Atoh7-independent specification of retinal ganglion cell identity
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Abstract
Retinal ganglion cells (RGCs), which relay visual information from the eye to the brain, are the first cell type generated during retinal neurogenesis. Loss of function of the transcription factor Atoh7 , which is expressed in multipotent early neurogenic retinal progenitor cells, leads to a selective and near complete loss of RGCs. Atoh7 has thus been considered essential for conferring competence on progenitors to generate RGCs. However, when apoptosis is inhibited in Atoh7- deficient mice by loss of function of Bax , only a modest reduction in RGC number is observed. Single-cell RNA-Seq of Atoh7;Bax -deficient retinas shows that RGC differentiation is delayed, but that RGC precursors are grossly normal. Atoh7;Bax -deficient RGCs eventually mature, fire action potentials, and incorporate into retinal circuitry, but exhibit severe axonal guidance defects. This study reveals an essential role for Atoh7 in RGC survival, and demonstrates Atoh7- independent mechanisms for RGC specification.
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###Reviewer #3:
The bHLH transcription factor Atoh7 has been studied as a critical regulator of retinal ganglion cell (RGC) generation in several species but, as the authors detail in the Introduction, it is not clear how or at what stage it acts. For example, some (most) data suggest it is required to specify the RGC fate in precursors, while other data suggest it may be required for RGC differentiation and/or survival following their generation. Here, Brodie-Kommit et al. use a well-established method to distinguish these possibilities, deleting Atoh7 in the absence of Bax, a powerful proapoptotic gene. Both naturally occurring and genetically-provoked apoptosis of many neuronal types, including RGCs, have been shown to be prevented in Bax mutants, which are viable and generally healthy.
The authors show that RGCs survive and are …
###Reviewer #3:
The bHLH transcription factor Atoh7 has been studied as a critical regulator of retinal ganglion cell (RGC) generation in several species but, as the authors detail in the Introduction, it is not clear how or at what stage it acts. For example, some (most) data suggest it is required to specify the RGC fate in precursors, while other data suggest it may be required for RGC differentiation and/or survival following their generation. Here, Brodie-Kommit et al. use a well-established method to distinguish these possibilities, deleting Atoh7 in the absence of Bax, a powerful proapoptotic gene. Both naturally occurring and genetically-provoked apoptosis of many neuronal types, including RGCs, have been shown to be prevented in Bax mutants, which are viable and generally healthy.
The authors show that RGCs survive and are functionally normal in the absence of both Atoh7 and Bax, but few axons leave the retina, so of course light-induced behaviors are greatly decreased. Single cell RNAseq demonstrates a delay in RGC differentiation in the absence of Atoh7 (with or without Bax) and a variety of gene expression changes.
As expected for a paper from two superb laboratories, the work is done to the highest standard and uses the best available methods. The result that blocking apoptosis rescues RGCs in the absence of Atoh7 is important and should help resolve controversies about its role, providing a strong argument against what is likely still the best-accepted model.
On the other hand, the paper does not go far beyond that simple result: it shows what Atoh7 does not do, but not what it does do, either to the RGCs that express it or to the RGCs that do not express it but nonetheless require it for survival. The physiological and histological data largely back up the survival result; the behavioral defects are sort of trivial once one knows that RGC axons fail to reach the brain; and the RNAseq data do not lead to substantial novel insights that shed light on either the presumably cell-autonomous or the clearly cell-nonautonomous mechanisms.
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###Reviewer #2:
In the present manuscript the authors reveal that RGC differentiation is largely rescued in the absence of Atoh7 when the pro apoptotic gene Bax is also removed in the developing retina. These rescued RGCs show some proper physiological responses but fail to develop proper connections to the brain. Retina vasculature is also affected by the absence of Atoh7 even when RGCs are "rescued". Finally by single cell analysis they reveal that Atho7 is required for proper timing of RGC differentiation but the expression of major markers for RGC can be independent from Aoh7 transcriptional activity. The paper is based on a series of very elegant genetic experiments and the single cell analysis is particularly illuminating in this context.
Major Points:
Cell death is only one of the RPC possible fates in the absence of Atoh7. …
###Reviewer #2:
In the present manuscript the authors reveal that RGC differentiation is largely rescued in the absence of Atoh7 when the pro apoptotic gene Bax is also removed in the developing retina. These rescued RGCs show some proper physiological responses but fail to develop proper connections to the brain. Retina vasculature is also affected by the absence of Atoh7 even when RGCs are "rescued". Finally by single cell analysis they reveal that Atho7 is required for proper timing of RGC differentiation but the expression of major markers for RGC can be independent from Aoh7 transcriptional activity. The paper is based on a series of very elegant genetic experiments and the single cell analysis is particularly illuminating in this context.
Major Points:
Cell death is only one of the RPC possible fates in the absence of Atoh7. Indeed the author and a vast amount of literature showed that in the absence of Atoh7 more adopt photoreceptor precursor fate among others. Is the block of apoptosis by Bax inactivation reducing this "ectopic differentiation phenotype" in addition to RGC fate restoration?
Linked to the previous point, does the single cell data reveal why some progenitors die in the absence of Atho7 while others change fate?
The authors should discuss this point in more detail.
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###Reviewer #1:
This manuscript challenges the notion that the transcription factor Atoh7 is required to confer neurogenic retinal progenitors the competence of generating retinal ganglion cells (RGCs), the first-born neurons of the retina. This idea is based on the evidence that Atoh7 inactivation in mice causes the loss of the majority of RGCs. Here the authors have generated a Atoh7Cre/Cre;Bax-/- mouse line to ask what happens if apoptotic cell death is prevented in Atoh7 null mice. Using a number of RGC markers, they show that in the adult retina a large number of RGCs are no longer lost and are functionally connected with other retinal cell types as the retinas generate light driven photic responses. However, the RGCs of the Atoh7Cre/Cre;Bax-/- mice cannot connect with brain targets as the axons (when present) do not exit the optic …
###Reviewer #1:
This manuscript challenges the notion that the transcription factor Atoh7 is required to confer neurogenic retinal progenitors the competence of generating retinal ganglion cells (RGCs), the first-born neurons of the retina. This idea is based on the evidence that Atoh7 inactivation in mice causes the loss of the majority of RGCs. Here the authors have generated a Atoh7Cre/Cre;Bax-/- mouse line to ask what happens if apoptotic cell death is prevented in Atoh7 null mice. Using a number of RGC markers, they show that in the adult retina a large number of RGCs are no longer lost and are functionally connected with other retinal cell types as the retinas generate light driven photic responses. However, the RGCs of the Atoh7Cre/Cre;Bax-/- mice cannot connect with brain targets as the axons (when present) do not exit the optic disk but grow in a disorganized manner within the fibre layer. As an additional feature, the hyoloid artery does not regress. In Atoh7Cre/Cre;Bax-/- embryos, RGC generation is delayed as determined by analysis of single cell RNA-seq. The authors conclude that Atoh7 is required for RGC survival but dispensable for their specification.
This is an interesting study that adds up to the existing literature related to the role of Atoh7 in RGC generation/differentiation. However, the conclusion seems rather stretched: do the cells generated in the absence of Atoh7 and Bax really have a (full) RGC identity as claimed in the title? Is the specification of ALL RGC really independent of Atoh7? Conclusions should be toned down and alternative interpretations should be offered. Indeed, preventing apoptosis does rescue the full number of RGCs (see for example melanopsin positive cells). The lack of Isl1 in Fig. 4 and the low number of Brn3a+ cells in Fig. S6 is rather striking and suggests more than a delay. Thus, at least a subset of RGCs seems to require Atoh7, likely early born RGCs. There are several studies indicating that RGCs secrete factors that regulate their own number (GDF11, Kim et al., 2005, Science, as an example). Lack of this feed-back at early stages may favour the generation of RGCs that are not full Atoh7-dependent, creating an imbalance between Atoh7-dependent (early) and Atoh7-independent (late) RGCs.
The second problem that remains unanswered is related to the "identity" of the RGCs present in Atoh7Cre/Cre;Bax-/- embryos. Are they really bona fide RGCs? These cells cannot connect properly with their brain target nor secrete the putative factors needed to induce hyaloid artery regression. These defects could perhaps be explained by asynchrony (cells are generated late to read the axon guidance cues, for examples) but they may also be interpreted as lack of full identity.
The authors need to consider these possibilities and further address the related points below:
The difference in cell number detected with RBPMS and Isl1 is puzzling (Fig. 1). Isl1 recognises RGCs but also amacrine cells, which should be increased in absence of Atoh7. How do the authors explain that Isl1+ cells are less than the RBPMS+ ones in Atoh7Cre/Cre;Bax-/- mice.
The sentence "Brn3b-positive ipRGCs differentiate normally in the absence of Atoh7" is an overstatement. Only 35% of them do, the others are presumably lost. Furthermore, the presence of a cell specific marker does not ensure that the cells are fully differentiated.
Line 435. Presumably a sentence describing the response of RGC in Atoh7Cre/Cre;Bax-/- is missing.
Lines 506-509. Failed vasculature regression: the authors state "...implies that Brn3b and Isl1 may activate expression of secreted factors that drive vascular regression". If this is the case why in Atoh7Cre/Cre;Bax-/- retinas the hyaloid artery is still present? The retinas do express levels of Isl1 and Brn3b so that these factors should be present.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
The three reviewers agree that the study is very elegant and well performed. However, they also find that the conclusions are rather stretched and there is no clear demonstration of what Atoh7 is needed for. Major concerns relate to the real identity of the rescued cells and the claimed independence of RGC specification from Atoh7. Unfortunately, the RNAseq data do not illuminate this issue or solve the cell-autonomous and non cell-non-autonomous mechanisms that are at the basis of the present observations.
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