Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

Purpose Cytoplasmic dynein-1 (henceforth dynein) moves cargo in conjunction with dynactin toward the minus end of microtubules. The dynein heavy chain, DYNC1H1, comprises the backbone of dynein, a retrograde motor. Deletion of Dync1h1 abrogates dynein function. The purpose of this communication is to demonstrate effects of photoreceptor dynein inactivation during late postnatal development and in adult retina. Methods We mated Dync1h1F/F mice with iCre75 and Prom1-CreERT2 mice to generate conditional rod and tamoxifen-induced knockout in rods and cones, respectively. We documented retina degeneration with confocal microscopy at postnatal day (P) 10 to P30 for the iCre75 line and 1 to 4 weeks post tamoxifen induction (wPTI) for the Prom1-CreERT2 line. We performed scotopic and photopic electroretinography (ERG) at P16 to P30 in the iCre75 line and at 1-week increments in the Prom1-CreERT2 line. Results were evaluated statistically using Student's t-test, two-factor ANOVA, and Welch's ANOVA. Results Cre-induced homologous recombination of Dync1h1F/F mice truncated DYNC1H1 after exon 23. rodDync1h1−/− photoreceptors degenerated after P14, reducing outer nuclear layer (ONL) thickness and combined inner segment/outer segment (IS/OS) length significantly by P18. Scotopic ERG a-wave amplitudes decreased by P16 and were extinguished at P30. Cones were stable under rod-knockout conditions until P21 but inactive at P30. In tamDync1h1−/− photoreceptors, the IS/OS began shortening by 3wPTI and were nearly eliminated by 4wPTI. The ONL shrank significantly over this interval, indicating rapid photoreceptor degeneration following the loss of dynein. Conclusions Our results demonstrate dynein is essential for the secretory pathway, formation of outer segments, and photoreceptor maintenance.

T ransport along microtubules is accomplished by cytoplasmic dynein, 1 which moves cargo toward the minus end of microtubules at the basal body. [2][3][4] In photoreceptors, the minus end is located at the basal body, serving as a microtubule organizing center (MTOC) (for review, see Baehr et al. 5 ). During early photoreceptor development, dynein cargo may include nuclei, 6 mitochondria, 4,7 membrane vesicles, 8,9 and other organelles (reviewed in Reck-Peterson et al. 10 ). After ciliogenesis, dynein plays a key role in the secretory pathway transporting membrane vesicles and membrane proteins from the endoplasmic reticulum to the periciliary ridge, where cargo is unloaded. 11 Dynein is composed of a pair of heavy chains (DYNC1H1) (Fig. 1A) and a set of noncatalytic accessory components termed intermediate, light intermediate, and light chains. 12-14 DYNC1H1 serves as a scaffold organizing the distribution of dynein subunits and the three-dimensional structure of the dynein megacomplex (Fig. 1B). 15,16 The N-terminal DYNC1H1 tail domain contains a dimerization domain 17 and binding sites for intermediate chains (DIC1 or DIC2) and light intermediate chains (DLIC1 or DLIC2). The C-terminal half contains the motor domain and micro-tubule interaction site. The dynein motor domain is built around a ring of six AAA+ (ATPases Associated with various Activities) of the heavy chain. The microtubule-binding domain sits at the tip of a coiled-coiled stalk emerging from AAA4. Dynactin and a cargo adaptor are essential for cytoplasmic dynein to move membrane vesicles along microtubules. 18 Numerous mutations in the human DYNC1H1 gene associate with spinal muscular atrophy and Charcot-Marie-Tooth disease. [19][20][21][22][23] Germline deletion of mouse Dync1h1 (truncation after exon 1) is lethal as embryos do not survive beyond embryonic day 8.5 (E8.5). 24 Four mouse mutantslegs at odd angles (Loa), 25,26 Cramping 1 (Cra1), 27,28 Sprawling (Swl), 29,30 and a model of Charcot-Marie-Tooth disease 31,32 -display autosomal dominant behavioral phenotypes with no reported retina phenotype. A DYNC1H1 nonsense mutation (Y3102X) underlies the cannonball (cnb) phenotype in zebrafish. 4 Green fluorescent protein (GFP)-tagged rhodopsin has been mislocalized in both the cannonball mutant and dync1h1 morphant cells. 4 Dynein is present prominently in photoreceptor inner segments (ISs), the outer plexiform layer (OPL), and the inner plexiform layer, as well as, to a lesser extent, within the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer. Dynein is responsible for retina lamination, nuclear positioning, and vesicular trafficking of photoreceptor membrane proteins. 13, 33 We previously reported that conditional knockout of Dync1h1 in mouse retina (Dync1h1 F/F ;Six3cre or ret Dync1h1 −/− ) resulted in rapid retinal degeneration within 2 postnatal weeks. 13 DYNC1H1 was undetectable in the ret Dync1h1 −/− IS area as early as postnatal day (P) 6, and photoreceptors did not elaborate IS, outer segment (OS), or functional synaptic terminals. In the P8 ret Dync1h1 −/− central retina, outer and inner nuclear layers were severely disorganized and lacked a recognizable OPL. 13 In this report, we depleted DYNC1H1 in rod photoreceptors during postnatal development and in both rods and cones of adult mice by tamoxifen induction. These knockouts permit analysis of the consequences of DYNC1H1 deletion during late postnatal development in rods and in photoreceptors of adult mice, respectively.

