Table 2.
Details of four differentially expressed genes prioritised for further characterisation with IHC. The bottom four rows refer to semiquantitative IHC co-localisation seen in Fig. 2, Fig. 3, Fig. 4, Fig. 5 p = - “adjusted p value”; n.s. – p > 0.05.
| Gene | Srm2 | Slf2 | Anxa7 | Cntn1 |
|---|---|---|---|---|
| Protein | Shroom 2 | Sulphatase 2 | Annexin a7 | Contactin 1 |
| Function | Actin cytoskeleton, axon guidance | HSPG metabolism | Membrane fusion, neurotransmitter release | Neuron projection development, synaptic remodelling |
| Comments | Regulates actin cyctoskeleton and therefore cell shape, axon sprouting and organelle location (essential for viral transduction, second messenger systems and neurotransmitter release) (Fairbank et al., 2006). | Extracellular endosulphatase. Removes sulphate residues from cell surface HSPG residues (Morimoto-Tomita et al., 2002) - important in the entry of AAV into cells (Summerford and Samulski, 1998) as well as retinal synaptic plasticity | Calcium dependant phospholipid binding protein implicated in synaptic neurotransmitter release and the bipolar cell light response (Caohuy and Pollard, 2002; Grewal et al., 2016; Hoque et al., 2014). PKCα (important in activation and termination of bipolar cell light response phosphorylates annexin a7 promoting membrane fusion and so neurotransmitter release (Hoque et al., 2014). PKCα expression was not altered in our gene array comparison. | A cell surface glycoprotein implicated in synaptic plasticity (Davisson et al., 2011) |
| Human disease caused defect in gene | Implicated in retinal degeneration with deafness (Fairbank et al., 2006) | Not reported | Not reported | Congenital Myopathy (Davisson et al., 2011) |
| Mouse knock outs | Knock down - failure of retinal lamination, full knock out - no retinal phenotype. Other Srm family members may compensate. (Fairbank et al., 2006) | Deficiencies in neural remodelling; no major developmental flaws or retinal defects reported (Masu, 2013) | No gross neurological phenotype, retina not examined (Grewal et al., 2016) | Impaired synaptic long-term depression (LTD) (Murai et al., 2002). Over-expression improves long term potentiation (LTP) (Gulisano et al., 2017). No retinal phenotype at P14 (Chatterjee et al., 2019) |
| Expression at P90 (gene array)in Pde6brd1/rd1vs Pde6bwt/wt (FDR adj’ p value) | Up (p = 0.042) | Up (p = 0.034) | Down (p = 0.024) | Down (p = 0.049) |
| Expected location of protein within cell | Cell membrane (Etournay et al., 2007) confirmed in cell culture (Fig. S1) & IHC (Fig. 2) | Cell surface, extracelluar (Morimoto-Tomita et al., 2002) Confirmed in cell culture (Fig. S1) & IHC (Fig. 3) | Cell membrane (Watson et al., 2004) confirmed in cell culture (Fig. S1) & IHC (Fig. 4) | Cell surface (Davisson et al., 2011), confirmed in cell culture (Fig. S1) & IHC (Fig. 5) |
| Previous IHC of Pde6bwt/wt retinae | Retinal pigment epithelium, bipolar cell bodies, inner plexiform layer (Etournay et al., 2007) | Bipolar cell bodies, outer plexiform layer photoreceptors synapses (Orlandi et al., 2018) | Not described previously in retina | Bipolar cell and outer plexiform layer (Davisson et al., 2011) |
| 2-way ANOVA – difference by genotype (Pde6brd1/rd1vs Pde6bwt/wt) | Yes F (1, 8) = 23.85; p = 0.0012 |
No F (1, 8) = 3.775; p = 0.0879 |
Yes F (1, 9) = 5.31; p = 0.0467 |
Yes F (1, 13) = 8.272; p = 0.0130 |
| 2-way ANOVA – difference by time | Yes F (3, 8) = 4.865; p = 0.0327 |
Yes F (3,8) = 19.9; p = 0.005 |
No F (3, 9) = 0.1829; p = 0.9053 |
No F (3, 13) = 0.168; p = 0.9161 |
| 2-way ANOVA - interaction genotype x time | Yes F (3,8) = 9.09; p = 0.0059 |
No F (3,8) = 2.718; p = 0.1148 |
No F (3,9) = 0.6712; p = 0.5909 |
Yes F (3,13) = 7.366; p = 0.0039 |
| Significant Post Hoc Tests |
Sidak P90 – p = 0.0136; p120 - p = 0.0026 Tukey Pde6brd1/rd1:P40 vs P150 p = 0.0307; P90 vs P150 p = 0.0157; Pde6bwt/wt:P40 vs P120 p = 0.0126); P120 vs P150 p = 0.0199 |
Tukey Pde6brd1/rd1:P40v s P90 p = 0.0083; P90 vs P150 p = 0.0028; P120 to P150 p = 0.0248; Pde6bwt/wt: P40 vs P150 p = 0.0146; P90 vs P150 p = 0.0052; P120 vs P150 p = 0.0116 |
Sidak Not significant at any individual timepoint |
Sidak P90 p = 0.0026 |