Retinal pigment epithelial integrity is compromised in the developing albino mouse retina

Abstract

In the developing murine eye, melanin synthesis in the retinal pigment epithelium (RPE) coincides with neurogenesis of retinal ganglion cells (RGCs). Disruption of pigmentation in the albino RPE is associated with delayed neurogenesis in the ventrotemporal retina, the source of ipsilateral RGCs, and a reduced ipsilateral RGC projection. To begin to unravel how melanogenesis and the RPE regulate RGC neurogenesis and cell subpopulation specification, we have compared the features of albino and pigmented mouse RPE cells during the period of RGC neurogenesis (embryonic day,E, 12.5 to 18.5) when the RPE is closely apposed to developing RGC precursors. At E12.5 and E15.5, although albino and pigmented RPE cells express RPE markers Otx2 and Mitf similarly, albino RPE cells are irregularly shaped and have fewer melanosomes compared with pigmented RPE cells. The adherens junction protein P-cadherin appears loosely distributed within the albino RPE cells rather than tightly localized on the cell membrane as in pigmented RPE. Connexin 43 (gap junction protein) is expressed in pigmented and albino RPE cells at E13.5 but at E15.5 albino RPE cells have fewer small connexin 43 puncta, and a larger fraction of phosphorylated connexin 43 at serine 368. These results suggest that the lack of pigment in the RPE results in impaired RPE cell integrity and communication via gap junctions between RPE and neural retina during RGC neurogenesis. Our findings should pave the way for further investigation of the role of RPE in regulating RGC development toward achieving a proper RGC axon decussation.

Activation of Wnt signaling reduces ipsilaterally-projecting retinal ganglion cells in pigmented retina

“…The albino RPE transcriptome was enriched in genes involved in cancer, signaling, promoter activity, cell differentiation, and cell cycle and proliferation. Gene sets associated with cytoskeleton and cell junction were enriched in albino compared with pigmented RPE, supporting our findings on disorganized RPE cell integrity in albino retina (Iwai-Takekoshi et al, 2016). Gene sets associated with signaling pathways and enriched in albino RPE included Hh, Notch, Bmp, Sfrp and Wnt (Fig.…”

“…For Cx43 and Sox2 (Wang et al, 2016) and Wif1, the unique sequence was amplified by PCR from E13.5 mouse RPE cDNA using primers generated from the mouse sequence. In some cases, to reduce the concentration of melanin in pigmented RPE, which masks the in situ hybridization signals, sections were bleached after color reaction of in situ hybridization as described previously (Foss et al, 1995;Iwai-Takekoshi et al, 2016;Orchard and Calonje, 1998). In brief, sections were incubated with 0.25% KMnO 4 (Sigma 23851) in PBS at room temperature for 30 min, washed in PBS and then incubated in 1% oxalic acid (Sigma, O-0376) at room temperature for 1 min and washed again in PBS.…”

“…In the developing albino mouse retina, ipsilateral RGC neurogenesis and subtype specification are disrupted (Bhansali et al, 2014;Herrera et al, 2003;Rebsam et al, 2012). During this period, albino RPE cells have irregular cell shape, fewer melanosomes and aberrant expression of junctional proteins (Iwai-Takekoshi et al, 2016). Because retinal neurons, including RGCs, are produced at the interface of the RPE and the neural retina, perturbed RPE cell integrity may result in aberrant RPE-neural retina communication and, in turn, altered ipsilateral RGC neurogenesis and subtype specification in albino mouse retina.…”

Retinal axon guidance at the midline: Chiasmatic misrouting and consequences

“…Further experiments will be needed to determine how L‐Dopa produced by RPE cells affects retinal neurogenesis. Finally, albino embryonic mouse retina has perturbed RPE cell integrity and altered gap junctions (Iwai‐Takekoshi et al, ), but whether these RPE deficits potentially impairing cell‐cell communications impact RGC neurogenesis remain to be determined. An interesting candidate downstream of the melanogenesis pathway is the glutamine transporter slc38a8 (SNAT8) (Poulter et al, ), as mutations in this gene cause the FHONDA syndrome, described as foveal hypoplasia and optic nerve decussation defects that are similar to those found in albinism but without any pigmentation defect (Al‐Araimi et al, ).…”

Conversations with Ray Guillery on albinism: linking Siamese cat visual pathway connectivity to mouse retinal development

“…These junctions could be important conduits for signaling molecules that regulate RPE integrity and the development of the neural retina, respectively (Jeffery, ; Cook & Becker, ), and that allow the passage of small molecules from cell to cell, such as ATP, which can control retinal proliferation as studied in wild type rodents (Pearson et al ., ). The localization and phosphorylation state of the gap‐junction forming protein Cx43 are defective in the embryonic albino RPE (Iwai‐Takekoshi et al ., ; Fig. ).…”