Generation of DYNC1H1 Conditional Knockout Mice
The dynein heavy chain DYNC1H1 (Fig. 1A) forms the backbone of cytoplasmic dynein, a mega complex of 1.5 mDa, composed of a pair of force-generating heavy chains and a set of accessory components termed intermediate, light intermediate, and light chains (Fig. 1B). The heavy chain serves as a scaffold organizing the distribution of dynein subunits. We previously generated a floxed Dync1h1 allele (Dync1h1 F/F ), in which loxP sites are placed in introns 23 and 25 (Fig. 1C). 13 Deletion of exons 24 and 25 truncates DYNC1H1 (red arrow, Fig. 1A, D) removing the C-terminal motor domain and the microtubule binding site. We mated Dync1h1 F/F mice with iCre75 transgenic mice to generate rod knockout mice (Dync1h1 F/F ;iCre75, abbreviated rod Dync1h1 −/− ) and with CreER T2 mice (Dync1h1 F/F ;Prom1-CreER T2 ) to generate rod/cone knockouts following tamoxifen induction ( tam Dync1h1 −/− ). In iCre75 mice, Cre is expressed under the control of a rhodopsin promoter, which drives Cre in the second postnatal week. 34 In Prom1-CreER T2 mice, CreERT2 expression occurs under the control of the Prom1 promoter driving expression in rods and cones. 35 Deletion of the Dync1h1 gene in rod Dync1h1 −/− mice was assessed by PCR with P10 retina DNA as template and primers X23-F and loxP-R to generate a 500-bp amplicon lacking exons 24/25 (Fig. 1E). Since dynein is expressed in all retina cells, a band at 1.6 kb corresponding to the Dync1h1 F allele is also observed. We did not perform an immunoblot as a blot using total retina tissue would be inconclusive due to the continued presence of DYNC1H1 in all nonrod retina cells. Genotyping with tail DNA confirmed the presence of loxP (indicating a Dync1h1 F allele) (Fig. 1F), iCre75 (Fig. 1G), EGFP-CETN2 (Fig. 1H), and Prom1-CreER T2 (Fig. 1I) in respective mice (see Methods for details).  rod photoreceptor degeneration progressed rapidly and was completed 2 weeks after onset. We assessed cone function in the DYNC1H1 rod knockout by assessing the photopic bwave. Photopic b-wave amplitudes of control and rod knockout mice were similar at P21 (Fig. 2E) but were extinguished at P30 (Fig. 2F). Assuming that cone bipolar cells are unaffected in the dynein rod knockout, the loss of photopic b-wave results from loss of cone function. This is consistent with previous findings in which deletion of rod-specific genes resulted in secondary loss of cones due to diminished rod-derived cone viability factor. 36 Examples of cone loss following loss of rods have been documented in animal models of dominant or recessive retinitis pigmentosa (RP), the rd1 mouse, 37 and bystander cone degeneration of human RP. 38

