Genome editing in the unicellular holozoan Capsaspora owczarzaki suggests a premetazoan role for the Hippo pathway in multicellular morphogenesis
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Evaluation Summary:
In this study, Phillips et al. report the ancient role of Hippo signaling nuclear effector YAP/TAZ/Yorkie ortholog (coYki) in a unicellular organism Capsaspora owczarzaki. Two major advances of this work include development of genome editing in Capsaspora owczarzaki and characterization of a key gene coYki. The authors found that different from Yki's role in multicellular organisms, the coYki does not contribute to cell proliferation. Instead, it affects cell adhesion to extracellular matrix to ensure the spherical shape of the aggregates. Overall, the presentation of this manuscript is clear with a nice logical flow, and this study should be of interest to the evolutionary cell biology field.
(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
Animal development is mediated by a surprisingly small set of canonical signaling pathways such as Wnt, Hedgehog, TGF-beta, Notch, and Hippo pathways. Although once thought to be present only in animals, recent genome sequencing has revealed components of these pathways in the closest unicellular relatives of animals. These findings raise questions about the ancestral functions of these developmental pathways and their potential role in the emergence of animal multicellularity. Here, we provide the first functional characterization of any of these developmental pathways in unicellular organisms by developing techniques for genetic manipulation in Capsaspora owczarzaki , a close unicellular relative of animals that displays aggregative multicellularity. We then use these tools to characterize the Capsaspora ortholog of the Hippo signaling nuclear effector YAP/TAZ/Yorkie (coYki), a key regulator of tissue size in animals. In contrast to what might be expected based on studies in animals, we show that coYki is dispensable for cell proliferation but regulates cytoskeletal dynamics and the three-dimensional (3D) shape of multicellular structures. We further demonstrate that the cytoskeletal abnormalities of individual coYki mutant cells underlie the abnormal 3D shape of coYki mutant aggregates. Taken together, these findings implicate an ancestral role for the Hippo pathway in cytoskeletal dynamics and multicellular morphogenesis predating the origin of animal multicellularity, which was co-opted during evolution to regulate cell proliferation.
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Author Response
Reviewer #3 (Public Review):
Phillips and colleagues present results obtained by generating loss-of-function mutations in the YAP/TAZ ortholog of the unicellular holozoan Capsaspora owczarzaki. In previous work published collaboratively by the Pan and Ruiz-Trillo labs, the authors had shown that Capsaspora has orthologs of yorkie (yki) and hippo (hpo) and that when these genes were expressed in Drosophila they functioned in a way that was consistent with the well-characterized function of the Hippo pathway in regulating cell proliferation.
Characterizing the role of the pathway in Capsaspora required the ability to manipulate gene expression in that organism. In this manuscript, the authors describe remarkable progress in that area. They generate lines that stably express fluorescent proteins. Excitingly, they are …
Author Response
Reviewer #3 (Public Review):
Phillips and colleagues present results obtained by generating loss-of-function mutations in the YAP/TAZ ortholog of the unicellular holozoan Capsaspora owczarzaki. In previous work published collaboratively by the Pan and Ruiz-Trillo labs, the authors had shown that Capsaspora has orthologs of yorkie (yki) and hippo (hpo) and that when these genes were expressed in Drosophila they functioned in a way that was consistent with the well-characterized function of the Hippo pathway in regulating cell proliferation.
Characterizing the role of the pathway in Capsaspora required the ability to manipulate gene expression in that organism. In this manuscript, the authors describe remarkable progress in that area. They generate lines that stably express fluorescent proteins. Excitingly, they are able to use CRISPR/Cas9 and generate loss-of-function alleles using a donor-template strategy. These accomplishments pave the way for the study of Capsaspora using molecular tools.
The authors then use these technologies to generate biallelic loss of function mutations in Capsaspora. They find no evidence of defects in cell proliferation either when these cells are cultured by themselves or when they are mixed with wild-type cells. However, they do find evidence of abnormalities in the cytoskeleton. They find that the cells themselves, and the multicellular aggregates that they form are more irregular in shape. The cells appear to adhere to substrates better than wild-type cells. They show surface blebbing that changes in the cell cortex with evidence for altered actin dynamics.
From these experiments, the authors conclude that the ancestral function of the Hippo pathway is to regulate the cytoskeleton and that its ability to regulate cell proliferation was acquired more recently in evolution.
The technical achievements are impressive, the experiments are well designed and executed, and are presented clearly. I have no issues with them. However, I feel that two of the main conclusions that the authors make are not justified by the results.
- The authors seem convinced that CoYki functions as a transcriptional regulator. They seem to suggest that it is primarily a regulator of cytoskeletal genes. There is a body of work from the Fehon laboratory that Yki has a function at the cell cortex in Drosophila that is independent of its function as a transcriptional regulator. See the work by Xu et al. 2018; PMID30032991 (not cited in this paper). In the absence of data that shows the localization of CoYki, I don't see how the authors can tell where it is working (in the nucleus or at the cell cortex) to regulate the cytoskeleton.
To provide support for asserting that coYki is transcriptional regulator, we have done the following:
We have cited previous results showing that coYki and its binding partner coSd can, when expressed together in the Drosophila eye, induce transcription of Hippo pathway genes, indicating a role for coYki in transcriptional regulation
We have examined the localization fluorescent fusions of coYki and a coYki (coYki 4SA) mutant predicted to be nonphosphorylatable by upstream Hippo pathway kinases. Enrichment of coYki at the cell cortex was not detected. However, the 4SA mutant showed increased localization in the nucleus relative to the WT coYki protein, arguing for a nuclear function of coYki.
These data are therefore consistent with the prevailing view of Yki/YAP/TAZ as a transcriptional regulator in other species. Nevertheless, we cannot formally exclude the possibility that coYki may also affect the cytoskeleton through a non-transcriptional manner as described by Xu et al., which we have now stated in the Results section of our manuscript.
- Capsaspora and animals such as ourselves are equally separated by time from our last common ancestor. There is no reason to think that the function of signaling pathways in the Capsaspora lineage has been frozen in time while ours have evolved. Indeed, the amazing diversity of protists is consistent with lots of evolution in every lineage. One could easily argue from the same data that the ancestral function of the Hippo pathway was to regulate cell proliferation and that this was lost in the lineage that led to Capsaspora. As we learn more about the function of the Hippo pathway in diverse organisms, we will be in a better position to guess what the ancestral function was.
We agree that the function of signaling pathways in modern protists and their ancestors may not necessarily be identical, and that studies of Hippo signaling in other organisms, especially unicellular holozoans, may clarify which functions may have been ancestral, as we make a point to state at the end of our discussion. However, given that in animals Hippo signaling regulates the cytoskeleton and proliferation, and we find that in Capsaspora coYki affects the cytoskeleton but apparently not proliferation, it seems reasonable to us to suggest a model where cytoskeletal regulation was an ancient function, and the pathway was later co-opted for regulation of proliferation. We have added a section in the Discussion pointing out that we cannot, from our results, definitively conclude an ancestral Hippo pathway function.
In summary, this manuscript describes technological innovations that will have a big impact on those who want to study this organism. They also provide convincing data to show that the Capsaspora Yorkie ortholog regulates cytoskeletal dynamics and not cell proliferation. However, as described above, the authors would need to tone down some of their conclusions.
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Evaluation Summary:
In this study, Phillips et al. report the ancient role of Hippo signaling nuclear effector YAP/TAZ/Yorkie ortholog (coYki) in a unicellular organism Capsaspora owczarzaki. Two major advances of this work include development of genome editing in Capsaspora owczarzaki and characterization of a key gene coYki. The authors found that different from Yki's role in multicellular organisms, the coYki does not contribute to cell proliferation. Instead, it affects cell adhesion to extracellular matrix to ensure the spherical shape of the aggregates. Overall, the presentation of this manuscript is clear with a nice logical flow, and this study should be of interest to the evolutionary cell biology field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive …
Evaluation Summary:
In this study, Phillips et al. report the ancient role of Hippo signaling nuclear effector YAP/TAZ/Yorkie ortholog (coYki) in a unicellular organism Capsaspora owczarzaki. Two major advances of this work include development of genome editing in Capsaspora owczarzaki and characterization of a key gene coYki. The authors found that different from Yki's role in multicellular organisms, the coYki does not contribute to cell proliferation. Instead, it affects cell adhesion to extracellular matrix to ensure the spherical shape of the aggregates. Overall, the presentation of this manuscript is clear with a nice logical flow, and this study should be of interest to the evolutionary cell biology field.
(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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Reviewer #1 (Public Review):
This study studies the cellular basis of the Yki ortholog using a unicellular organism Capsaspora owczarzaki. The authors made a genetic model for the loss-of-function of coYki and found distinct roles of this ortholog, which is unlike its roles in metazoans. These findings should enhance our understanding of the ancient roles of this factor. The tools they developed could also be useful for other people to use this unicellular organism as a model.
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Reviewer #2 (Public Review):
This manuscript reports two important advances: (1) the establishment of genome editing in Capsaspora and (2) the characterization of a Yki mutant. Genome editing in Capsaspora is an important advance that will no doubt greatly increase the experimental tractability of this organism, which sits on a key position in the tree of life. It was a particularly suitable target for the study of Yki function, as it contains many components of the Hippo pathway that are conserved in animals but missing from choanoflagellates. Most of the data are convincing and well documented. Particularly striking is the result in Fig. 4E, in which treatment of Yki mutants with blebbistatin leads to the rescue of the normal round aggregate phenotype.
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Reviewer #3 (Public Review):
Phillips and colleagues present results obtained by generating loss-of-function mutations in the YAP/TAZ ortholog of the unicellular holozoan Capsaspora owczarzaki. In previous work published collaboratively by the Pan and Ruiz-Trillo labs, the authors had shown that Capsaspora has orthologs of yorkie (yki) and hippo (hpo) and that when these genes were expressed in Drosophila they functioned in a way that was consistent with the well-characterized function of the Hippo pathway in regulating cell proliferation.
Characterizing the role of the pathway in Capsaspora required the ability to manipulate gene expression in that organism. In this manuscript, the authors describe remarkable progress in that area. They generate lines that stably express fluorescent proteins. Excitingly, they are able to use …
Reviewer #3 (Public Review):
Phillips and colleagues present results obtained by generating loss-of-function mutations in the YAP/TAZ ortholog of the unicellular holozoan Capsaspora owczarzaki. In previous work published collaboratively by the Pan and Ruiz-Trillo labs, the authors had shown that Capsaspora has orthologs of yorkie (yki) and hippo (hpo) and that when these genes were expressed in Drosophila they functioned in a way that was consistent with the well-characterized function of the Hippo pathway in regulating cell proliferation.
Characterizing the role of the pathway in Capsaspora required the ability to manipulate gene expression in that organism. In this manuscript, the authors describe remarkable progress in that area. They generate lines that stably express fluorescent proteins. Excitingly, they are able to use CRISPR/Cas9 and generate loss-of-function alleles using a donor-template strategy. These accomplishments pave the way for the study of Capsaspora using molecular tools.
The authors then use these technologies to generate biallelic loss of function mutations in Capsaspora. They find no evidence of defects in cell proliferation either when these cells are cultured by themselves or when they are mixed with wild-type cells. However, they do find evidence of abnormalities in the cytoskeleton. They find that the cells themselves, and the multicellular aggregates that they form are more irregular in shape. The cells appear to adhere to substrates better than wild-type cells. They show surface blebbing that changes in the cell cortex with evidence for altered actin dynamics.
From these experiments, the authors conclude that the ancestral function of the Hippo pathway is to regulate the cytoskeleton and that its ability to regulate cell proliferation was acquired more recently in evolution.
The technical achievements are impressive, the experiments are well designed and executed, and are presented clearly. I have no issues with them. However, I feel that two of the main conclusions that the authors make are not justified by the results.
The authors seem convinced that CoYki functions as a transcriptional regulator. They seem to suggest that it is primarily a regulator of cytoskeletal genes. There is a body of work from the Fehon laboratory that Yki has a function at the cell cortex in Drosophila that is independent of its function as a transcriptional regulator. See the work by Xu et al. 2018; PMID30032991 (not cited in this paper). In the absence of data that shows the localization of CoYki, I don't see how the authors can tell where it is working (in the nucleus or at the cell cortex) to regulate the cytoskeleton.
Capsaspora and animals such as ourselves are equally separated by time from our last common ancestor. There is no reason to think that the function of signaling pathways in the Capsaspora lineage has been frozen in time while ours have evolved. Indeed, the amazing diversity of protists is consistent with lots of evolution in every lineage. One could easily argue from the same data that the ancestral function of the Hippo pathway was to regulate cell proliferation and that this was lost in the lineage that led to Capsaspora. As we learn more about the function of the Hippo pathway in diverse organisms, we will be in a better position to guess what the ancestral function was.
In summary, this manuscript describes technological innovations that will have a big impact on those who want to study this organism. They also provide convincing data to show that the Capsaspora Yorkie ortholog regulates cytoskeletal dynamics and not cell proliferation. However, as described above, the authors would need to tone down some of their conclusions.
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