In addition, we reasoned that disruption of ciliary gates would have a profound impact on the localization of inner segment membrane proteins, because of the physical properties of the outer segment (i.e. large size, high membrane content, and continuous disc renewal) and its tendency to house membrane proteins [1,25]. Therefore, we examined (mis)trafficking of not only outer segment-resident proteins but also various inner segment membrane proteins. severely truncated proteins. In mouse Cep290, we noticed that omitting exon 36 in the Cep290 D allele prevents frameshift ( Figure 1A). To test whether basal exon skipping or nonsense-associated altered splicing occurs, we analyzed Cep290 mRNAs from Cep290 fl/fl ;Creand Cep290 fl/fl ;Cre + retinas. Reverse transcription PCR (RT-PCR) with primers specific to exons 34 and 41 (forward and reverse primers, respectively) produced a single fragment in controls but 3 fragments in Cep290 fl/fl ;Cre + retinas ( Figure   1C). Sequence analyses of the PCR products revealed that the largest fragment (F1; 1224 bp) was derived from unexcised alleles and contained all exons between exons 34 and 41 ( Figure 1D). F2 (872 bp), which was the main PCR product from the Cep290 fl/fl ;Cre + retinas, contained exons 35, 36, and 39 but not 37 and 38. This indicates that F2 is derived from the Cep290 D allele. F3 (672 bp) was devoid of exon 36 in addition to exons 37 and 38, indicating that F3 was derived from the Cep290 D allele but exon 36 was skipped during splicing. Since no smaller PCR product was detected in control retinas, our data suggest that exon 36 skipping is a result of nonsense-associated altered splicing rather than basal exon skipping. Loss of exons 36-38 (i.e. 552 bp) causes an in-frame deletion of 184 amino acids (~20 kDa), which are part of the region deleted in rd16 mutants ( Figure 1E). RT-PCR data also suggest that CREmediated excision of exons 37 and 38 occurs before P21 in the vast majority of rods. In addition, it is noteworthy that F2 is at least 12-fold more abundant than F3 but the quantity of protein products derived from these transcripts (black and red arrowheads in Figure 1B) are comparable. Therefore, the nearfull-length in-frame deletion mutant (p.D1606_K1789del) appears to be significantly more stable than the truncated mutant (p.L1673HfsX6). In summary, our data show that Cep290 D is a hypomorphic allele encoding two species of mutant proteins, in which the myosin-tail homology domain is disrupted.

Localization of outer segment-resident proteins in Cep290 fl/fl ;Cre + retinas
The myosin-tail homology domain of CEP290 was previously shown to have a microtubule-binding activity [32] and interact with Raf-1 kinase inhibitory protein (RKIP) [33]. CEP290-associated ciliopathy patients with mutations in this domain present with more severe phenotypes than predicted by a model based on the total quantity of full-length or near-full-length CEP290 proteins [29]. In addition, disruption of this domain in rd16 mice causes outer segment biogenesis defects and rapid degeneration of photoreceptors [14]. These findings suggest that the myosin-tail homology domain is essential for CEP290's function and possibly involved in the trafficking and confinement of outer segment proteins. Therefore, we examined localization of outer segment-resident proteins in Cep290 fl/fl ;Cre + conditional mutant mice.
To this end, we probed localization of RHO, PRPH2, ROM1, ABCA4, PDE6B, GUCY2D, ATP8A2, and CNGA1 in Cep290 +/fl ;Cre + (control) and Cep290 fl/fl ;Cre + retinas. Whilst RHO localizes to both disc membranes and outer segment plasma membranes [34, 35], PRPH2, ROM1, ABCA4, GUCY2D, and ATP8A2 specifically localize to disc membranes [36][37][38][39][40][41]. CNGA1 specifically localizes to the outer segment plasma membrane [42], which is contiguous with the inner segment plasma membrane (hereafter our use of the term inner segment encompasses all parts of photoreceptors including ellipsoid, myoid, soma, axon, and synaptic terminal but excluding the outer segment). At P11, when the connecting cilium assembly is completed and the outer segment is rapidly growing [43], no significant differences were observed between control and Cep290 fl/fl ;Cre + retinas with respect to the localization of outer segment-resident proteins, including RHO (Supplementary Figure S1). At P20, most outer segment-resident proteins examined still showed normal localization to the outer segment (Figure 2).
RHO was the only exception and significant mislocalization was observed in inner segments (Figure   2A), indicating that RHO mislocalization is one of the earliest events that occur upon disruption of the myosin-tail homology domain.
