Glia regulate local retinoic acid levels to specify neuronal specialisation for high-acuity vision
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eLife Assessment
This important study investigates how retinoic acid signaling and retinal support cells called Müller glia might contribute to regional differences in the morphology and function of cone photoreceptors - light-sensitive neurons in the zebrafish retina - and may have broader implications for understanding how non-neuronal cells such as glia shape neuronal cell specialization in other regions of the nervous system. Despite well-executed imaging and pharmacological experiments, the evidence remains incomplete because the proposed mechanism is not established as specific to Müller glia, and broader effects on retinal development, cell growth, maturation, and/or differentiation could explain the observed shortening of light-sensitive structures. The work will interest researchers studying vision, nervous system development, and interactions between neurons and their supporting cells.
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Abstract
Neurons of the same type can be differentially tuned to perform distinct functional roles, yet how such specialisation arises during development remains poorly understood. Here, we show that in the zebrafish retina, heterogeneity within a single glial population generates local gradients of retinoic acid (RA) signalling that fine-tune neural function within a single neuronal type. The acute zone (AZ), a ventro-temporal region of the retina, is specialised for prey capture in the upper frontal visual field. In this region, UV cones exhibit enhanced light sensitivity through elongation of their outer segments (OS), the cellular compartment responsible for phototransduction. We found that cyp26 genes, which encode RA-degrading enzymes, are expressed in Müller glia in a region-specific manner, and that this expression pattern closely matches the spatial variation in cone OS length. Inhibition of Cyp26 activity prevented OS elongation in the AZ. This effect is mediated by RA signalling, as direct activation of RA receptors using RARα agonists similarly reduced OS length in this region. Notably, inhibition of Cyp26 predominantly activated RA signalling in Müller glia rather than in cones, indicating that RA signalling regulates cone OS length in a cell-non-autonomous manner via Müller glia. Consistent with this model, blocking Müller glia genesis abolished cone specialisation across all regions of the developing retina. Together, these findings identify Müller glia as key regulators of cone functional specialisation through the spatial control of RA signalling.
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eLife Assessment
This important study investigates how retinoic acid signaling and retinal support cells called Müller glia might contribute to regional differences in the morphology and function of cone photoreceptors - light-sensitive neurons in the zebrafish retina - and may have broader implications for understanding how non-neuronal cells such as glia shape neuronal cell specialization in other regions of the nervous system. Despite well-executed imaging and pharmacological experiments, the evidence remains incomplete because the proposed mechanism is not established as specific to Müller glia, and broader effects on retinal development, cell growth, maturation, and/or differentiation could explain the observed shortening of light-sensitive structures. The work will interest researchers studying vision, nervous system development, …
eLife Assessment
This important study investigates how retinoic acid signaling and retinal support cells called Müller glia might contribute to regional differences in the morphology and function of cone photoreceptors - light-sensitive neurons in the zebrafish retina - and may have broader implications for understanding how non-neuronal cells such as glia shape neuronal cell specialization in other regions of the nervous system. Despite well-executed imaging and pharmacological experiments, the evidence remains incomplete because the proposed mechanism is not established as specific to Müller glia, and broader effects on retinal development, cell growth, maturation, and/or differentiation could explain the observed shortening of light-sensitive structures. The work will interest researchers studying vision, nervous system development, and interactions between neurons and their supporting cells.
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Reviewer #1 (Public review):
Summary:
The authors examine the role of retinoic acid signaling in the formation of lengthened outer segments in UV cones in a localized region within the ventral temporal zebrafish retina. The authors demonstrate that the formation of this region of increased sensitivity depends on the degradation of retinoic acid by Cyp26, which is expressed in MG. Inhibiting Cyp26 or stimulating RA expression reduces OS length and visual sensitivity.
