AGS3 antagonizes LGN to balance oriented cell divisions and cell fate choices in mammalian epidermis
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Descovich et al examine the important decision between proliferative (planar) and differentiation (perpendicular) divisions in the basal layers of the skin and find a key promoter of perpendicular divisions is inhibited by its paralog to specify planar divisions. The authors use sophisticated mouse genetics and imaging and discover that LGN and its paralog AGS3 function antagonistically in determining perpendicular vs. planar divisions. Some statements need to be tamed or further backed up, but overall this study provides a significant advance in the field.
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Abstract
Oriented cell divisions balance self-renewal and differentiation in stratified epithelia such as the skin epidermis. During peak epidermal stratification, the distribution of division angles among basal keratinocyte progenitors is bimodal, with planar and perpendicular divisions driving symmetric and asymmetric daughter cell fates, respectively. An apically restricted, evolutionarily conserved spindle orientation complex that includes the scaffolding protein LGN/Pins/Gpsm2 plays a central role in promoting perpendicular divisions and stratification, but why only a subset of cell polarize LGN is not known. Here, we demonstrate that the LGN paralog, AGS3/Gpsm1, is a novel negative regulator of LGN and inhibits perpendicular divisions. Static and ex vivo live imaging reveal that AGS3 overexpression displaces LGN from the apical cortex and increases planar orientations, while AGS3 loss prolongs cortical LGN localization and leads to a perpendicular orientation bias. Genetic epistasis experiments in double mutants confirm that AGS3 operates through LGN. Finally, clonal lineage tracing shows that LGN and AGS3 promote asymmetric and symmetric fates, respectively, while also influencing differentiation through delamination. Collectively, these studies shed new light on how spindle orientation influences epidermal stratification.
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Author Response
Reviewer #1 (Public Review):
The model put forward by the authors in this manuscript is a simple and exciting one, explaining the function of AGS3 as a negative regulator of LGN, acting as a 'dominant-negative' version of LGN. Overall, the results support the model very well, and the results shown in Fig 6, which clearly reveal the functional relevance of AGS3, add strength to the paper.
We thank the reviewer for their enthusiasm regarding our finding that AGS3 acts as an endogenous dominant-negative to inhibit LGN. We appreciate their assertion that the results support the model and that the functional relevance to epidermal stratification is a strength.
In Figures 3A and B, the authors claim that AGS3 overexpression leads to depolarization of LGN in epidermal stem cells. However, in the example provided in Figure …
Author Response
Reviewer #1 (Public Review):
The model put forward by the authors in this manuscript is a simple and exciting one, explaining the function of AGS3 as a negative regulator of LGN, acting as a 'dominant-negative' version of LGN. Overall, the results support the model very well, and the results shown in Fig 6, which clearly reveal the functional relevance of AGS3, add strength to the paper.
We thank the reviewer for their enthusiasm regarding our finding that AGS3 acts as an endogenous dominant-negative to inhibit LGN. We appreciate their assertion that the results support the model and that the functional relevance to epidermal stratification is a strength.
In Figures 3A and B, the authors claim that AGS3 overexpression leads to depolarization of LGN in epidermal stem cells. However, in the example provided in Figure 3A, the LGN signal appears to be stronger than the control, with more LGN still on the apical side (many would categorize this as 'apically polarized'). In the scoring shown in Figure 3B, I am not sure if 'eyeballing' is the right way to decide whether it is polarized/depolarized/absent. The authors should come up with a bit more quantitative method to quantify the localization/amount of LGN and explain the method well in the manuscript. A similar concern regarding the determination of the LGN localization pattern applies to the rest of figure 3 as well.
We agree with this important critique about the methodology used to assess LGN expression patterns. While we have historically included categorical analyses like those used in Fig. 3A,B in past publications (Williams et al, NCB 2014; Lough et al eLife, 2019), we have also now performed additional, unbiased, quantitative measures of LGN fluorescent intensity, as described in greater detail above. We added these new data in Fig. 4C-J, while the data previously in Fig. 3A,B have now been redistributed between Fig. 3E,F (overexpression) and Fig. 4A,B (knockdown).