To test whether outer segment protein localization deteriorates as photoreceptors degenerate, we examined the localization of the above 8 proteins at P40 (Figure 2). Twenty days (from P20 to P40) is sufficient for the outer segment to renew entirely in mouse retinas [2], and approximately 30-40% of photoreceptors were lost by P40 in Cep290 fl/fl ;Cre + retinas. However, no significant mislocalization of PRPH2, ROM1, ABCA4, PDE6B, GUCY2D, ATP8A2, and CNGA1 was observed. In addition, the length of the outer segment was not significantly reduced despite the progression of degeneration and cell loss. This observation sharply contrasts with the rapid shortening of the outer segment in Ift88 fl/fl ;Cre + mice, in which intraflagellar transport (IFT) to maintain the outer segment is ablated, between P22 (when RHO mislocalization is first noticeable) and P40 (red bracket; Figure 2I and J). Therefore, most outer segment proteins except RHO appear to be properly transported and confined to the outer segment in Cep290 fl/fl ;Cre + rods.

Accumulation of inner segment membrane proteins in the outer segment upon disruption of the CEP290 myosin-tail homology domain
We then examined whether disruption of the myosin-tail homology domain affected confinement of inner segment membrane proteins. STX3 is a SNARE (Soluble N-ethymaleimide-sensitive factor Attachment protein REceptor) protein with a single transmembrane domain at its C-terminus and facilitates membrane/vesicle fusion for exocytosis. In normal photoreceptors, STX3 is found in the plasma membrane throughout the inner segment but not in the outer segment [44,45] (Figure 3A; left). Since STX3 and its interacting partner STXBP1 mislocalize to the outer segment in Bardet-Biedl syndrome (BBS) mutant retinas [45-47], we speculated that these proteins might need an intact ciliary gate for their inner segment-restricted localization. Indeed, STX3 and STXBP1 were found mislocalized to outer segments in Cep290 fl/fl ;Cre + retinas at P20 (Figure 3A and B; also see Figure 3E for quantification).
These results prompted us to examine the localization of other inner segment-specific membrane proteins. SNAP25 and VAMP2 are SNARE proteins that interact with STX3. Although both SNAP25 and VAMP2 are restricted to the inner segment, SNAP25 is a t-SNARE protein localizing to the plasma membrane while VAMP2 is a v-SNARE protein present on secretory vesicles. Also, while SNAP25 is almost evenly distributed throughout the inner segment, VAMP2 is highly enriched at synaptic terminals  HCN1 (K + /Na + hyperpolarization-activated cyclic nucleotide-gated channel 1) and ATP1A3 (Na + /K +transporting ATPase1 subunit a3) are transmembrane proteins localizing to the inner segment plasma membrane [53-55]. Within the inner segment, HCN1 and ATP1A3 are enriched within the myoid and ellipsoid zones (Figure 4B, C, and F). Mild mislocalization of HCN1 was observed throughout the outer segment in Cep290 fl/fl ;Cre + retinas. ATP1A3 also exhibited mild mislocalization to outer segments but there was a considerable cell-to-cell variation with respect to the severity of mislocalization. SYP (synaptophysin) and LAMP1 (lysosome-associated membrane glycoprotein 1) are integral membrane proteins that specifically localize to synaptic vesicles and lysosomes, respectively. As expected, SYP was found at synaptic terminals and LAMP1 was in the ellipsoid zone in normal photoreceptors.
Localization of these endomembrane proteins was not altered in Cep290 fl/fl ;Cre + retinas ( Figure 4D and To test whether mislocalization of inner segment proteins becomes more prevalent or severe as retinal degeneration progresses, we examined the localization of the aforementioned proteins at P40 (Figures Stx3 and Stxbp1 are expressed in not only rods but also cones and residual signals in the synaptic terminals (which are included in quantification as a part of the inner segment) are mostly from cones, in which Cre is not expressed (see Supplementary Figure S3). SNAP25 showed a moderate increase in mislocalization at P40, but the signal intensity in the outer segment did not exceed that in the inner segment. Mislocalization of other inner segment proteins was not appreciably exacerbated from what was observed at P20. These data show that the myosin-tail homology domain of CEP290 is essential for confinement of inner segment membrane proteins. Our data also suggest that proteins on the plasma membranes are primarily subject to diffusion and that STX3 and STXBP1 are particularly susceptible to accumulation in the outer segment. (control) and Cep290 fl/fl ;Cre + mice indicated that there was a slight to moderate (12-25%) reduction of photoreceptor-specific proteins (RHO, GRK1, PRPH2, and PDE6A) in Cep290 fl/fl ;Cre + retinas ( Figure   5A). This is consistent with some loss of photoreceptors by this age. BBSome components (BBS2 and BBS7) and LZTFL1 showed similar levels of reduction. These data suggest that reduction of BBS proteins is proportionate to the loss of photoreceptors and that BBS protein levels are not affected by impaired CEP290 functions in Cep290 fl/fl ;Cre + retinas. We then examined BBSome assembly by immunoprecipitation. As shown in Figure 5B, all BBSome components tested were similarly pulled down by BBS7 antibodies in control and Cep290 fl/fl ;Cre + retinas. These data indicate that BBSome assembly is not altered in Cep290 fl/fl ;Cre + retinas.

