Blocking the action of CD44 and IMPG1 in pluripotent stem cell derived retinal organoids affected the development of photoreceptors, their inner/outer segments and connecting cilia and disrupted IPM formation, with IMPG1 having an earlier and more significant impact. Together, our data suggest an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation during human retinogenesis.
ExperimentName: Blocking IMPG1 in retinal organoids disrupts early development of IPM, photoreceptors and inner/outer segments, and connecting cilia.
NonPatientCell: UBERON:0000966 (retina, specifically IPSC-derived retinal organoids) or CL:0009004 (retinal cell)
GeneFunction: GO:0033165 (interphotoreceptor matrix)
GeneAlteration: Alteration was not performed at the genetic level, but rather a blocking antibody to IMPG1 protein was added to the culture media of the developing retinal organoid at days 90 and 150 of the differentiation process. After incubation, the effects were assessed by IHC and qRT-PCR detecting key markers of differentiation (Figure 6, Figures S12-S15).
FunctionalEvidence: Expression of RCVRN (a marker of photoreceptor and bipolar cells) and proportion of recoverin-positive cells were reduced (Figs. 6B, S12B, S14B, S14C, S15B). Expression of ARL13B (marker of photoreceptor connecting cilia) was reduced (Figs. 6B, S15B). Expression of RBP3 (marker of IPM formation) was changed only at the later time point (Figs. 6B, S12B, S15B). Expression of OPN1LW/OPN1MW (cone marker) was down-regulated (Fig. S14B). Expression of RHO (rod cell marker) and proportion of rhodopsin-positive cells were reduced, and the typical pattern of staining was replaced with a punctate pattern at the surface of the organoids (Fig. 6B, 6C). Staining for WGA (rod inner/outer segment marker) was reduced, indicating disrupted development of these structures (Fig. 6B, 6C). The authors conclude that IMPG1 is important for development of photoreceptors, inner and outer segments, connecting cilia, and IPM.
FAVariants: No variant in IMPG1 was used, but rather an inhibitory antibody.