bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Ift172 conditional knockout mice exhibit rapid retinal degeneration and
protein trafficking defects
Priya R Gupta1,2, Nachiket Pendse1, Scott H Greenwald1, Mihoko Leon1, Qin
Liu1, Eric A Pierce1, Kinga M Bujakowska1
1Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Department of
Ophthalmology, Harvard Medical School, Boston, Massachusetts, 02114, USA
2Weill Cornell Medical College, New York, New York, 10021, USA
Corresponding author:
Kinga M Bujakowska:
Massachusetts Eye and Ear, 243 Charles Street, Boston, MA 02114
[email protected], Tel.: (617)-391-5933, Fax: (617)-573-
6901
1 bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Abstract
Intraflagellar transport (IFT) is a bidirectional transport process that occurs along
primary cilia and specialized sensory cilia, such as photoreceptor outer-
segments. Genes coding for various IFT components are associated with
ciliopathies. Mutations in IFT172 lead to diseases ranging from isolated retinal
degeneration to severe syndromic ciliopathies. In this study, we created a mouse
model of IFT172-associated retinal degeneration to investigate the ocular
disease mechanism. We found that depletion of IFT172 in rod photoreceptors
leads to a rapid degeneration of the retina, with severely reduced
electroretinography responses by one month and complete outer-nuclear layer
degeneration by two months. We investigated molecular mechanisms of
degeneration and show that IFT172 protein reduction leads to mislocalization of
specific photoreceptor outer-segments proteins (RHO, RP1, IFT139), aberrant
light-driven translocation of alpha transducin and altered localization of glioma-
associated oncogene family member 1 (GLI1). This murine model recapitulates
the retinal phenotype seen in patients with IFT172-associated blindness and can
be used for in vivo testing of ciliopathy therapies.
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INTRODUCTION
Primary cilia are non-motile microtubule-based organelles that are present in the
majority of vertebrate cell types. They are involved in cellular signaling that is
crucial for the development and functioning of most organs (1). Mutations in
genes coding for ciliary proteins lead to ciliopathies, rare genetic disorders that
may affect the retina, brain, olfactory epithelium, heart, liver, kidney, bone,
gonads and adipose tissues, in isolation or as part of specific syndromes (2–4).
Vision loss is a common feature of ciliopathies because the photoreceptor outer-
segment (OS) is a highly specialized sensory cilium that is critical for
phototransduction (5–7).
The assembly and maintenance of the cilium requires intraflagellar transport
(IFT), the bidirectional protein trafficking that occurs along the axoneme that is
necessary for the growth and maintenance of the cilium (8). IFT is dependent on
two large protein complexes: IFT-B is responsible for anterograde transport to
the ciliary tip and IFT-A is responsible for retrograde transport and return of
materials to the basal region (9). Mutations in many of these IFT components,
including IFT172, have been associated with disease (10–21).
IFT172 is a peripheral IFT-B complex protein that is thought to mediate the
transition from anterograde to retrograde transport at the tip of the cilium (22–24).
Therefore, even though it is associated with the anterograde machinery, its
temperature sensitive mutants in Chlamydomonas display phenotypes typical of
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retrograde proteins, including elongation of the cilium (24, 25). Elongation of the
cilium was also observed in cultured fibroblasts obtained from patients with
IFT172 associated disease (10). In contrast, node cells of mouse embryos
harboring a homozygous p.Leu1564Pro mutation in Ift172 (wim mutants) lacked
all cilia (26). Mice homozygous for loss of function Ift172 alleles wim and slb, or
the hypomorphic avc1 allele, die in utero or at birth respectively (26–28),
therefore photoreceptor degeneration in a murine Ift172 model has not been
described to date. However, homozygous ift172 zebrafish mutants have
abnormal photoreceptor development, where photoreceptor cilia fail to extend, in
addition to severe multi-systemic defects (29, 30).
Given IFT172’s role in the retinal disease, we created rod photoreceptor-specific
Ift172 knock-out mice by crossing mice that have a conditional Ift172 allele (31)
with mice that express Cre driven by the Rhodopsin promoter (32). We
generated and validated an IFT172-specific antibody, which allowed us to
monitor reduction of the IFT172 protein, and to demonstrate that depletion of
IFT172 in the mouse retina causes a rapid retinal degeneration. We also show
that decreasing levels of IFT172 lead to aberrant localization of specific proteins.