Strengths:
A key strength is the use of several experimental strategies to support conclusions. For example, the authors localize key enzymes in Müller glia (MG) and, in a separate experiment, to highlight the overall importance of MG, they ablate them. High acuity zones are critical visual specializations, and understanding their formation is important for vision research, …
Reviewer #1 (Public review):
Summary:
The authors examine the role of retinoic acid signaling in the formation of lengthened outer segments in UV cones in a localized region within the ventral temporal zebrafish retina. The authors demonstrate that the formation of this region of increased sensitivity depends on the degradation of retinoic acid by Cyp26, which is expressed in MG. Inhibiting Cyp26 or stimulating RA expression reduces OS length and visual sensitivity.
Strengths:
A key strength is the use of several experimental strategies to support conclusions. For example, the authors localize key enzymes in Müller glia (MG) and, in a separate experiment, to highlight the overall importance of MG, they ablate them. High acuity zones are critical visual specializations, and understanding their formation is important for vision research, retinal disease, and regenerative medicine. The manuscript also addresses a fundamental question in neuroscience: What are the specific signaling mechanisms that occur between neurons and glia during development?
Weaknesses:
The primary weakness is the use of inhibitors, which have systemic effects. This caveat has been appropriately addressed in the discussion.
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Reviewer #2 (Public review):
Summary:
This manuscript by Lahne et al. connects retinoic acid (RA) signaling regulation to the length of outer segments (OS) in UV-sensitive cone photoreceptors located in a specialized region of the zebrafish retina called the high acuity zone (AZ). The authors provide potentially compelling, but as it stands, incomplete, evidence that the Müller glia (possibly through expression of the Cyp26 RA-degrading enzyme) may act to regulate spatially restricted RA availability to UV cones in the AZ, supporting these UV cones to grow longer OS. While the evidence presented is indeed suggestive, it is not directly linked and is insufficient to support the full conclusions presented.
Strengths:
(1) The experiments make outstanding use of the strengths offered by the zebrafish model, including excellent reporter …
Reviewer #2 (Public review):
Summary:
This manuscript by Lahne et al. connects retinoic acid (RA) signaling regulation to the length of outer segments (OS) in UV-sensitive cone photoreceptors located in a specialized region of the zebrafish retina called the high acuity zone (AZ). The authors provide potentially compelling, but as it stands, incomplete, evidence that the Müller glia (possibly through expression of the Cyp26 RA-degrading enzyme) may act to regulate spatially restricted RA availability to UV cones in the AZ, supporting these UV cones to grow longer OS. While the evidence presented is indeed suggestive, it is not directly linked and is insufficient to support the full conclusions presented.
Strengths:
(1) The experiments make outstanding use of the strengths offered by the zebrafish model, including excellent reporter lines for RA signaling and Müller glia visualization, fluorescent in situ hybridization, high resolution fluorescent microscopy, and pharmacological manipulations; tools which are all well selected and utilized.
(2) The pharmacological manipulations allow temporally controlled modulation of RA signaling and Cyp26 enzyme activity at developmental timepoints of interest.
(3) Manipulation of RA signaling impacts cone OS lengths.
(4) The Müller glia express mRNA transcripts encoding the RA-degrading enzyme Cyp26 and respond to RA signaling modulation in a manner consistent with a role in regulating RA signaling.
(5) The use of DAPT to prevent Müller glia differentiation in the retina is used to attempt to implicate the Muller glia in the regulation of RA signaling and UV cone OS length.
Weaknesses:
(1) The experiments cannot rule out a role for other cell types in driving the outcomes studied (UV cone OS length). The expression of Cyp26 transcripts by the Müller glia does not specifically align with the high acuity zone, and other cell types show signal for these transcripts. Impacts on RA signaling, as well as Cyp26 expression can be detected in regions potentially implicating cell types other than the Müller glia. One region of Cyp26 transcript signal is consistent with the Retinal Pigment Epithelium (RPE), and cells in this same region also show RA signaling changes in response to pharmacological treatments.
(2) The analysis following pharmacological manipulations lacks quantification of UV cone numbers and their morphology after the treatments, which could indicate broader impacts on cones that may manifest in their outer segments.
(3) The cell-type assignment of RARE:GFP-positive ONL cells is central to the proposed non-cell-autonomous mechanism, yet representative colocalization images supporting the quantification are not fully shown.