Mislocalization of inner segment membrane proteins in
To assess the quantity of BBS proteins within the outer segment, we isolated outer segments from Cep290 +/fl ;Cre ? and Cep290 fl/fl ;Cre + retinas and conducted immunoblot analyses ( Figure 5C). After normalization to total protein quantities, no significant differences were observed between normal and CEP290-deficient outer segments with respect to the quantity of outer segment-resident proteins.
BBSome components, BBS2 and BBS7, also showed no significant differences. Interestingly, however, there was a more than 2-fold increase in LZTFL1 quantity in outer segments from Cep290 fl/fl ;Cre + retinas.
Consistent with the mislocalization of STX3 and STXBP1 in the outer segment, there was a large increase of these proteins in Cep290 fl/fl ;Cre + outer segments. SNAP25 showed ~3 fold increase in Cep290 fl/fl ;Cre + outer segments.
We then examined the localization of BBS proteins in normal and Cep290 fl/fl ;Cre + photoreceptors. While CEP290 is localized to the connecting cilium, BBS5 was previously shown to localize along the axoneme in the outer segment [67]. BBS5 was also detected within the connecting cilium at a lower intensity. Very similar localization patterns were observed with BBS8 and LZTFL1 (Figure 5D and E).
LZTFL1 immunoreactivity was also detected around the basal body but this staining persisted in Lztfl1 mutant [45] retinas, indicating that signals around the basal bodies are from cross-reacting protein(s) ( Figure 5E; arrowheads). Localization of BBS8 and LZTFL1 in Cep290 fl/fl ;Cre + retinas was comparable to that of normal photoreceptors (Figure 5D and E). These data indicate that, despite some quantitative changes in LZTFL1 in the outer segment, overall localization patterns of the BBSome and LZTFL1 are not altered in Cep290 fl/fl ;Cre + retinas. Therefore, we conclude that the accumulation of STX3 and STXBP1 in the outer segment in Cep290 fl/fl ;Cre + retinas is unlikely due to alterations in BBSome functions.

Accumulation of inner segment membrane proteins in the outer segment in rd16 mice
We next examined whether similar protein mislocalization was observed in rd16 mice ( Figure 6). As previously described [14], retinal degeneration in rd16 mice was already evident at P15 and outer segments were rudimentary, suggesting that outer segment biogenesis is severely impaired in rd16 mice. RHO mislocalization was also evident in the inner segment ( Figure 6B). In addition, presumably due to the outer segment biogenesis defect, low level mislocalization of outer segment proteins was detected in general. However, the highest immunoreactivity of all outer segment-resident proteins examined was detected in the outer segment, suggesting that these proteins are still delivered to the outer segment in rd16 mice. Mislocalization of CNGA1 was relatively obvious compared to other outer segment proteins and predominantly restricted to the distal (ellipsoid) portion of the inner segment as opposed to being dispersed throughout the inner segment like RHO. (Figure 6H).
We then examined the localization of inner segment membrane proteins in rd16 mice. Proteins that exhibited significant mislocalization in Cep290 fl/fl ;Cre + retinas (STX3, STXBP1, SNAP25, and IMPG2) all showed obvious mislocalization to the outer segment in rd16 retinas (Figure 6). In contrast, localization of VAMP2, ATP1A3, HCN1, SYP, and LAMP1 was not noticeably altered in rd16 retinas Our study shows that the myosin-tail homology domain is essential for CEP290's function as a ciliary gatekeeper, particularly to prevent diffusion of inner segment membrane proteins into the outer segment.