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RESULTS
Targeted disruption of Ift172 in rod photoreceptors leads to Ift172 protein
depletion
A rod photoreceptor-specific Ift172 knock-out mouse was generated by crossing
mice carrying a conditional knock-out allele (Ift172fl) (31) with transgenic mice
harbouring Cre recombinase under a Rhodopsin promoter (iCre) (32) (Figure
1A). Expression of iCre in the Ift172 knockout mouse (Ift172fl/fl/iCre) was specific
to the outer nuclear layer (ONL) and expressed in the majority of the rod
photoreceptors at postnatal day 25 (PN25) (Figure 1B). iCre is necessary for
generating the Ift172 knock-out allele (Ift172fliCre) and as a consequence for
depletion of the IFT172 protein. Therefore, the time-course of IFT172 protein
depletion was monitored by immunostaining of mouse retinas with a specific anti-
IFT172 antibody, developed to the C-terminus of the protein (Figure 1C).
Complete depletion of the IFT172 protein in rod photoreceptors was observed at
PN28 (Figure 1D).
IFT172 deficient mouse retinas degenerate rapidly
Retinas of the Ift172fl/fliCre mice showed thinning of the ONL at one month and
complete degeneration of the ONL by two months, whereas age-matched
littermate controls did not show this phenotype (Figure 2A-C, Supplementary
Figure 1). Electroretinography (ERG) testing concordantly demonstrated that at
one month Ift172fl/fliCre mice showed a significantly reduced (84% reduction) rod-
driven b-wave amplitude (32.3 µV ±12 µV) compared to the wild-type controls
(196.9 µV ±12 µV). Mixed rod/cone and cone-isolated responses were reduced
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by half at the same age, indicating a secondary cone degeneration (Figure 2D).
By two months of age, the ERG was undetectable in Ift172fl/fliCre mice across the
stimulus conditions (Figure 2D). Mice homozygous for the floxed allele (Ift172fl/fl)
or heterozygous retina specific knock-out mice (Ift172wt/fliCre) were followed up to
6 months and maintained normal retinal function and structure (Figure 2D,
Supplementary Figure 1).
Ultrastructural analyses of the retina in the Ift172fl/fliCre mice showed no signs of
photoreceptor degeneration at PN21 (Figure 3). However, at PN25, shortening of
the OS was observed, and at PN28, OS disc disorganization and accumulation of
extracellular debris was seen (Figure 3). At these stages, no changes in
axoneme length were observed. At PN31 the outer-segments were disorganized
and they appeared to have lost the connection with the axoneme and the inner
segment of the photoreceptor cell (Figure 3). Based on these results, further
studies were performed on mice up to PN28.
Depletion of IFT172 leads to mislocalization of Rp1 and Ift139
Disruption of the outer-segment structure led us to investigate the localization of
the axoneme-associated proteins RP1 and acetylated tubulin (acTub)(33). AcTub
showed no differences in localization between the mutant and the control retinas
up to PN28. RP1 staining, however, showed shortening of the signal relative to
acTub at PN28 in mutant mice compared with the controls (Figure 4A), and it
was observed to mislocalize to the synaptic terminals (Figure 4B).
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Since IFT172 is thought to be involved in the switching between the anterograde
and retrograde transport, mislocalization of the IFT components is expected. For
example, before the complete cessation of IFT, accumulation of the IFT particles
at the tip of the cilium would be likely, as seen in Chlamydomonas (24, 25). We
therefore tested localization of two components of the retrograde IFT (IFT43 and
IFT139) and a Bbsome protein (Bbs9). Of these three, only IFT139 showed
mislocalization at the synaptic terminals of rods, however no changes in location
at the axoneme were seen (Figure 4B, Supplementary Figure 2).
Mislocalization of photoreceptor outer segment proteins in Ift172fl/fliCre
mice
Depletion of a component of the intraflagellar transport machinery is expected to
affect IFT overall and thus alter trafficking of the outer-segment proteins.
Localization of four different outer-segment proteins was investigated: two
phototransduction proteins dependent on IFT (rhodopsin (Rho) and guanylate
cyclase-1 (Gc-1)) (34–36), one protein supporting disk structure (peripherin
(Prph2))(37) and one phototransduction protein independent of IFT (transducin)
(38, 39). Early signs of RHO mislocalization became apparent at PN21 and
PN25, where the protein was detected around the nuclei. This pattern became
stronger at PN28 (Figure 5). Depletion of IFT172, however, had no visible effect
on localization of GC-1 and PRPH2 (Figure 5).