Reviewer #2 (Public Review):
To date, only a handful of studies have addressed the importance of AGS3, a paralog of the relatively well-characterized spindle orientation factor LGN. The authors now show that AGS3 acts as a negative regulator of LGN and propose that this activity could work through competition for binding partner(s). Remarkably, regulation is temporally restricted in such a way that the conserved role played by LGN in metaphase spindle orientation is unaffected. Instead, AGS3 regulates a post-metaphase function for LGN, namely Telophase Correction. The article is well-written, the experiments are performed at a high level, and the claims are generally supported by the data. Two main points of confusion are raised in the current version. 1) The authors show that AGS3 regulates cortical localization of LGN, but would need to clarify how LGN is being affected. 2) The authors propose in the discussion that AGS3 might exert its regulatory effect through competition for NuMA, an important binding partner for LGN, but would need to clarify how and why NuMA would be involved in Telophase Correction.
We thank the reviewer for appreciating the novelty of our findings regarding the understudied LGN/pins paralog AGS3. In regards to the first point, as described earlier, we have added additional quantitative analyses of how AGS3 affects cortical LGN fluorescent intensity in Fig. 4C-J. We now show that AGS3 loss leads to broader and higher expression levels throughout mitosis, and therefore we have amended our model to soften the claim that AGS3 primarily operates during telophase correction. This renders the second point somewhat moot, but we nonetheless have expanded our Discussion to note that NuMA can be cortically recruited to the anaphase cortex independent of LGN (lines 531-542). We also contextualize our findings with the Reviewer’s own recent study which proposes a “threshold model” of cortical Insc as a determinant of spindle orientation (Neville et al, 2023), and speculate that a similar model could apply in our system, perhaps with AGS3 binding and sequesting Insc rather than NuMA (lines 543-556).
Reviewer #3 (Public Review):
This paper examines the mechanisms that control division orientation in the basal layers of the epidermis. Previous work established LGN as a key promoter of divisions where one of the siblings populates the differentiated layers (perpendicular). This work addresses two important, related issues - the mechanisms that determine whether a particular division is planar vs perpendicular, and the function of AGS3, and LGN paralog that has been enigmatic. A central finding is that AGS3 is required for the normal distribution of planar and perpendicular divisions (roughly equal) such that in its absence the distribution is skewed towards the perpendicular. Interestingly, however, the authors find that AGS3 has no detectable effect on orientation if the orientation is measured at anaphase. This timing aspect builds upon previous work from this group demonstrating a phenomenon they term "telophase correction" in which the orientation changes at the latest phases of division (and possibly post division?). Thus AGS3 seems to exert its effect using these later mechanisms and this is supported by further analysis by the authors. Importantly, the authors show that AGS3 acts through LGN, based on localization data and an epistasis analysis. The function of AGS3 has been highly enigmatic so resolving this issue while providing a useful step towards understanding how the division orientation decision is made, makes for exciting progress towards an important problem. I found the overall narrative and presentation to be quite good and especially appreciated the thoughtful discussion section that did an excellent job of putting the results in context and speculating how unknown aspects of the mechanism might work based on current clues. With that said, I think there are some important issues that should be resolved.
We thank the Reviewer for this excellent summary of our findings and appreciation of the significance of the issues that our study addresses.
Regarding the orientation measurements, the authors should specify how the midbody marker was used to mark sibling cells, especially given the midbody can move following division. For example, how can the authors be confident that the siblings in the middle panel of 1A are correct and not an adjacent cell? Regarding quantification, it would be useful for the authors to comment on how the following would influence their measurements: 1) movements along the z-axis, and 2) movement of the nucleus within the cell
We have used this methodology for over a decade, and while it is not flawless, we have included several safeguards to ensure that sibling cells are correctly identified. We have added additional details to the Methods section (lines 867-869, 873-879).
A similar question is how much telophase correction really happens in telophase. How confident are the authors that the process actually occurs during division and not subsequent to it? What is drawn in their previous paper and in Figure 7A implies that post-division movements may be important. It would be useful for the authors to comment on whether they can make the distinction and whether or not it might be important.