A subset of inner segment membrane proteins rapidly accumulate in the outer segment upon disruption of the myosin-tail homology domain. STX3 and STXBP1, in particular, show striking accumulation in outer segments as degeneration progresses. Other inner segment membrane proteins tested show various degrees of accumulation in the outer segment. SNAP25 and IMPG2 exhibit significant accumulation in the outer segment but their density in the outer segment does not exceed that in the inner segment. It is tantalizing to speculate that there are additional factor(s) that induce STX3 and STXBP1 enrichment in the outer segment. Mislocalization of ATP1A3 and HCN1 is mild, and localization of VAMP2, LAMP1, and SYP is not affected by the loss of CEP290 myosin-tail homology domain.
In contrast, the myosin-tail homology domain appears to be dispensable for the trafficking and confinement of most outer segment-resident proteins except RHO. In rd16 mice, the myosin-tail homology domain is constitutively disrupted and the outer segment biogenesis is severely impaired.
Most outer segment proteins, however, manage to be delivered to the rudimentary outer segment. The low level mislocalization of outer segment proteins is likely secondary to the outer segment biogenesis defects including small size and disorganized disc structures. In Cep290 fl/fl ;Cre + retinas, in which the myosin-tail homology domain is disrupted after the connecting cilium is formed, mislocalization of outer segment-resident proteins is not observed except for RHO. Furthermore, the length of the outer segment does not change significantly for 20 days while degeneration is progressing. Zebrafish cep290 fh297/fh297 mutants, which have a nonsense mutation (p.Q1217*) before the myosin-tail homology domain, exhibit partial mislocalization of RHO but not of rhodopsin kinase GRK1 or a tranducin subunit GNB1 at 6 months post fertilization [24]. Although degradation of mis-trafficked proteins in the inner segment could contribute to the apparent lack of mislocalized proteins, these data suggest that most outer segment proteins do not require the myosin-tail homology domain for their outer segment-specific localization. It remains to be determined whether the residual part of CEP290, which is expressed in  Figure S2B). These data suggest that IMPG2 immunoreactivity detected by our IMPG2 antibody in the retina is from full-length IMPG2 in the secretory pathway (including endoplasmic reticulum and Golgi) or the C-terminal cleavage product that localizes to the plasma membrane. In contrast, localization of endomembrane proteins (VAMP2, SYP, and LAMP1) is not affected by the loss of the myosin-tail homology domain. These proteins are actively sorted and targeted to their destinations upon synthesis and during recycling, and therefore not subject to diffusion along the plasma membrane. ATP1A3 and HCN1 localize to the inner segment plasma membrane but exhibit only mild mislocalization. We speculate that these proteins may be targeted and retained in the inner segment plasma membrane by unknown mechanisms. Indeed, contrary to STX3, STXBP1, and SNAP25, which are evenly distributed throughout the inner segment in normal photoreceptors, ATP1A3 and HCN1 are significantly enriched in the myoid and ellipsoid zones, implying that they do not freely diffuse within the inner segment. Taken together, our data suggest that a subset of inner segment plasma membrane proteins are liable to mislocalization to the outer segment upon disruption of the myosin-tail homology domain.
Based on our study, we propose that failure of protein confinement at the connecting cilium and consequent accumulation of inner segment plasma membrane proteins in the outer segment combined with insufficient RHO delivery underlies retinal degeneration in CEP290-associated ciliopathies (Figure   7). In normal photoreceptors, CEP290 is a part of the ciliary gate that confines inner segment plasma membrane proteins to the inner segment. The myosin-tail homology domain is crucial for this function.
CEP290 is also required for the trafficking and/or confinement of RHO to the outer segment. In photoreceptors with compromised CEP290 functions, ciliary gates are impaired, allowing diffusion of select inner segment plasma membrane proteins into the outer segment. Abundance of membranes in the outer segment is likely to contribute to the accumulation of inner segment membrane proteins.