Localization of transducin in rods is light dependent, where in the darkness,
transducin is predominantly present in the outer-segments and upon bright light
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stimulation it is translocated to the inner-segment (40, 41). This light-driven
translocation is an adaptation of rods to be able to respond to different light
conditions and it is widely believed to occur by diffusion (38, 39, 42–44). To
establish if depletion of IFT172 had an effect on the translocation of the alpha
subunit of transducin (Gαt), we tested the localization of this protein in dark-
adapted and in photobleached retinas from Ift172fl/fliCre and Ift172fl/fl control
littermates at PN21 and PN25 (Figure 6). These two time-points were chosen
because the mice show only early signs of retinal degeneration in which
alteration of the photoreceptor structure and early signs of RHO mislocalization
(Figure 3 and 5) are mild or absent. At PN21 the location of Gαt followed the
expected pattern, with the protein detected only in the outer-segments of the
dark-adapted photoreceptors and distributed throughout the photoreceptors in
the light-adapted tissue (Figure 6A,B). At PN25 however, Gαt is detectable in the
cell bodies of the dark-adapted photoreceptors of the Ift172fl/fliCre to a greater
extent than in the Ift172fl/fl control mice, however after Bonferroni-Sidak mutli-
comparison corrections, this difference was not statistically significant (Figure 6C,
E). In the light, more Gαt protein was seen in the inner-segment of the
Ift172fl/fliCre than the control mice (Figure 6D). Quantification of the relative
fluorescence signal in the outer-segments compared to the total fluorescence of
the retina, revealed that the Gαt content in outer-segment is lower in the mutant
mice compared to the controls by 36.3% (p=0.0003) (Figure 6E).
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IFT172 deficient mouse retinas show early mislocalization of hedgehog
signaling pathway protein Gli1
Primary cilia that modulate developmental signaling events, such as Hedgehog
(Hh) signaling and all three hedgehog signaling proteins (Sonic, Desert, and
Indian), have been shown to be expressed by the retinal ganglion cells or the
retinal pigment epithelium in the developing and adult murine eye (45). Moreover,
depletion of IFT172 has been shown to affect expression of Hh components in
the developing brain (28). We therefore investigated the role of Hh signaling in
the degenerating photoreceptors due to abnormal IFT. Using
immunofluorescence analyses, we investigated the localization of smoothened
(Smo) and the glioma-associated oncogene (Gli) family members 1, 2, and 3 (46,
47). Of the four proteins, only Gli1 showed differing localization in the Ift172fl/fliCre
retinas compared to the control mice. In control mice, Gli1 was predominantly
detected in the outer segments, while in Ift172fl/fliCre mice Gli1 localized to the
inner-segments at PN25 and at PN28. At PN28, the mutant photoreceptors had a
substantially reduced expression of Gli1, as well (Figure 7). Smo localized to the
base of the photoreceptor cilia in the control and the mutant mice, Gli2 showed a
diffuse inner-segment staining, and Gli3 expression was seen in the inner
nuclear layer and ganglion cells but not in the photoreceptors (Supplementary
Figure 3).
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DISCUSSION
We have generated a murine model of retinal degeneration due to mutations in
IFT172, a gene previously implicated in a syndromic and a non-syndromic retinal
degeneration (10, 11, 21). Since general disruption of Ift172 is embryonic lethal
(27, 28), we used post-natal Cre-mediated excision of exons 2 and 3 in rods. Cre
recombinase under control of the Rho promoter (iCre) showed increasing
expression through to PN25, which led to undetectable levels of the IFT172
protein by PN28. Of note, the iCre recombinase expression was observed later
than previously reported (32), which may be due to a lower copy of the transgene
caused by numerous mouse crosses and due to chromatin modifications that
may affect the transgene expression timeline (48–50).
The mutant Ift172 mice showed a rapid retinal degeneration with severely
reduced retina thickness and ERG responses by one month and absent
photoreceptor cells by two months. The first signs of photoreceptor outer-
segment shortening were seen before the complete IFT172 depletion, at PN25
and within six days, by PN31, the outer-segments were fully degenerated. The
degeneration occurs therefore faster than the normal 10 day cycle of the outer-
segment regeneration in mice (51). This indicates that shortening of the outer-
segments is not only due to the cessation of transport of the proteins necessary
for the outer-segment regeneration, but implies an additional function of IFT in
the photoreceptors.
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Other selected outer-segment/cilia related proteins, such as RP1 and IFT139
showed mislocalization, which may indicate a general protein trafficking
disturbance in the cell. RHO accumulation in the rod cell bodies of the mutant
mice was first observed at PN21, before any visible signs of structural changes to
the outer-segments. This was the first symptom of the failing IFT machinery that
was observed in the Ift172fl/fliCre mice. RHO mislocalization has been reported in
other murine models, where anterograde complex B (Ift88) and retrograde
complex A (Ift140) genes were perturbed (35, 52). The result obtained in this
study adds further evidence supporting IFT’s role in opsin transport.
Localization of two other outer-segment proteins, GC1 and PRPH2 was not
altered in the mutant mice up to PN28 (Figure 5). PRPH2, is a structural protein,
necessary for the outer-segment disc rim formation (42). Previous studies have
indicated that PRPH2 and RHO utilize separate methods of transport to reach
the outer segment, which is in agreement with our findings (37, 42, 53). The lack
of GC1 mislocalization was, however, surprising since it requires RHO for
trafficking to the outer-segments (53). One of the reasons for this result could be
a low abundance of the GC1 protein and the low sensitivity of the
immunohistochemical assay. Of note, GC1 accumulation was also absent in the
rod cell bodies of the Rho-/- retinas (53).