Our intent in coining the term “telophase correction” was to imply that this process initiates, rather than completes, during telophase. We apologize for this confusion and have clarified this in the text (lines 80-82). Since most mammalian cells complete M phase in ~1h, with the longest time spent in prophase, in the absence of direct evidence to the contrary, it may be prudent to assume that telophase, like metaphase and anaphase, is relatively short, on the order of minutes. Since we cannot directly observe reformation of the nuclear membrane in our movies, we cannot be sure when telophase ends. Likewise, we do not currently have a suitable marker of the spindle midbody for live-imaging, so cannot be sure when cytokinesis completes. That said, we feel confident that most of the reorientation is occurring prior to cytokinesis, because we have previously reported that the greatest changes in daughter cell positioning occur within the first 10-15 minutes of anaphase onset, when a gap in membrane-GFP/TdTomato is still visible (Lough et al, eLife, 2019). However, while we feel that there are many interesting questions that our work raises about the timing or reorientation relative to specific mitotic stages—e.g. is the midbody asymmetrically positioned, inherited, or ejected?—these questions are beyond the scope of the present study.
Does the division angle in the AGS3 OE experiment (Figure 1D) correlate with AGS3 levels within the cell?
This is an interesting question, and indeed, we our hypothesis would predict that it would. However, it is not straightforward to quantify AGS3 or mRFP1 levels, and as we explain in a new section of the Results (lines 212-237), we have some concerns that N-terminally tagged AGS3 may not be fully functional. We have added new data with C-terminally tagged AGS3-mKate2, which we feel provides even stronger evidence that mKate2+ cells show a planar shift compared to mKate2- cells (Fig. 3C,D). In the future, we could test this hypothesis at the population level by comparing division orientation profiles for AGS3-mKate2+ cells carrying either a non-targeting scramble or Gpsm11147 shRNA. We would predict that knocking down endogenous AGS3 while overexpressing AGS3-mKate2 should give an intermediate phenotype.
I found the localization data to be the weakest part of the paper and feel that some reconsideration and reanalysis are warranted. First, the quantifications in Figures 2C, 3B, and 3F are unnecessarily vague scoring-based metrics. In 2C, "Localization pattern" should be replaced with membrane/cytoplasm ratio or an equivalent quantification. In 3B "LGN localization" should be replaced with apical/cytoplasmic and apical/basal ratios or equivalents. In 3F, "Polarized LGN frequency" should be replaced with apical/basal ratio or equivalent. It seems to me that non-AI processed data would be most appropriate for these quantifications unless such processing can be justified.
This issue was raised by the previous two Reviewers and has been addressed by new data added to Figure 4.
Second, it is important to note that the cytoplasmic localization of AGS3 does not allow one to conclude that AGS3 is not on the membrane. Unfortunately, high cytoplasmic signal can preclude the determination of membrane-bound signal.
We agree with the Reviewer and have softened our language throughout the text.
Finally, I had difficulty reconciling the images of LGN shown in Figure 3 with the conclusions made by the authors.
We have added additional, representative images of LGN expression in control and AGS3 KD cells in Figure 4C-E.
The challenge of the localization data is troubling because an important conclusion of the paper is that AGS3 acts via LGN. The localization data provided one leg of support for this conclusion and the other is provided by an epistasis analysis. Unfortunately, this data seems to be right on the edge because it is based on the difference between the solid and dashed blue lines in Figure 5B not being significant. However, we can see how close this is by comparing the solid and dashed red lines in the adjacent 5C, which are significantly different. Between the localization data, which doesn't seem clear cut, and the epistasis experiment, which is on the razor's edge, I'm concerned that the conclusion that AGS3 acts through LGN may be going beyond what the data allows.
We appreciate the Reviewer’s comments about the importance of these two lines of experimentation: 1) AGS3’s effect on LGN localization, and 2) epistasis experiments between AGS3/Gpsm1 and LGN/Gpsm2. We feel we have significantly strengthened this first pillar with the additional data presented in Fig. 4C-J. Regarding the second point, we would like to emphasize that we present three lines of evidence for the existence of an epistatic relationship between LGN and AGS3: 1) the static division orientation data comparing LGN single KOs to both LGN KO + AGS3 KD and AGS3+LGN dKOs (Fig. 6B); 2) live imaging division orientation/telophase correction comparing LGN KOs to AGS3+LGN dKOs (Fig. 6C-E); 3) lineage tracing data comparing LGN KOs to AGS3+LGN dKOs (Fig. 7H,I). Further, we think the reviewer may have misconstrued the data presented in Fig. 5C (now Fig. 6C). The dashed lines indicate orientation at anaphase and solid lines 1h after anaphase, so the shift between dashed and solid lines indicates telophase correction, which occurs to similar (and statiscially significant) degrees in both LGN single mutants and AGS3+LGN dKOs. Comparisons between the single and double mutant would be between red and magenta solid lines or red and magenta dashed lines, and neither of these are statistically significant. We realize that our use of dashed lines in Fig. 5B (now Fig. 6B), which we normally only use to refer to anaphase entry in live imaging data, may have caused this confusion. Therefore, we have changed all plots to solid lines¬ in Fig. 6B, and use light and dark magenta, respectively, to differentiate between LGN KO + AGS3 KD and AGS3+LGN dKOs.