Rapid Rod Degeneration in the Absence of DYNC1H1
While DYNC1H1 was present in all retina cells, the highest concentrations were found in the photoreceptor IS, OPL, and, to a lesser extent, the ONL (Fig. 3, row A). Our iCre75 mice were maintained on the Egfp-Cetn2 + background, in which centrin 2 (CETN2) inhabited the connecting cilium tethering the IS to OS (Fig. 3A, see P14 inset) and aided to identify photoreceptor compartments. Although the Dync1h1 gene was knocked out in rods by P10 (Fig. 1E), DYNC1H1 protein persisted in the rod ONL/IS (Fig. 3, row B), and thus rod Dync1h1 −/− morphology was indistinguishable from controls at P14. DYNC1H1 levels were reduced in the IS beginning at P16, but some DYNC1H1 was still detectable at P30, presumably in cone inner segments (Fig. 3, rows B). DYNC1H1 in the OPL persisted as distal processes of rod bipolar cells were unaffected. rod Dync1h1 −/− IS/OS regions and ONL began to shrink after P16 (Figs. 3E, F). The mutant ONL revealed three to four layers of nuclei at P21, and only a single nuclear layer (presumably cones) was evident at P30. Unexpectedly, we observed little rhodopsin accumulation or mislocalization in the rod Dync1h1 −/− ONL (Fig. 3D).

OS Protein PDE6 and Synaptic Protein CtBP2/RIBEYE in Rod Knockouts
We determined the fates of rod PDE6 and RIBEYE, both of which are not directly dependent on dynein for transport to their destinations (OS and synapse, respectively). Rod PDE6 is a heterotetrameric protein consisting of isoprenylated PDE6α and PDE6β catalytic subunits and two PDE6γ inhibitory subunits (reviewed in Cote 39 ). Subunit delivery to the OS is dependent on diffusion by interaction with PDE6D, a solubilization factor. In the P16 rod Dync1h1 −/− retina, PDE6 content was reduced significantly based on OS shortening (Fig. 4, row B). As observed with rhodopsin, PDE6 never accumulated in the IS during OS shrinking at P16 and P18.
RIBEYE, expressed in retina via a tissue-specific promoter within intron 1 of the CtBP2 (C-terminal binding protein 2) gene, 40 is a major structural protein of rod/cone synaptic contacts and delineates the OPL of controls (Fig. 4, row C). RIBEYE is not a transmembrane protein, and its transport to the synaptic terminal (the microtubule plus end) is not expected to be dependent on dynein. Mutant photoreceptor synaptic termini have normal levels of RIBEYE at P14 and P16 but are nearly devoid of CtBP2/RIBEYE at P21 (Fig. 4, row D), consistent with attenuated scotopic electroretinography (ERG) measurements. RIBEYE-immunoreactive puncta of the P21 OPL likely belong to cone pedicles, as the photopic ERG is near normal at P21. RIBEYE is undetectable at P30, consistent with absence of functional photoreceptors and "flat" rod and cone ERG amplitudes.