Unexpectedly, decreasing levels of IFT172 affected light-driven translocation of
alpha transducin at PN25. Specifically, transducin showed increased localization
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within the inner segment and around the photoreceptor nuclei after light
adaptation (Figure 6). It is thought that light-mediated translocation of transducin
and the reciprocal translocation of arrestin occur via diffusion and thus are IFT-
independent (38–44). Therefore, it is not clear how perturbed IFT can alter
protein diffusion through the transition zone. It is plausible that at the PN25 time
point, the structure of the axoneme starts to disintegrate, forming a barrier
against diffusion. A similar conclusion has been drawn from studies in mouse
retinal explants, where drug-mediated disruption of actin filaments and
microtubules led to impaired distribution of transducin and arrestin during the
dark adaptation (54). Involvement of cytoskeleton in light-mediated protein
translocation was also observed in Xenopus laevis (55, 56). Another possible
explanation is that the transducin mislocalization observed in our model is
secondary to the impaired trafficking of rhodopsin. Current evidence suggests
that light activated rhodopsin and intact disc membranes are needed to act as
“sinks” for arrestin/transducin transport (43, 57, 58). Therefore it is plausible that
perturbations in rhodopsin transport would lead to secondary mislocalization of
transducin. The timeline of this theory also matches our findings of rhodopsin
mislocalization at PN21, preceding transducin mislocalization detectable at
PN25.
Numerous studies have shown the importance of ciliogenesis and IFT on Hh
signaling during development; however, little is known about how the Hh
signaling pathway functions in cilia after development and during degeneration
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(26, 59–63). Generally, activation of the Hh pathway in vertebrate cells involves
binding of the ligand (Sonic, Desert or Indian Hedgehog) by the Patched
(PTCH1) receptor present at the ciliary membrane and subsequent activation
and translocation of SMO to the cilium. SMO subsequently blocks GLI repressors
(mostly GLI3) and triggers GLI transcriptional activators (mostly GLI2 and GLI1),
which translocate from the cilium to the nucleus (62, 63). Double mutant mouse
experiments have demonstrated that IFT acts downstream of the Hh receptor
PTCH1 and SMO, but upstream of the GLI effectors (26, 60, 62, 63). We
therefore used immunofluorescence analyses to determine the localization of key
Hh pathway components in the degenerating retina. Lack of differences in
localization of SMO, GLI2, and GLI3 indicate that Hh pathway was not activated
in the degenerating retina of the Ift172fl/fliCre mice. Immunofluorescence analysis
of GLI1 however, gave contrary results, as this protein translocated from the
cilium to the inner segments and the peri-nuclear region of the degenerating
photoreceptors. Functional differences between GLI1 and GLI2/GLI3
transcription factors have been noted before. For example in contrast to GLI2
and GLI3, GLI1 lacks a repressor domain and homozygous Gli1 knock-out mice
develop normally (46, 64). GLI1 has also been reported to be activated in a Hh-
independent fashion (65), which is likely the case in the present study. Currently,
the role of GLI1 in developed photoreceptors is unknown. Whether the observed
translocation of GLI1 from the outer segment has a protective function or if it
enhances photoreceptor degeneration remains to be determined. Further
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research will be necessary to fully answer the question about its role in normal
and degenerating retinas.
The results of this study highlight the crucial nature of transition from anterograde
to retrograde IFT within the rod photoreceptors and expand our understanding of
IFT’s role in protein trafficking within these cells. A profound degeneration of the
outer segments occurred within just a matter of days after IFT172 was depleted,
which was shown by a thorough characterization of the degeneration between
PN21 and PN28. Phenotypic and molecular results from this time-course
analysis indicate that the hypomorphic condition present in patients with IFT172
associated retinal degeneration is most accurately modeled at PN25, when
depletion of some but not all of the IFT172 protein leads to the first signs of
photoreceptor degeneration. This model is therefore useful to understand the
IFT-associated disease and for the development of potential treatments for
patients with the IFT172-associated disease.
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MATERIALS AND METHODS
Ethics Statement: The in-vivo experiments performed on mice were performed
according to protocols approved by the Animal Care Committee from the
Massachusetts Eye and Ear. All procedures were performed to minimize
suffering in accordance with the animal care rules in our institution in compliance
with the Animal Welfare Act, the Guide for the Care and Use of Laboratory
Animals, and the Public Health Service Policy on Humane Care and Use of
Laboratory Animals.