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**eLife assessment
**
Descovich et al examine the important decision between proliferative (planar) and differentiation (perpendicular) divisions in the basal layers of the skin and find a key promoter of perpendicular divisions is inhibited by its paralog to specify planar divisions. The authors use sophisticated mouse genetics and imaging and discover that LGN and its paralog AGS3 function antagonistically in determining perpendicular vs. planar divisions. Some statements need to be tamed or further backed up, but overall this study provides a significant advance in the field. -
Reviewer #1 (Public Review):
The model put forward by the authors in this manuscript is a simple and exciting one, explaining the function of AGS3 as a negative regulator of LGN, acting as a 'dominant-negative' version of LGN. Overall, the results support the model very well, and the results shown in Fig 6, which clearly reveal the functional relevance of AGS3, add strength to the paper.
In Figures 3A and B, the authors claim that AGS3 overexpression leads to depolarization of LGN in epidermal stem cells. However, in the example provided in Figure 3A, the LGN signal appears to be stronger than the control, with more LGN still on the apical side (many would categorize this as 'apically polarized'). In the scoring shown in Figure 3B, I am not sure if 'eyeballing' is the right way to decide whether it is polarized/depolarized/absent. The …
Reviewer #1 (Public Review):
The model put forward by the authors in this manuscript is a simple and exciting one, explaining the function of AGS3 as a negative regulator of LGN, acting as a 'dominant-negative' version of LGN. Overall, the results support the model very well, and the results shown in Fig 6, which clearly reveal the functional relevance of AGS3, add strength to the paper.
In Figures 3A and B, the authors claim that AGS3 overexpression leads to depolarization of LGN in epidermal stem cells. However, in the example provided in Figure 3A, the LGN signal appears to be stronger than the control, with more LGN still on the apical side (many would categorize this as 'apically polarized'). In the scoring shown in Figure 3B, I am not sure if 'eyeballing' is the right way to decide whether it is polarized/depolarized/absent. The authors should come up with a bit more quantitative method to quantify the localization/amount of LGN and explain the method well in the manuscript. A similar concern regarding the determination of the LGN localization pattern applies to the rest of figure 3 as well.
-
Reviewer #2 (Public Review):
To date, only a handful of studies have addressed the importance of AGS3, a paralog of the relatively well-characterized spindle orientation factor LGN. The authors now show that AGS3 acts as a negative regulator of LGN and propose that this activity could work through competition for binding partner(s). Remarkably, regulation is temporally restricted in such a way that the conserved role played by LGN in metaphase spindle orientation is unaffected. Instead, AGS3 regulates a post-metaphase function for LGN, namely Telophase Correction.
The article is well-written, the experiments are performed at a high level, and the claims are generally supported by the data. Two main points of confusion are raised in the current version. 1) The authors show that AGS3 regulates cortical localization of LGN, but would need …
Reviewer #2 (Public Review):
To date, only a handful of studies have addressed the importance of AGS3, a paralog of the relatively well-characterized spindle orientation factor LGN. The authors now show that AGS3 acts as a negative regulator of LGN and propose that this activity could work through competition for binding partner(s). Remarkably, regulation is temporally restricted in such a way that the conserved role played by LGN in metaphase spindle orientation is unaffected. Instead, AGS3 regulates a post-metaphase function for LGN, namely Telophase Correction.
The article is well-written, the experiments are performed at a high level, and the claims are generally supported by the data. Two main points of confusion are raised in the current version. 1) The authors show that AGS3 regulates cortical localization of LGN, but would need to clarify how LGN is being affected. 2) The authors propose in the discussion that AGS3 might exert its regulatory effect through competition for NuMA, an important binding partner for LGN, but would need to clarify how and why NuMA would be involved in Telophase Correction.
