Briefly, 15ng of cDNA and 0.
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Each reaction was run as technical duplicates and a minimum of 4 biological replicates was used per group. Prss56 Cre mice were bred to tdTomato reporter mice R26 tdTomato to generate offsprings with one copy of each of tdTomato and Cre recombinase under the control of the Prss56 promoter.
The eyes were enucleated at both embryonic and postnatal time points. The eyes were processed, sectioned and visualized for tdTomato fluorescence as described below. Our design ensured uniform copy number of tdTomato and Prss56 Cre , allowing a direct comparison of tdTomato expressing cells between Prss56 mutant and heterozygous control eyes. Cryosections were then washed and subject to signal amplification and detection using fast red substrate, counterstained and mounted for subsequent imaging.
Tamoxifen T, Sigma, St. Each experimental mouse received a single intraperitoneal injection of tamoxifen 0. Eight equivalent areas of the retina were consistently selected for each whole mount. Two independent observers masked to genotypes manually quantified the relative distribution of the two types of endfeet. The retinal cell suspension was used for flow cytometry. Amira software was used for 3D visualization and analysis. We obtained illustrative biometric data from a nanophthalmic patient affected with a homozygous missense variant p. In brief, ocular dimensions measured via ultrasound biomicroscopy A- and B-scan techniques revealed a very small globe bilaterally featuring crystalline lenses that were normally positioned, but large in size relative to that of the eye.
The choroid was also observed to be diffusely thickened. Power for a two-tailed two-sample t-tests was calculated using a range of means and standard deviation values of axial length that could be reasonably expected based on published data and our initial assessment [ 26 ]. Although the difference between the two group means and within-group standard deviation are statistically independent parameters, in many biological data sets they show various levels of collinearity.
Where logistically possible, and partly as a precaution against the possibility of some failed experiments, we collected data from more samples. Number of eyes, mean and standard deviation of all measurements in each group are presented in S1 — S3 Tables.
D Representative optical coherence tomography images of 2 months old eyes. C , D tdTomato expression was not detected in the iridocorneal angle, ciliary body CB , cornea, lens, sclera, choroid or retinal pigment epithelium RPE.
Müller glia-derived PRSS56 is required to sustain ocular axial growth and prevent refractive error
Top panel: A representative image of P10 retina showing Prss56 expression predominantly in the peripheral region of the retina. At P15, increased Prss56 expression was detected in the inner nuclear layer of the retina in mutant mice compared to their wild-type littermates. D Representative images of retinal sections showing absence of immunolabeling when using rabbit or goat IgG isotypes as negative controls. A Prss56 alleles and targeting construct. Top: Wild-type Prss56 allele exons indicated as solid black rectangles. Middle: Targeting vector containing 2 LoxP sites black triangles flanking Prss56 exons 3 and 4 and a Neomycin selection cassette Neo flanked by 2 Frt sites green triangles.
The Prss56 F allele expresses normally and behaves as the wild-type Prss56 allele in the absence of Cre recombinase activity. Bottom: Cre recombinase activation causes deletion of exons 3 and 4 resulting in a frameshift mutation and premature stop codon, rendering the Prss56 gene inactive. PCR analyses using various primer combinations gray triangles in A are shown. PCR reactions using the F1R1 primer pair gives a product that is about 34 bp longer in mice carrying the Prss56 F allele compared to wild-type mice.
PCR reactions using the primer pair F1R2 give rise to product sizes of bp and bp for the wild-type and Prss56 F alleles, respectively. We are grateful to Garett Cheung and Eric Dang for assistance with genotyping. We thank Dorenda Itzkowitz for mouse colony management and Suling Wang for assistance with figure preparation. We would also like to thank Yien-Ming Kuo for histological sectioning and staining. Abstract A mismatch between optical power and ocular axial length results in refractive errors.
Author summary Refractive errors mainly occur when changes in ocular size ocular axial length prevent light from focusing directly on the retina. Introduction A central feature of organogenesis is the intrinsic ability to faithfully determine final organ size and shape.
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Table 1. Ocular biometry of an individual carrying a PRSS56 mutation. Prss56 ocular expression is restricted to the neural retina and is first detected in a pool of late retinal progenitor cells. Fig 2. Lineage tracing of Prss56 expressing cells during ocular development. Fig 3. Earliest Prss56 expression occurs in late retinal progenitor cells.
Molecular regulation of visual system development: more than meets the eye
Fig 4. Early requirement for PRSS56 during ocular axial growth. Fig 5. Early requirement for PRSS56 in ocular size determination. Fig 6. Sustained PRSS56 activity is required for ocular axial growth. Fig 7. PRSS56 activity is required during both the vision-independent and dependent stages of ocular growth.
Genetic inactivation of PRSS56 rescues ocular axial elongation in a mouse model of myopia.
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Discussion The molecular and cellular mechanisms involved in ocular size regulation and refractive development are poorly understood. Modulation of Prss56 expression in response to mutations in gene s involved in ocular size determination. Findings from our mouse study suggest a role for PRSS56 in pre- and post-natal human ocular growth.
Identification of PRSS56 as a potential target for therapeutic intervention to slowdown myopia progression. Mouse lines Mutant mice. Reporter line. Cre lines. Ocular biometry Ocular biometry was performed using optical coherence tomography or a digital Vernier caliper. Refraction measurement Ocular refractions were acquired using an automated infrared photorefractor as described previously with some minor modifications [ 28 ].
Tamoxifen injection Tamoxifen T, Sigma, St. Human ocular biometry We obtained illustrative biometric data from a nanophthalmic patient affected with a homozygous missense variant p. Supporting information. S1 Fig. S2 Fig.
Prss56 ocular expression is restricted to the retina. S3 Fig. Prss56 expression in Prss56 mutant and control retina. S4 Fig. Generation of conditional Prss56 mutant mice. S5 Fig. S6 Fig. Increase in ocular anterior chamber depth following Prss56 ablation at P S7 Fig. S1 Table. Summary of ocular measurements in Prss56 mutant mice across ages. S2 Table.