Mouse husbandry and genotyping: Mice were housed in the animal facility of
the Schepens Eye Research Institute, provided a standard rodent diet and
housed in standard 12 hour alternating light/dark cycles.
Genomic DNA was isolated from ear biopsies (Allele-In-One reagent, Allele
Biotechnology, San Diego, CA, USA). Two genotyping PCR reactions (Hot
FirePol DNA polymerase, Solis Biodyne, Tartu, Estonia) were performed for the
Ift172 allele (F: 5’-GAAGAGTTGGGTGTAAGAAATGC -3’ and R: 5’-
CTGGAGCTACATCAAAGACAG-3’) and the iCre allele (F: 5’-
TCAGTGCCTGGAGTTGCGCTGTGG-3’ and R: 5’-
CACAGACAGGAGCATCTTCCAG-3’). Both PCR reactions were performed in
0 2.0mM MgCl2 with the thermocycling protocol as follows: 95 C for 15 minutes; 35
cycles of 940C for 30 seconds, 600C for iCre/620C for Ift172 for 30 seconds; 720C
for 1 minute; final extension: 720C for 5 minutes. Gel electrophoresis was
performed on the PCR products. The Ift172 floxed allele produced a 380 base
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pair band, whereas the wild type ift172 allele produced a 176 base pair band.
Presence of the iCre allele produced a 200 base pair band.
Electroretinography (ERG): Full-field ERGs were performed on mice at 1, 2, 3
and 6 months of age to assess rod and cone photoreceptor function (66). Mice
were dark adapted overnight, then approximately 20 minutes before procedure
they were anesthetized by intraperitoneal injection of ketamine and xylazine
diluted in sterile saline, and the eyes were dilated by topical application of 1%
tropicamide. ERG was recorded simultaneously from both eyes in response to 4-
ms broadband stimuli with the use of gold ring electrodes (Diagnosys, Lowell,
MA) and the ColorDome Ganzfeld system (Diagnosys), as reported before (67).
In dark-adapted mice, rod-driven ERGs were recorded in response to a 0.01
cd·s/m2 light stimulus (10 flashes at 0.2 Hz), and mixed rod/cone ERGs were
recorded in response to a 10 cd·s/m2 light stimulus (three flashes at 0.03 Hz).
Next, mice were light-adapted by exposure to a steady, rod-suppressing
background light (30 cd/m2) for 10 minutes. This background light remained
during the acquisition of cone-driven ERGs that were recorded in response to a
20 cd·s/m2 light stimulus (20 flashes at 0.5 Hz). The magnitude of the ERG b-
wave was measured as the absolute voltage change from the trough of the a-
wave (or from the voltage measured at the expected implicit time of that trough,
should the a-wave be undetectable) to the b-wave peak (67).
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Optical Coherence Tomography (OCT): OCT was performed on mice at 1, 2,
and 3 months of age. After anesthesia and pupil dilation (as above), cross-
sectional retinal images were acquired with the InVivoVue OCT system
(Bioptogen, Morrisville, NC). A rectangular and radial OCT volume centered on
the optic nerve head was captured for both eyes. A B-scan located approximately
200 µm temporal and nasal of the ventral optic nerve head was selected for
measurement. Retinal thickness was measured as the distance between the
nerve fiber layer and the hyporeflective boundary between the retinal pigment
epithelium (RPE) and choroid.
Histology: The mice at 1, 2, 3 and 6 months of age were sacrificed by
transcardial perfusion (4% paraformaldehyde, approximately 30ml per mouse).
After perfusion, each eye was enucleated and placed in 4% PFA in PBS
overnight for post-fixation. After dehydration with graded ethanol solutions, the
eyes were embedded in glycol methacrylate (GMA) (Technovit 7100, Heraeas
Kulzer GmBH, Wehrheim, Germany) and 3µm thick sections were cut at the level
of the optic nerve head (LKB Historange microtome). The sections were stained
with hematoxylin and eosin and imaged by bright field microscopy (Eclipse Ti,
Nikon, Tokyo, Japan).
Immunofluorescence: The pups at ages PN18 to PN31 were sacrificed by CO2
asphyxiation and the eyes were enucleated. Since many of the cilia antibodies
are incompatible with tissue fixation, one eye was fresh frozen and one fixed. For
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the non-fixed tissue, the eye was immediately embedded (OCT reagent, Tissue-
Tek, Fisher Scientific, USA) within molds and snap-frozen using 200 proof
ethanol over dry ice. For the fixed tissues, the cornea and the lens were
dissected and the eyes were placed in 4% paraformaldehyde (PFA) for 1 hour
post-fixation, and the eyecups were cryopreserved (30% sucrose in phosphate
buffered saline buffer (PBS), overnight at 4°C) (Sigma-Aldrich, USA) and
embedded (OCT reagent). The eyes were cryosectioned into 10 µm slices and
then processed using the following procedure: blocking and permeabilization
(0.05% Triton X100, 1% Bovine Serium Albumin (BSA) in PBS, 1 hour at RT);
primary antibody staining (dilutions listed below, 0.3% Triton X100 in PBS,
overnight at 4°C); incubation of the secondary antibodies conjugated with a
fluorophore (Alexa Fluor 488, AlexaFluor 555, and AlexaFluor 647)
(ThermoFisher, Waltham, MA, USA) (1:500 dilution, 0.3% Triton X100 in PBS, 2
hours at RT). The nuclei were counterstained with Hoechst after the secondary
antibody incubation (1:1000 dilution in PBS for 15 minutes at RT). In between
the steps the retina sections were washed four times with PBS. The slides were
mounted with coverslips with Fluoromount medium (Electron Microscopy
Sciences, Hatfield, PA, USA). The images were acquired in a sequential manner,
with pictures taken every 0.5µm in the z-plane using confocal microscopy (SP5,
Leica Microsystems, USA). Figure 5 images were taken with a fluorescent
microscope (Eclipse Ti, Nikon, Tokyo, Japan).
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Primary antibodies used on 4% PFA fixed tissues were: mouse-anti-Cre (1:500,
Millipore, MAB3120); mouse-anti-Rhodopsin (1:1000, Millipore, MAB5356);
mouse-anti-Transducin (1:100, BD BioSciences, 610589). Primary antibodies
used on nonfixed tissue after brief postfixation on the slide (1% PFA in PBS for
15 minutes prior to staining) were: guinea pig-anti-GLI2 (1:1000, kind gift from Dr.
Eggenschwiler); rabbit-anti-GLI3 (1:200, Thermo Fisher, PS5-28029); mouse-
anti-acetylated tubulin (1:200, Sigma-Aldrich, T6793-100uL); rabbit-anti-IFT139
(1:500, custom). Primary antibodies used with nonfixed tissue were rabbit-anti-
IFT43 (1:100, Proteintech, 24338-1-AP); mouse-anti-PRPH2 (1:15, kind gift from
Dr. Molday); rabbit-anti-GLI1 (1:200, abcam, ab92611); rabbit-anti-SMO (1:1000,
kind gift from Dr. Anderson); rabbit-anti-BBS9 (1:500, Sigma-Aldrich,
HPA021289-100UL); mouse-anti-Gc1 (1:1000, kind gift from Dr. Baer); chicken-
anti-RP1 (1:1000, custom); rabbit-anti-IFT172 (1:1000, custom).
Ift172 Antibody Development: A custom affinity-purified rabbit anti-IFT172
polyclonal antibody was generated through a comercial provider (SC1676
PolyExpress Premium Service, GenScript USA Piscataway, NJ, USA). For rabbit
immunizations a 488 amino acid peptide from the C terminus of the human
IFT172 protein was chosen:
EEYEREATKKGARGVEGFVEQARHWEQAGEYSRAVDCYLKVRDSGNSGLAE
KCWMKAAELSIKFLPPQRNMEVVLAVGPQLIGIGKHSAAAELYLNLDLVKEAIDA
FIEGEEWNKAKRVAKELDPRYEDYVDQHYKEFLKNQGKVDSLVGVDVIAALDL
YVEQGQWDKCIETATKQNYKILHKYVALYATHLIREGSSAQALALYVQHGAPAN
19 bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
PQNFNIYKRIFTDMVSSPGTNCAEAYHSWADLRDVLFNLCENLVKSSEANSPA
HEEFKTMLLIAHYYATRSAAQSVKQLETVAARLSVSLLRHTQLLPVDKAFYEAGI
AAKAVGWDNMAFIFLNRFLDLTDAIEEGTLDGLDHSDFQDTDIPFEVPLPAKQH
VPEAEREEVRDWVLTVSMDQRLEQVLPRDERGAYEASLVAASTGVRALPCLIT
GYPILRNKIEFKRPGKAANKDNWNKFLMAIKTSHSPVCQDVLKFISQWCGGLPS
TSFSFQ
Transmission Electron Microscopy (TEM) Materials & Methods: Mice were
sacrificed by transcardial perfusion with half strength Karnovsky’s fixative (2%
formaldehyde + 2.5% glutaraldehyde, in 0.1 M sodium cacodylate buffer, pH 7.4;
Electron Microscopy Sciences, Hatfield, Pennsylvania) at room temperature.
The eyes were enucleated and after cornea and lens dissection they were placed
back into the half strength Karnovsky’s fixative for a minimum of 24 hours under
refrigeration. After fixation, samples were rinsed with 0.1M sodium cacodylate
buffer, post-fixed with 2% osmium tetroxide in 0.1M sodium cacodylate buffer for
1.5 hours, en bloc stained with 2% aqueous uranyl acetate for 30 minutes, then
dehydrated with graded ethyl alcohol solutions, transitioned with propylene oxide
and resin infiltrated in tEPON-812 epoxy resin (Tousimis, Rockville, Maryland)
utilizing an automated EMS Lynx 2 EM tissue processor (Electron Microscopy
Sciences, Hatfield, Pennsylvania.) The processed samples were oriented into
tEPON-812 epoxy resin inside flat molds and polymerized in a 60oC oven.
Semi-thin sections were cut at 1 µm thickness through the mid-equatorial plane
traversing the optic nerve in the posterior eyecup samples and stained with 1%
20 bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
toluidine blue in 1% sodium tetraborate aqueous solution for assessment by light
microscopy. Ultrathin sections (80 nm) were cut from each sample block using a
Leica EM UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL, USA) and
a diamond knife, then collected using a loop tool onto either 2x1 mm, single slot
formvar-carbon coated or 200 mesh uncoated copper grids and air-dried. The
thin sections on grids were stained with aqueous 2.5% aqueous gadolinium
triacetate hydrate and Sato’s lead citrate stains using a modified Hiraoka grid
staining system. Grids were imaged using a FEI Tecnai G2 Spirit transmission
electron microscope (FEI, Hillsboro, Oregon) at 8o kV interfaced with an AMT
XR41 digital CCD camera (Advanced Microscopy Techniques, Woburn,
Massachusetts) for digital TIFF file image acquisition. TEM imaging of retinas
were assessed and digital images captured at 2kx2k pixel, 16-bit resolution.
Transducin Translocation experiment
In this experiment, the mice were distributed in 2 different groups. For each
group, three Ift172fl/fliCre mice and littermate Ift172fl/fl control mice were used.
Group 1: The mice were dark-adapted overnight, euthanized in darkness with
CO2 and their eyes were enucleated under dark conditions, fixed in (4% PFA in
PBS) and prepared for cryosectioning as described in previous section. Group 2:
The pupils of mice were dilated as described above in ERG section, and mice
were exposed to saturating light conditions (1400lux) for 20 min. CO2 was used
to euthanize the mice, and their eyes were enucleated and prepared from
dryosectioning as before. The eyes from different conditions were sectioned in
one block and they were immonolabeled with mouse-anti-Transducin antibody
21 bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
(1:100, BD BioSciences, 610589) on the same slide. The images were taken with
confocal microscopy (SP5, Leica Microsystems, USA). Image J software was
used to analyse signal intensities, where signal form the OS was divided by the
total signal form the whole retina. The analysis conditions were kept exact for test
and control mice. Two-way ANOVA and a post-hoc multiple t-test with
Bonferroni-Sidak correction were used for statistical analysis (Graphpad, Prism 7
software).
Acknowledgments
This work was supported by grants from the Fight for Sight (2016 International
Retinal Research Foundation Grant-In-Aid Award to KMB), Research to Prevent
Blindness Medical Student Research Fellowship (PRG), National Eye Institute
[EY012910 (EAP) and P30EY014104 (MEEI core support), P30EYE003790
(SERI core support)], the Foundation Fighting Blindness (USA, EAP).
Conflict of Interest Statement: The authors declare no conflict of interest
22 bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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27 bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Legends to Figures
Figure 1. Generation of the Ift172fl/fliCre Mouse Model and of the IFT172
Antibody. A. Schematic representation of the targeted Ift172 allele (Ift172fl/fl)
and the knck-out allele (Ift172fl/fliCre). B. Expression of Cre recombinase (red) in
the ONL at postnatal days 18, 21 and 25. Nuclei are counterstained with Hoechst
(blue). C. Schematic representation of human IFT172 protein, containing
repetitive W40 and TPR motifs. 488 carboxyl terminal amino acid residues were
chosen for the rabbit polyclonal antibody generation. D. Staining for IFT172 (red)
and RP1 (green) showing at postnatal days 21, 25 and 28, showing depletion of
IFT172 from rods by P28 in Ift172fl/fliCre mice.
Figure 2. Ift172fl/fliCre mice display rapid retinal degeneration. A. OCT data
obtained at one, two, and three months of age in Ift172fl/fliCre and wild-type
control mice, showing diminishing ONL in the Ift172fl/fliCre mice. B. Graphical
representation of the neural retina and the ONL thickness measured from OCT
images in Ift172fl/fliCre and control genotypes. C. Representative histology
sections from Ift172fl/fliCre and control mice. D. b-wave amplitudes form dark-
adapted and light-adapted ERGs performed on Ift172fl/fliCre and control
genotypes.