Deletion of DYNC1H1 by Tamoxifen Induction
To explore consequences of DYNC1H1 deletion in the adult mouse, we deleted Dync1h1 in Dync1h1 F/F ;Prom1-CreER T2 1-month-old mice using tamoxifen induction. Prom1 expression in adult mice is seen in rods and cones along with cells within the brain, pancreas, intestine/colon, kidney, lung, and reproductive system (male and female). 41 Dync1h1 F/F ;Prom1-CreER T2 , Dync1h1 F/F , and Dync1h1 F/+ ;Prom1-CreER T2 mice were injected with tamoxifen for 5 consecutive days, with the first injection occurring between P21 and P30 (Figs. 1C, D). Uninjected Dync1h1 F/F ;Prom1-CreER T2 mice served as an additional control. Injected Dync1h1 F/F ;Prom1-CreER T2 mice are abbreviated as tam Dync1h1 −/− (Figs. 5, 6). Intraperitoneal injection of tamoxifen for 5 consecutive days induced the nuclear translocation of Cre, and the degeneration rate was assessed in retina cryosections of eyes harvested 1, 2, 3, and 4 weeks after the first injection. At 1 and 2 weeks post tamoxifen induction (wPTI), DYNC1H1 levels were indistinguishable among injected tam Dync1h1 −/− , uninjected Dync1h1 F/F ;Prom1-CreER T2 , and injected Dync1h1 F/F or Dync1h1 F/+ ;Prom1-CreER T2 mice (Fig. 5, rows A and B). At 3wPTI, DYNC1H1 was nearly eliminated in half of the tam Dync1h1 −/− mice (see Fig. 5 legend). In tam Dync1h1 −/− mice in which elimination of DYNC1H1 was seen, the ONL thickness was reduced by 50% and the OS + IS length was reduced to ∼30% that of control (Figs. 5C, E). In the other half of the tam Dync1h1 −/− mice, tamoxifeninduced knockout of DYNC1H1 was inefficient and ONL and OS + IS measurements were normal or slightly reduced. DYNC1H1 antibody fluorescence in the OPL was undisturbed as DYNC1H1 levels in bipolar cells were unaffected in the tamoxifen-induced knockout (Fig. 5C). At 4wPTI, ONL thickness was reduced by 80% in mice with efficient tamoxifen injections, but DYNC1H1 was still detectable in the deteriorating mutant IS (Fig. 5D, E), reflecting failure to clear DYNC1H1 completely. In these mice, the average OS + IS length was reduced to about 12% of control (Fig. 5D, F).
Scotopic a-wave and photopic b-wave amplitudes as a function of light intensity did not display significant changes at 1wPTI for any of the light intensities (Fig. 8A). At 2wPTI, induced knockout (KO) did show as statistically different from control uninjected mice but not from the control injected mice. Control injected and control uninjected did not differ from each other at 2wPTI (Fig. 8B). At 3wPTI, scotopic a-wave and photopic b-wave amplitudes in the induced KO were reduced, but this reduction was only statistically significant at the photopic 1.4 log cd s·m −2 intensity (Fig. 8C). At 4wPTI, the reduction in scotopic a-wave and photopic b-wave amplitudes for induced KO was significant at most intensities (Fig. 8D). These results indicate that in mice in which loss of DYNC1H1 was efficiently induced by tamoxifen injection, rod and cone degeneration began between 2 and 3wPTI, with further retinal degeneration occurring between 3 and 4wPTI.

DISCUSSION
In our previous study, 13 we conditionally knocked out DYNC1H1 in retina using Six3Cre that initiates ablation of DYNC1H1 after embryonic day 9. We showed that segregation of the ONL/INL was absent at P6 and P8, and mutant photoreceptors did not elaborate inner and outer segments. Here, we show that deletion of DYNC1H1 in mouse photoreceptors after ciliogenesis led to loss of function (Fig. 2), shortening of the inner and outer segments, and shrinking of the ONL (Figs. 3, 4). In wild-type (WT) photoreceptors, dynein, in conjunction with dynactin, transports cargo toward the minus end of microtubules at the MTOC or basal body (for a recent review, see Dahl and Baehr 14 ). Once the basal body has docked to the IS cortex, inner segment biosynthetic activity is directed toward synthesis of outer segment proteins, transport of OS proteins to the periciliary ridge by dynein, movement through the connecting cilium either by diffusion or intraflagellar transport, and incorporation into nascent discs. As mouse photoreceptor outer segments are renewed every 10 days, 42,43 sustained delivery of large amounts of transmembrane and peripheral membrane-attached and soluble proteins is essential.
When dynein is absent in photoreceptors before ciliogenesis (<P6), the ONL never forms correctly, and OS/synaptic structures are absent. 13 In Dync1h1 rod knockouts (this communication), the process was interrupted in the second postnatal week (>P16) (Figs. 3B, D; Figs. 4B, D). Interruption occurred in tamoxifen-induced knockouts between the second and third week postinduction (Figs. 5C, D; Figs. 6C, D). In the absence of dynein, delivery of OS proteins ceases and disc morphogenesis declined. In rod knockouts beginning at P16, rod Dync1h1 −/− IS/OS shortened and were lost by P30, consistent with supply chain interruption of the secretory pathway. OS structures became unstable and essentially died by starvation. In tamoxifen-induced knockouts, rods and cones appeared to be stable for 2 weeks postinduction. As observed previously, 13 impaired DYNC1H1 protein clearance delayed the onset of degeneration in rod (Fig. 3, row C) and tamoxifen-induced (Figs. 5C, D) knockouts of Dync1h1.
The most abundant OS membrane protein, rhodopsin, is synthesized at the endoplasmic reticulum, posttranslation- ally modified in the Golgi, and transported to the OS base via the secretory pathway. 11,44 Newly synthesized rhodopsin is predicted to accumulate in the ONL in the absence of dynein, as COPII vesicle transport from the ER to Golgi is impaired.
Unexpectedly, we observed little rhodopsin accumulation or mislocalization in the rod Dync1h1 −/− ONL (Fig. 3D), an observation that could mean that rhodopsin biosynthesis terminates. Alternatively, newly synthesized rhodopsin  At 1wPTI (A), average scotopic and photopic ERGs amplitudes did not differ significantly between induced knockout and control. At 2wPTI (B), the average scotopic a-wave amplitude was slightly reduced compared to controls. Photopic ERGs did not differ. At 3wPTI (C), induced KO shows reduced average ERG amplitudes for both scotopic a-wave and photopic b-wave, but this reduction was only statically significant for the photopic 1.4 log cd s·m −2 intensity. At 4wPTI (D), the reduction in average ERG amplitude for both scotopic a-wave and photopic b-wave for the induced KO was statically significant for most ERG flash intensities. *P < 0.05 (unbalanced two-factor ANOVA, Tukey's honestly significant difference criterion). may be cleared immediately if it cannot exit the ER or if rhodopsin-containing vesicles cannot attach to dynein after ER exit.
PDE6 is a key protein in the phototransduction cascade regulating the levels of cGMP, the internal transmitter of phototransduction. 45 Rod PDE6 is peripherally membrane attached using prenyl side chains as anchors. 39 PDE6D (PDEδ) acts as a trafficking chaperone to move PDE6 to the OS by diffusion. 46 In the absence of PDE6D, PDE6 still manages to reach the OS, presumably hitching a ride with rhodopsin-bearing vesicles in the secretory pathway powered by dynein. 47 It is unclear whether PDE6 transport in the absence of PDE6D depends on dynein. Mouse RIBEYE is a ribbon-specific protein, expressed under its own promoter within the CtBP2 gene. 40 It has the capability to build the synaptic ribbon scaffold via multiple RIBEYE-RIBEYE interactions. The B domain of RIBEYE is a homolog of 2-hydroxyacid dehydrogenase, binding NAD +40 and UNC119. 48 RIBEYE is presumably not a membraneassociated protein, and while its transport mode to the synaptic region is unknown, it is not likely to be dynein dependent. Both PDE6 and RIBEYE disappear as rod photoreceptors degenerate rapidly.
Our study currently is limited to evaluation of photoreceptor degeneration by confocal microscopy and electroretinography. We show that photoreceptor OS, IS, and ONL nearly simultaneously shrink in rod knockouts beginning at P16 and in tamoxifen-induced knockouts by the third week postinjection. OS likely shrink by failed delivery of newly synthesized proteins to the periciliary ridge near the basal body. It will be interesting to evaluate the status of the IS microtubule cytoskeleton in the absence of dynein by immunohistochemistry with anti-tubulin antibodies, the effect on disc morphogenesis as protein delivery ceases, and potential changes in synaptic structures by transmission electron microscopy.

Generation of Rod Knockout Mice
Dync1h1 F/F mice 13 were crossed with iCre75 transgenic mice 34 kept on an Egfp-Cetn2 background to generate Dync1h1 F/+ ;iCre75;Egfp-Cetn2 mice. Mice were then backcrossed to Dync1h1 F/F to generate experimental animals. Expression of Egfp-Cetn2 allows connecting cilium and centriole identification without use of a specific antibody.

Generation of Knockouts by Tamoxifen Induction
Dync1h1 F/F mice were bred to Prom1-CreER T2 mice 41 to generate Dync1h1 F/+ ;Prom1-CreER T2 mice. In these mice, expression of CreER T2 is driven by the prominin 1 (Prom1) promoter/enhancer. Dync1h1 F/+ ;Prom1-CreER T2 mice were bred to Dync1h1 F/F mice to generate mice for tamoxifen experiments. Tamoxifen was administered via intraperi-toneal injection in adult mice (P21-P30 at time of first injection). Tamoxifen was dissolved in corn oil to a stock solution of 20 mg/mL. Mice were dosed with 150 mg/kg body weight for 5 consecutive days according to their weight on the first day of injections (i.e., 7.5 μL of 20 mg/mL tamoxifen solution per gram weight). ERG and collection of eyes for confocal immunolocalization were performed 1 to 4 weeks after the first injection.