Perhaps, accumulation of inner segment membrane proteins in nascent discs combined with insufficient delivery of RHO disturbs the disc morphogenesis process and causes or contributes to the outer segment biogenesis defects observed in rd16 mice. Inner segment mislocalization of RHO is likely another key pathomechanism that triggers photoreceptor death, as mutations disrupting RHO trafficking commonly cause retinal degeneration ( [68] and references therein). Precise roles of CEP290 for outer segment protein trafficking and confinement as well as the mechanisms by which the myosin-tail homology domain blocks inner segment membrane proteins remain to be determined. It also should be noted that both CEP290 mouse models used in the present study possess hypomorphic alleles not a null. Cep290 null mutants completely lack the connecting cilium and the outer segment [15]. Therefore, although not fully understood, one should bear in mind that CEP290 has additional roles in the connecting cilium biogenesis and that the N-terminal 2/3 of CEP290 might be necessary for outer segment protein trafficking and confinement. Finally, ciliary gates are composed of multiple proteins of the "NPHP" and "MKS modules" [5][6][7][8]69]. Loss-of-function mutations in these genes/proteins commonly cause retinal degeneration. Similar pathomechanisms may underlie retinal degenerations in these diseases. Pde6b +/+ ;Crb1 +/+ in B6;129S6 mixed backgrounds. Cep290 rd16 mice were maintained in a BXD;B6 mixed background to obtain heterozygous littermates (i.e. Cep290 +/rd16 ) as a control. All primers for genotyping were from Integrated DNA Technologies and their sequences are described in Table 1. All animals were maintained in 12-hour light/dark cycles and fed ad libitum standard mouse chow. PCR protocols are available upon request.

RNA extraction and Reverse Transcription (RT)-PCR
Mice were euthanized by CO2 asphyxiation followed by cervical dislocation. Eyes were enucleated and the anterior segment was removed using micro-dissecting scissors. The neural retina was separated from the rest of the ocular tissues with forceps and snap-frozen in liquid nitrogen. Upon completion of retina collection, frozen retinas were placed on ice, immersed in 1 ml of TRI Reagent (Sigma), and homogenized with PT1200E Polytron homogenizer (Kinematica). Total RNAs were extracted following the manufacturer's instruction. One µg of total RNA was used for cDNA synthesis using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) and random hexamers (Thermo Fisher Scientific) following the manufacturer's instruction. Cep290 cDNA fragments between exons 34 and 41 were PCR amplified with Universe High-Fidelity Hot Start DNA polymerase (Bimake) and two primers (F-Cep290-e34: 5'-GCCGAAATCTCATCACACAATG-3' and R-Cep290-e41: 5'-GCTTCTCCTTCCTTCTCCTTTAG-3'). PCR products were separated in a 1.2% agarose gel and purified with a Gel/PCR DNA Fragment Extraction kit (IBI Scientific). Purified DNAs were sequenced by Sanger sequencing using the aforementioned two primers (F-Cep290-e34 and R-Cep290-e41).

Immunofluorescence microscopy
After euthanasia by CO2 asphyxiation followed by cervical dislocation, mouse eyes were enucleated and immersed in 4% (wt/vol) paraformaldehyde (PFA)/PBS fixative. A small puncture was created between the lens and the sclera using a 26-G needle. After 5 minutes of fixation, the lens and the anterior chamber were removed with micro-dissecting scissors. The eye-cups were further fixed in 4% PFA/PBS for 3 hours at 4 °C. After washing with PBS (3 times, 10-min each), eye-cups were infiltrated and embedded in acrylamide as previously described [71]. using ImageJ to calculate the ratio of fluorescence intensities in the outer segment. Two-tailed, twosample t-tests assuming unequal variances were used for statistical analyses. P values smaller than 0.01 were regarded as statistically significant.

Outer segment isolation and immunoblotting
Photoreceptor outer segments were isolated as previously described [45]. Briefly, eyes were enucleated from Cep290 +/fl ;Cre ? and Cep290 fl/fl ;Cre + mice at P25-28, and the anterior segment and the lens were removed using micro-dissecting scissors. The neural retina was separated from the pigmented retinal epithelium with forceps, collected in 63% sucrose/PBS, snap-frozen in liquid nitrogen, and stored in -80 °C until needed. Fifteen to twenty animals were used per genotype per preparation. Retinas with same genotypes were pooled in 1.5-ml tubes on ice and 63% sucrose/PBS was added to 1 ml. Retinal suspensions were gently pipetted up and down 10 times using a P1000 tip with a 1.5-2 mm orifice, and further vortexed for 30 seconds. Homogenates were centrifuged at 100 x g for 3 minutes, and supernatants were transferred to fresh 1.5-ml tubes and spun at 2,350 x g for 10 minutes at 4 °C.
Supernatants were transferred to ultracentrifuge tubes, and layers of 42%, 37%, and 32% sucrose/PBS solutions (1.1 ml each) were overlaid. Homogenates were centrifuged at 116,000 x gave for 1 hour at 4°C using a Sorvall TH-660 rotor. Outer segments were collected at the 32%-37% sucrose interface, and an equal volume of ice-cold PBS was added to the collected outer segment fractions.

Antibodies
The following primary antibodies were used for immunofluorescence microscopy and immunoblotting: