Fish primary embryonic pluripotent cells assemble into retinal tissue mirroring in vivo early eye development
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Evaluation Summary:
The experimental system characterized in this paper opens up new avenues for studying mechanisms of retinal patterning and morphogenesis. The data presented make a compelling case for the emergence of complex multicellular structures upon re-aggregation of embryonic teleost cells, but open questions remain regarding the basic underlying principles.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Organoids derived from pluripotent stem cells promise the solution to current challenges in basic and biomedical research. Mammalian organoids are however limited by long developmental time, variable success, and lack of direct comparison to an in vivo reference. To overcome these limitations and address species-specific cellular organization, we derived organoids from rapidly developing teleosts. We demonstrate how primary embryonic pluripotent cells from medaka and zebrafish efficiently assemble into anterior neural structures, particularly retina. Within 4 days, blastula-stage cell aggregates reproducibly execute key steps of eye development: retinal specification, morphogenesis, and differentiation. The number of aggregated cells and genetic factors crucially impacted upon the concomitant morphological changes that were intriguingly reflecting the in vivo situation. High efficiency and rapid development of fish-derived organoids in combination with advanced genome editing techniques immediately allow addressing aspects of development and disease, and systematic probing of impact of the physical environment on morphogenesis and differentiation.
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Reviewer #3 (Public Review):
Zilova et al. investigate cell differentiation in aggregates made from cells of early medaka and zebrafish embryos upon culture in defined media. Using reporter lines and immunostaining, they find evidence for retinal differentiation and morphogenesis in these aggregates, the extent of which depends on the size of the aggregates. This dependence of patterning and morphogenesis on aggregate size indicates that these processes are at least partially controlled by cell-cell interactions in the population. The authors also perform experiments with cells from genetic mutants that indicate similar genetic control of retinal morphogenesis in aggregates and intact fish embryos.
This work is a nice example of morphogenesis of differentiated cell types upon dissociation and re-aggregation of early embryonic cells. The …
Reviewer #3 (Public Review):
Zilova et al. investigate cell differentiation in aggregates made from cells of early medaka and zebrafish embryos upon culture in defined media. Using reporter lines and immunostaining, they find evidence for retinal differentiation and morphogenesis in these aggregates, the extent of which depends on the size of the aggregates. This dependence of patterning and morphogenesis on aggregate size indicates that these processes are at least partially controlled by cell-cell interactions in the population. The authors also perform experiments with cells from genetic mutants that indicate similar genetic control of retinal morphogenesis in aggregates and intact fish embryos.
This work is a nice example of morphogenesis of differentiated cell types upon dissociation and re-aggregation of early embryonic cells. The similar behaviour of aggregates from evolutionarily distant species reported in the manuscript underscores the generality of the findings. Organoid formation from teleost cells recapitulates species-specific timescales and is therefore faster than organoid generation from mammalian cells which constitutes a potential technical advantage of this system. The major advance of this work lies in providing a clear example that organoids consisting of early neural and retinal cells can be formed in non-mammalian species. Such an approach can open up new avenues for describing basic principles of cell differentiation and pattern formation during embryogenesis, and can thereby be useful to the community.
While the reported observations are highly interesting, the level of quantitative analysis currently does not fully support all of the author's interpretations and conclusions.
The authors variably interpret their observations as the result of self-assembly or self-organization. At the moment, the data does not allow distinguishing whether the observed phenomena result from cells following largely cell-autonomous differentiation paths and come together through cell sorting, or whether dissociation and aggregation generates a condition that leads to (spatially restricted) retinal differentiation in cells that would not normally adopt this fate. I would say that the first scenario is consistent with self-assembly, while the second one is more self-organized in the sense that the new cell-cell interactions resulting from the aggregation result in emergent cellular behaviours. A first step to distinguish between these possibilities would be to quantitatively demonstrate that aggregation biases cell differentiation towards neural and retinal fates at the expense of other cell types, compared to the intact embryo. The examples shown in Figure 2 and 3d seem to indicate an overrepresentation of neural cells, but it would be good to see a quantitative comparison to the embryo.
The authors use the term "primary embryonic stem cells" for the early embryonic cells that they aggregate. I find this problematic as some cells in this population may already have lineage bias and not have true multi-lineage potential. I also understand there is a difference between the cells that are used in this study and the teleost embryonic stem cells referenced in lines 49 and 50, in the sense that the latter were established as true self-renewing cell lines. But correct me if I have missed something here.
The authors claim that their system is highly reproducible. Unfortunately, they do not give an indication of the success rate of aggregate formation in figure 1. Figure 4 shows the most complex patterns, but I realize that there is quite a bit of variability in between the aggregates - they are just as likely to have one or two Rx2-expressing areas (panel b). I also could not find information how many aggregates show the patterns in panels e and f, and from how many aggregates the data in panels g - i has been collected.
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Reviewer #2 (Public Review):
This study shows that dissociated blastula cells from teleost fishes (medaka and zebrafish) reaggregate to form optic vesicle-like organoids if cultured in the presence of extracellular matrix molecules. Notably, cell number is critical for a reaggregation with movements that resemble those observed in vivo. These organoids acquire dorso-ventral polarity and can differentiate into different retinal cell types.
This is well written manuscript describing a technological advance: the generation of an organoid from teleost cells. Some of the images are impressive as since blastula cells seem to reproduce an organized forebrain with bilateral optic vesicles. Still these vesicles are rudimentary when compared with those obtained from mouse or human cells (see work from Eiraku team).
There are no critiques to the …
Reviewer #2 (Public Review):
This study shows that dissociated blastula cells from teleost fishes (medaka and zebrafish) reaggregate to form optic vesicle-like organoids if cultured in the presence of extracellular matrix molecules. Notably, cell number is critical for a reaggregation with movements that resemble those observed in vivo. These organoids acquire dorso-ventral polarity and can differentiate into different retinal cell types.
This is well written manuscript describing a technological advance: the generation of an organoid from teleost cells. Some of the images are impressive as since blastula cells seem to reproduce an organized forebrain with bilateral optic vesicles. Still these vesicles are rudimentary when compared with those obtained from mouse or human cells (see work from Eiraku team).
There are no critiques to the work per se, which is technically impeccable, well illustrated and quantified. However, one wonder what happens to the RPE cells in the differentiation process. In Fig 4, the authors show that the optic vesicle organoids are organized as in vivo with cells expressing RPE markers. These cells are no longer present in Fig 5. What happens to them? There is no mention of this problem in the text.
The discussion is generally informative but somehow fails to provide real advantages of using teleost organoids vs the fish per se or vs for example human organoids. Indeed, obtaining a fish organoid is faster that a human one, but more expensive and time consuming than using fish embryos.
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Reviewer #1 (Public Review):
In this manuscript, Zilova et al. show that primary embryonic cells derived from blastula-stage Medaka and Zebrafish embryos can self-organize into retinal organoids. When aggregates of 1000-2000 primary embryonic cell are embedded in Matrigel addition, they form a neuroepithelium under the control of Rx3 which develops into a retinal organoid. The process mirrors some aspects of embryo development. Moreover, another interesting finding is that Rx3 expression is initiated in the absence of Matrigel at day 0, which indicates that the retinal fate occurs by default and is not dependent on extracellular matrix components. The authors compare the ability of cells from Mesaka and zebra fish and show that both are competent to form organoids, though each does it with the time scale of the embryo of origin. The …
Reviewer #1 (Public Review):
In this manuscript, Zilova et al. show that primary embryonic cells derived from blastula-stage Medaka and Zebrafish embryos can self-organize into retinal organoids. When aggregates of 1000-2000 primary embryonic cell are embedded in Matrigel addition, they form a neuroepithelium under the control of Rx3 which develops into a retinal organoid. The process mirrors some aspects of embryo development. Moreover, another interesting finding is that Rx3 expression is initiated in the absence of Matrigel at day 0, which indicates that the retinal fate occurs by default and is not dependent on extracellular matrix components. The authors compare the ability of cells from Mesaka and zebra fish and show that both are competent to form organoids, though each does it with the time scale of the embryo of origin. The authors show that by reducing the number of Medaka cells to aggregate (500-800 cells), Rx2 and Rx3 are expressed only in restricted regions of the small aggregates, presumably where they organize into discrete circular Rx2 and Rx3 positive neuroepithelial units that develop into structure resembling retinal epitjhelia with some diversity of retinal cell types including amacrine, ganglion, photoreceptor, bipolar and horizontal cells.
This is a novel and original piece of work that reveals the capacity of fish primary embryonic pluripotent cells to behave like mammalian embryonic stem cells and organize optic cup organoids.
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Evaluation Summary:
The experimental system characterized in this paper opens up new avenues for studying mechanisms of retinal patterning and morphogenesis. The data presented make a compelling case for the emergence of complex multicellular structures upon re-aggregation of embryonic teleost cells, but open questions remain regarding the basic underlying principles.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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