Eye Collection Methods
Mice were sacrificed by cervical dislocation. Eyes were enucleated and immersion-fixed for 1 hour using 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Anterior segments were removed after 10 minutes of fixation. Eyecups were equilibrated in 15% sucrose in PBS, equilibrated overnight in 30% sucrose in PBS, embedded in Optimal Cutting Temperature (OCT) compound, frozen on dry ice, and stored at −80°C. Blocks containing eyecups were equilibrated to −20°C prior to sectioning. Transverse sections, including or adjacent to the optic nerve, were cut at a 14-μm thickness using a (Leica, Buffalo Grove, IL) cryostat and transferred to charged slides (Thermo-Fisher, Waltham, Massachusetts). Slides were stored at −80°C. Control retinas included Dync1h1 F/F , Dync1h1 F/+ , or Dync1h1 F/+ ;iCre75 littermates for rod Dync1h1 −/− experiments. Control retinas for tam Dync1h1 −/− experiments were uninjected Dync1h1 F/F ;Prom1-ETCre, injected Dync1h1 F/F , and injected Dync1h1 F/+ ;Prom1-CreER T2 mice.

Immunohistochemistry
Sections were encircled using a PAP pen (Ted Pella, Redding, CA), warmed for 30 minutes at 37°C, and then rehydrated by washing 10 minutes three times in 1× PBS. All sections used were from the central retina near the optic nerve. Sections were blocked in 5% normal goat serum/0.1% TritonX-100 in 1× PBS for 1 hour. Antibodies, dilutions, and sources follow: , applied to the sections, and sections were incubated in the dark for 1 hour at room temperature. Slides were washed for 10 minutes three times in PBS. Slides were dipped briefly in deionized H 2 O and coverslipped using Fluoromount-G Mounting Medium (Southern Biotech, Southern Biotech Birmingham, AL). Images were acquired using the 40× objective of a Zeiss (Zeiss Jena 07745, Germany) LSM800 confocal microscope. All genotypes of a given age and antibody were imaged at a single z-plane using identical settings for laser intensity and master gain. Digital gain was 1 for all images. Pinhole size was set for 1 AU on the red channel (39 μm for the 40× objective). Postprocessing of nonsaturated images consisted of equal adjustments to brightness and contrast of control and knockout images using Adobe Photoshop (Adobe Photoshop San Jose, CA) but without affecting the conclusions made.

Electroretinography
Scotopic and photopic ERG measurements were performed using P16, P21, or P30 mice for the iCre75 experiments and at 1, 2, 3, or 4 weeks after tamoxifen induction for the Prom1-CreER T2 experiments. Prior to ERG, the mice were dark-adapted overnight and anesthetized with an intraperitoneal injection of 1% ketamine/0.1% xylazine at 10 μL/g body weight. The mice were kept warm during ERG by using a temperature-controlled stage. Scotopic and photopic responses were recorded as described 52 using a UTAS BigShot Ganzfeld system (LKC Technologies, Gaithersburg, MD, USA). Scotopic single-flash responses were recorded at stimulus intensities of −4.5 to 2.4 log cd s·m −2 . Mice were light-adapted under a background light of 1.48 log cd s·m −2 for 5 to 10 minutes prior to measuring photopic responses. Photopic single-flash responses of control and knockout were recorded at stimulus intensities of −0.1 to 1.9 log cd s·m −2 .

Statistical Analysis
We performed an unbalanced two-factor ANOVA to compare experimental and control animals for their quantified aand b-wave ERG response across multiple ages or weeks after tamoxifen induction. Post hoc multiple comparison was performed using Tukey's honestly significant difference criterion. Statistical significance was determined using an α value of P < 0.05. All electroretinography statistics were computed using statistical toolbox "anovan" and "multcompare" functions in MATLAB (MathWorks, Natick, MA, USA).
We performed a Student's t-test assuming unequal variances to compare iCre75 control and KO ONL thickness or IS + OS length. Retina measurements used for these calculations were determined based on an average of three measurements per retina. Microsoft Excel (Microsoft, Redmond, WA, USA) function "T.TEST assuming unequal variances" was used to calculate the P value, with statistical significance determined using an α value of P < 0.05. Since we had unequal sample sizes and could not assume homogeneity of variances, we preformed Welch's ANOVA test to compare control injected, control uninjected, and tam Dync1h1 −/− ONL thickness or IS + OS length using the Xrealstats data analysis add-on to Microsoft Excel. Statistical significance was determined using a P value of <0.05.