(4) The SyGCaMP6f activity reporter experiments are a nice addition, but the results are not necessarily directly connected to OS length or to the Müller glia.
(5) The experiments using DAPT to prevent Muller glia development are not sufficiently executed to support the conclusions presented, and their efficacy in inhibiting Müller glia development, as well as UV-cone-specific impacts, are not demonstrated. These treatments could have broad impacts on photoreceptor maturation, retinal organization, or trophic support.
(6) The title refers to glial regulation of "local retinoic acid levels." However, the study measures: A synthetic RARE reporter, RA-responsive genes and pharmacological responses.
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Reviewer #3 (Public review):
Summary:
The manuscript titled "Glia regulate local retinoic acid levels to specify neuronal specialisation for high-acuity vision" by Lahne et al. examines the interaction of cellular and signaling mechanisms involved in neuronal specialization. While there have been rapid increases in the discovery and cataloguing of different cell types based on transcriptional signatures that can be uncovered by single-cell approaches, there is much less known about how variations in cell morphology within a cell type are governed. The highly stereotypical specializations in photoreceptor morphology found in high acuity zones of various species are an excellent example. This study implicates retinoic acid signaling as a functional driver of these photoreceptor specializations in addition to its previously described role …
Reviewer #3 (Public review):
Summary:
The manuscript titled "Glia regulate local retinoic acid levels to specify neuronal specialisation for high-acuity vision" by Lahne et al. examines the interaction of cellular and signaling mechanisms involved in neuronal specialization. While there have been rapid increases in the discovery and cataloguing of different cell types based on transcriptional signatures that can be uncovered by single-cell approaches, there is much less known about how variations in cell morphology within a cell type are governed. The highly stereotypical specializations in photoreceptor morphology found in high acuity zones of various species are an excellent example. This study implicates retinoic acid signaling as a functional driver of these photoreceptor specializations in addition to its previously described role in establishing the spatial positioning of high acuity zones within the neural tissue. Furthermore, evidence is presented that retinoic acid does not directly signal through photoreceptors, but instead this signal is received by Müller glia to non-cell-autonomously affect the photoreceptors. Overall, this study identifies a potential mechanism that integrates the broad patterning role of retinoic acid with its local effects on specializations within a cell type and suggests that glial cells are integral to this mechanism.
Strengths:
The major strengths of this study are the well-executed experiments and clearly communicated results. The application of pharmacological agents is useful for precise timing of target inhibition and to prevent effects on other retinoic acid-mediated processes.
Weaknesses:
There are two issues with the study that weaken the conclusions that are drawn. The first is that the primary evidence that Mueller glia are the cells receiving the retinoic acid signal is that the RARE reporter is active in these cells. However, it should be considered whether this reporter is capable of responding to all retinoic acid signaling. Is it possible that the construct's binding site sequences and/or their configuration will limit the ability to detect other receptor complexes that respond to retinoic acid? For example, will complexes that contain RorBeta or RxrGamma (two established retinoic acid receptors) in complex with thyroid receptors be able to activate this reporter? If that is the case, then it could be that the Mueller glia are shaping the retinoic acid availability in the local environment of cones through their expression of the Cyp26 genes, but the cones as well as the Müller glia are directly responding to the retinoic acid signaling. If there are methods to test this, such as directly activating/repressing retinoic acid signaling in cones or MG with some cell type specificity, that would greatly strengthen the argument that it is a non-cell autonomous effect. One experiment that does argue in favor of this is that the loss of MG leads to a small outer segment, but this is where the second issue should be considered. The study uses three methods (including genetic loss of MG) that block the acuity zone-specific elongation of the photoreceptors, but one concern is that the shortening of these outer segments is a non-specific effect. In all three of the treatments (Figures 3, 5, 9), the segments look somewhat disorganized and not simply like a conversion of the Acuity zone outer segments to the Dorsal state. It would be powerful to complement these approaches with a retinoic acid-related manipulation that does the opposite - that converts Dorsal outer segments into a longer Acuity zone-like morphology. That would be strong support for the proposed hypothesis.
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