Afadin-deficient retinas exhibit severe neuronal lamination defects but preserve visual functions

Reviewer #1 (Public review):

Summary:

The question of how central nervous system (CNS) lamination defects affect functional integrity is an interesting topic, though it remains a subject of debate. The authors focused on the retina, which is a relatively simple yet well-laminated tissue, to investigate the impact of afadin - a key component of adherens junctions on retinal structure and function. Their findings show that the loss of afadin leads to significant disruptions in outer retinal lamination, affecting the morphology and localization of photoreceptors and their synapses, as illustrated by high-quality images. Despite these severe changes, the study found that some functions of the retinal circuits, such as the ability to process light stimuli, could still be partially preserved. This research offers new insights into the relationship between retinal lamination and neural circuit function, suggesting that altered retinal morphology does not completely eliminate the capacity for visual information processing.

Strengths:

The retina serves as an excellent model for investigating lamination defects and functional integrity due to its relatively simple yet well-organized structure, along with the ease of analyzing visual function. The images depicting outer retinal lamination, as well as the morphology and localization of photoreceptors and their synapses, are clear and well-described. The paper is logically organized, progressing from structural defects to functional analysis. Additionally, the manuscript includes a comprehensive discussion of the findings and their implications.

Weaknesses:

While this work presents a wealth of descriptive data, it lacks quantification, which would help readers fully understand the findings and compare results with those from other studies. Furthermore, the molecular mechanisms underlying the defects caused by afadin deletion were not explored, leaving the role of afadin and its intracellular signaling pathways in retinal cells unclear. Finally, the study relied solely on electrophysiological recordings to demonstrate RGC function, which may not be robust enough to support the conclusions. Incorporating additional experiments, such as visual behavior tests, would strengthen the overall conclusions.

Reviewer #2 (Public review):

Summary:

Ueno et al. described substantial changes in the afadin knockout retina. These changes include decreased numbers of rods and cones, an increased number of bipolar cells, and disrupted somatic and synaptic organization of the outer limiting membrane, outer nuclear layer, and outer plexiform layer. In contrast, the number and organization of amacrine cells and retinal ganglion cells remain relatively intact. They also observed changes in ERG responses and RGC receptive fields and functions using MEA recordings.

Strengths:

The morphological characterization of retinal cell types and laminations is detailed and relatively comprehensive.

Weaknesses:

(1) The major weakness of this study, perhaps, is that its findings are predominantly descriptive and lack any mechanistic explanation. As afadin is key component of adherent junctions, its role in mediating retinal lamination has been reported previously (see PMCID: PMC6284407). Thus, a more detailed dissection of afadin's role in processes, such as progenitor generation, cell migration, or the formation of retinal lamination would provide greater insight into the defects caused by knocking out afadin.

(2) The authors observed striking changes in the numbers of rods, cones, and BCs, but not in ACs or RGCs. The causes of these distinct changes in specific cell classes remain unclear. Detailed characterizations, such as the expression of afadin in early developing retina, tracing cell numbers across various early developmental time points, and staining of apoptotic markers in developing retinal cells, could help to distinguish between defects in cell generation and survival, providing a better understand of the underlying causes of these phenotypes.

(3) Although the total number of ACs or RGCs remains unchanged, their localizations are somewhat altered (Figures 2E and 4E). Again, the cause of the altered somatic localization in ACs and RGCs is unclear.

(4) One conclusion that the authors emphasise is that the function of RGCs remains detectable despite a major disrupted outer plexiform layer. However, the organization of the inner plexiform layer remains largely intact, and the axonal innervation of BCs remains unchanged. This could explain the function integrity of RGCs. In addition, the resolution of detecting RGCs by MEA is low, as they only detected 5 clusters in heterozygous animals. This represents an incomplete clustering of RGC functional types and does not provide a full picture of how functional RGC types are altered in the afadin knockout.

Minor Comments:

(1) Line 56-67: "Overall, these findings provide the first evidence that retinal circuit function can be partially preserved even when there are significant disruptions in retinal lamination and photoreceptor synapses" There is existing evidence showing substantial adaption in retinal function when retinal lamination or photoreceptor synapses are disrupted, such as PMCID: PMC10133175.

(2) Line 114-115: "we focused on afadin, which is a scaffolding protein for nectin and has no ortholog in mice." The term "Ortholog" is misused here, as the mouse has an afadin gene. Should the intended meaning be that afadin has no other isoforms in mouse?