-
Reviewer #3 (Public Review):
This paper examines the mechanisms that control division orientation in the basal layers of the epidermis. Previous work established LGN as a key promoter of divisions where one of the siblings populates the differentiated layers (perpendicular). This work addresses two important, related issues - the mechanisms that determine whether a particular division is planar vs perpendicular, and the function of AGS3, and LGN paralog that has been enigmatic. A central finding is that AGS3 is required for the normal distribution of planar and perpendicular divisions (roughly equal) such that in its absence the distribution is skewed towards the perpendicular. Interestingly, however, the authors find that AGS3 has no detectable effect on orientation if the orientation is measured at anaphase. This timing aspect builds …
Reviewer #3 (Public Review):
This paper examines the mechanisms that control division orientation in the basal layers of the epidermis. Previous work established LGN as a key promoter of divisions where one of the siblings populates the differentiated layers (perpendicular). This work addresses two important, related issues - the mechanisms that determine whether a particular division is planar vs perpendicular, and the function of AGS3, and LGN paralog that has been enigmatic. A central finding is that AGS3 is required for the normal distribution of planar and perpendicular divisions (roughly equal) such that in its absence the distribution is skewed towards the perpendicular. Interestingly, however, the authors find that AGS3 has no detectable effect on orientation if the orientation is measured at anaphase. This timing aspect builds upon previous work from this group demonstrating a phenomenon they term "telophase correction" in which the orientation changes at the latest phases of division (and possibly post division?). Thus AGS3 seems to exert its effect using these later mechanisms and this is supported by further analysis by the authors. Importantly, the authors show that AGS3 acts through LGN, based on localization data and an epistasis analysis. The function of AGS3 has been highly enigmatic so resolving this issue while providing a useful step towards understanding how the division orientation decision is made, makes for exciting progress towards an important problem. I found the overall narrative and presentation to be quite good and especially appreciated the thoughtful discussion section that did an excellent job of putting the results in context and speculating how unknown aspects of the mechanism might work based on current clues. With that said, I think there are some important issues that should be resolved.
Regarding the orientation measurements, the authors should specify how the midbody marker was used to mark sibling cells, especially given the midbody can move following division. For example, how can the authors be confident that the siblings in the middle panel of 1A are correct and not an adjacent cell?
Regarding quantification, it would be useful for the authors to comment on how the following would influence their measurements: 1) movements along the z-axis, and 2) movement of the nucleus within the cell.
A similar question is how much telophase correction really happens in telophase. How confident are the authors that the process actually occurs during division and not subsequent to it? What is drawn in their previous paper and in Figure 7A implies that post-division movements may be important. It would be useful for the authors to comment on whether they can make the distinction and whether or not it might be important.
Does the division angle in the AGS3 OE experiment (Figure 1D) correlate with AGS3 levels within the cell?
I found the localization data to be the weakest part of the paper and feel that some reconsideration and reanalysis are warranted.
First, the quantifications in Figures 2C, 3B, and 3F are unnecessarily vague scoring-based metrics. In 2C, "Localization pattern" should be replaced with membrane/cytoplasm ratio or an equivalent quantification. In 3B "LGN localization" should be replaced with apical/cytoplasmic and apical/basal ratios or equivalents. In 3F, "Polarized LGN frequency" should be replaced with apical/basal ratio or equivalent. It seems to me that non-AI processed data would be most appropriate for these quantifications unless such processing can be justified.
Second, it is important to note that the cytoplasmic localization of AGS3 does not allow one to conclude that AGS3 is not on the membrane. Unfortunately, high cytoplasmic signal can preclude the determination of membrane-bound signal.
Finally, I had difficulty reconciling the images of LGN shown in Figure 3 with the conclusions made by the authors.
The challenge of the localization data is troubling because an important conclusion of the paper is that AGS3 acts via LGS. The localization data provided one leg of support for this conclusion and the other is provided by an epistasis analysis. Unfortunately, this data seems to be right on the edge because it is based on the difference between the solid and dashed blue lines in Figure 5B not being significant. However, we can see how close this is by comparing the solid and dashed red lines in the adjacent 5C, which are significantly different. Between the localization data, which doesn't seem clear cut, and the epistasis experiment, which is on the razor's edge, I'm concerned that the conclusion that AGS3 acts through LGN may be going beyond what the data allows.
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