June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Cross-species comparison of non-canonical Wnt pathway expression in the developing outer plexiform layer
Author Affiliations & Notes
  • ROSANNA CALDERON
    Developmental, Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, California, United States
    Ophthalmology, Children's Hospital of Los Angeles, Los Angeles, California, United States
  • Kayla Stepanian
    Ophthalmology, Children's Hospital of Los Angeles, Los Angeles, California, United States
  • Angela Ferrario
    Ophthalmology, Children's Hospital of Los Angeles, Los Angeles, California, United States
  • G. Esteban Fernandez
    Cellular Imaging Core, The Saban Research Institute, Children's Hospital of Los Angeles, Los Angeles, California, United States
  • Aaron Nagiel
    Ophthalmology, Children's Hospital of Los Angeles, Los Angeles, California, United States
    Developmental, Stem Cell and Regenerative Medicine, University of Southern California, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   ROSANNA CALDERON None; Kayla Stepanian None; Angela Ferrario None; G. Esteban Fernandez None; Aaron Nagiel None
  • Footnotes
    Support  none
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1371 – F0302. doi:
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      ROSANNA CALDERON, Kayla Stepanian, Angela Ferrario, G. Esteban Fernandez, Aaron Nagiel; Cross-species comparison of non-canonical Wnt pathway expression in the developing outer plexiform layer. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1371 – F0302.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Photoreceptor axons terminate in the outer plexiform layer (OPL) of the retina, where they form synapses with the bipolar cells located in the inner nuclear layer (INL). The purpose of this study is to investigate the developmental expression of non-canonical Wnt signaling components in the OPL as potential mediators of circuit assembly in the outer retina.

Methods : Publicly available single-cell RNA sequencing (scRNAseq) data sets (GSE118614 and GSE138002) were analyzed for expression of non-canonical Wnt mediators, ligands, and receptors at various developmental timepoints in mouse and human retina. To validate expression of candidate genes, mouse (P6-P12 and adult, n=3), human fetal retina (human fetal week (HFW) 12-17, n=1) and human retinal organoid (HRO, n=3) sections were prepared using an RNase-free protocol and mRNA transcripts were detected using fluorescence in situ hybridization (FISH). FISH signal was quantified in the ONL and INL areas using the Find Maxima function on ImageJ. ANOVA analysis was performed to determine statistically significant changes between selected timepoints.

Results : Analysis of scRNAseq for 34 selected non-canonical Wnt pathway genes revealed 6 mediators, 3 Frizzled receptors and 4 Wnt ligands expressed in mouse and human retina during early OPL development. In both mouse and human retina, FISH confirmed expression of non-canonical Wnt mediators Dvl1, Celsr3, and Ryk in all nuclear layers, and of Vangl2 in the INL. The Wnt receptors Fzd3 and Fzd5 were expressed in the INL as early as P6 in mouse retina and GW17.4 in human fetal retina. The Wnt ligand Wnt5a was detected in the INL at P10 and showed a significant decrease in adults (p<0.001). Mouse Wnt2b co-localized with the amacrine cell marker ChAT. WNT10A expression was human-specific and restricted to the ONL (scRNAseq shows predominant rod expression).

Conclusions : Non-canonical Wnt signaling pathway components are expressed in the developing mouse and human outer retina during synaptic maturation of the OPL. Importantly, we found previously undescribed expression patterns (e.g., mouse Wnt2b in amacrine cells) and human-specific expression (e.g., Wnt10a in the ONL). This provides the basis for further studies to dissect the functional role of non-canonical Wnt genes in outer retinal development.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Figure 1: Confocal fluorescent in situ of Fzd3 transcripts in mouse, human fetal retina, and HRO.

Figure 1: Confocal fluorescent in situ of Fzd3 transcripts in mouse, human fetal retina, and HRO.

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