Figure 3. Transmission Electron Microscopy Data Ultrastructure of
Ift172fl/fliCre and control mice showing OS shortening starting at PN25 and
28 bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
cellular debris at PN28 and PN31 (black arrows). Intact transition zone
axonemes are visible at PN28 but not at PN31, where severe photoreceptor
degeneration occurs. Scale bar on the lower magniticatin picture represents 2µm
and on the higher magnification picture 500nm.
Figure 4. Cilia-associated Protein Localization. A. Ciliary axoneme staining
with RP1 (green) and acetylated tubulin (red), showing shortening of the Rp1
signal relative to acetylated tubulin in Ift172fl/fliCre mice compared to controls at
PN28. RP1 mislocalization to the inner segment is visible in Ift172fl/fliCre mice
starting at PN25. B. Staining of IFT139 (red) at the base of the ciliary axoneme,
makerd with Rp1 staining (green). C. IFT139 (red) and RP1 (green) both
mislocalize to the synaptic region in Ift172fl/fliCre mice, but not in controls, starting
at PN25. Nuclei in A, B and C are counterstained with Hoechst (blue).
Figure 5. Outer Segment Protein Localizations. A. Rhodopsin (red)
mislocalization to the ONL is present at PN21 and progresses over time in
Ift172fl/fliCre mice compared with Ift172fl/fl controls. B and C. Guanyl cyclase 1
(green) and Peripherin2 (red) localize appropriately to the outer segment at all
tested time points in both Ift172fl/fliCre mice and Ift172fl/fl controls.
29 bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 6. Transducin translocation kinetics are affected in the absence of
IFT172. A. Retina cryosections from dark-adapted mice at PN21 stained for
transducin-Gαt (green), DAPI (blue) and Cre (magenta). Transducin is
predominantly localized in rod OS region in Ift172fl/fliCre (bottom panel) and the
littermate Ift172fl/fl controls (top panel). B. Retina cryosections from light-adapted
mice at PN21 stained with transducin-Gαt (green), DAPI (blue) and Cre
(magenta). In both Ift172fl/fliCre (bottom panel) and littermate Ift172fl/fl controls
(top panel), transducin is translocated to rod IS and inner retinal region.
C. Retina cryosections from dark-adapted mice at PN25 showing predominant
transducin-Gαt localization to rod OS, in both genotypes. In addition, presence of
fl/fl transducin-Gαt in the IS of the Ift172 iCre mice but not the controls was-
observed. D. Retina cryosections from light-adapted mice at PN25, showing
fl/fl significantly higher transducin-Gαt staining in the IS of Ift172 iCre mice
compared to Ift172fl/fl littermate controls (white arrowhead). E. Relative
fluorescence intensity of the OS signal in relation to the total retina signal plotted
for Ift172fl/fliCre and Ift172fl/fl control in dark and light conditions at PN21 and
PN25.
Figure 7. GLI1 Localization. Photoreceptor localization of GLI1 (red) at PN21-
28, showing mislocalization in the photoreceptor inner segment at PN25.
RP1(green) marks the tranzision zone and nuclei are counterstained with
Hoechst (blue).
30 bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Supplementary Figure 1. Additional Control Genotype OCT data. OCT data
of additional control genotypes (Ift172wt/wt Ift172fl/wtiCre, Ift172fl/fl ,) obtained at
one, two, and three months of age, with no signs of degeneration.
Supplementary Figure 2. Additional Cilia- associated Protein Localization
A. Immunostaining of Bbs9 (red), showing no differences in localization between
Ift172fl/fliCre and control mice. B. Immunostaining of IFT43 (red) ), showing no
differences in localization between Ift172fl/fliCre and control mice. Nuclei in A and
B are counterstained with Hoechst (blue) and RP1 is immunolabelled (green).
Supplementary Figure 3. Immunostaining of SMO, GLI2, or GLI3 (red), showing
no differences in localization between Ift172fl/fliCre and control mice. Nuclei in
are counterstained with Hoechst (blue) and RP1 is immunolabelled (green). Size
bar in the top panel represents 2µm and in the lower panel 30µm.
Abbreviations
Intraflagellar Transport (IFT), Outer segments (OS), Outer Nuclear Layer (ONL),
Inner Nuclear Layer (INL), Inner segments (IS), Retinal Pigment Epithelium
(RPE), Neural Retina (NR), Transmission Electron Microscopy (TEM), Light
Microscopy (LM), Peripherin 2 (Prph2), Guanyl-cyclase 1 (GC-1), Acetylated
Tubulin (AcTub), microliters (µL), millimolar (mM), micromolar (µM), Bovine
Serum Albumin (BSA), Paraformaldehyde (PFA), Phosphate Buffered Saline
(PBS), glioma-associated oncogene family member 1 (GLI1).
31 bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/210617; this version posted October 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.