FGF8 induces chemokinesis and regulates condensation of mouse nephron progenitor cells

  1. Abhishek Sharma
  2. Marco Meer
  3. Arvydas Dapkunas
  4. Anneliis Ihermann-Hella
  5. Satu Kuure
  6. Seppo J. Vainio
  7. Dagmar Iber
  8. Florence Naillat

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Abstract

Kidneys develop via iterative branching of the ureteric epithelial tree and subsequent nephrogenesis at the branch points. Nephrons form in the cap mesenchyme as the metanephric mesenchyme (MM) condenses around the epithelial ureteric buds (UBs). Previous work has demonstrated that FGF8 is important for the survival of nephron progenitor cells (NPCs), and early deletion of Fgf8 leads to the cessation of nephron formation, which results in post-natal lethality. We now reveal a previously unreported function of FGF8. By combining transgenic mouse models, quantitative imaging assays and data-driven computational modelling, we show that FGF8 has a strong chemokinetic effect and that this chemokinetic effect is important for the condensation of NPCs to the UB. The computational model shows that the motility must be lower close to the UB to achieve NPC attachment. We conclude that the FGF8 signalling pathway is crucial for the coordination of NPC condensation at the UB. Chemokinetic effects have also been described for other FGFs and may be generally important for the formation of mesenchymal condensates.

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  1. Version published to 10.1242/dev.201012
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    Reply to the reviewers

    Manuscript number: RC-2022-01288R

    Corresponding author(s): Florence Naillat, Seppo Vainio, Dagmar Iber

    1. General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    We would like to thank the reviewers for their positive and constructive reviews. We have already addressed their major concerns by including additional data, especially in the new Figure 3. We also detail the planned experiments that we propose to perform to address their remaining comments. Some points are mentioned in both section 2 and 3 of the revision plan.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Major Comments:

    1. Fig 3, I don't understand why the control lost Six2 expression (Fig 3A). Fig 3E is not consistent withFig 3d, which showed that the treatment of Fgf8 antibody significantly decreases the number of live cells. Lastly, the 3D culture matrix experiments did not provide evidence on the role of Fgf8 for NPC condensation.

    The figure 3A showed a reduced expression of SIX2 expression (Red) in the NPC population in the kidney. When the NPC population is cultured without any FGF ligands, the SIX2 expression in the NPCs disappeared. Our results are similar to Dapkunas et al 2019 (Arvydas Dapkunas, Ville Rantanen, Yujuan Gui, Maciej Lalowski, Kirsi Sainio, Satu Kuure, and Hannu Sariola. Simple 3d culture of dissociated kidney mesenchyme mimics nephron progenitor niche and facilitates nephrogenesis wnt-independently. Scientific reports, 9(1):1–10, 201).

    The figure 3D showed that the antibody against FGF8 prevented the SIX2 expression in the NPC population from the nephrosphere experiment. We have modified the legend of figure 3D.

    We agreed with the reviewer that this result might confuse the reader. We are carrying another set of experiment for the Figure 3E where nephrospheres will be treated with and without FGF8 ligand instead of culturing a full kidney with and without FGF8 ligand for 24 hours as mentioned in figure 3E.

    We would like to mention that Dapkunas et al. 2019 demonstrated that dissociated kidney mesenchyme which contain the NPC population formed spontaneously self-organized spheres with the addition of FGF2 ligand and PP2 a Src inhibitor. By staining with Pax2 antibody which is a marker for progenitors and early nephron precursors we could show similarly as in Dapkunas et al that ectopic FGF8 ligand induced PAX2 expression whereas the antibody against FGF8 did not induce PAX2 expression in the cultured nephrosphere (Figure 3E-G)

    At the end of "A model based on Fgf8-induced motility leads to robust condensation of NPC", there is not a conclusive sentence.

    We have modified the text. We have written “We conclude from these simulation results that the chemokinetic effect of FGF8 enables the niche-wide distribution of NPCs. This allows them to reach the vicinity of the UB and also to enter the sphere of influence of epithelial factors that support the immobilization of NPCs. The corresponding motility gradient that appeared in the simulations (Supplementary Fig. Sup3) is in agreement with experimental observations Combes et al. 2016 (Alexander N. Combes, James G. Lefevre, Sean Wilson, Nicholas A. Hamilton, and Melissa H. Little. Cap mesenchyme cell swarming during kidney development is influenced by attraction, repulsion, and adhesion to the ureteric tip. (2016) Developmental Biology, 418(2):297–306. The simulations also show that excess FGF8 can override the guidance of epithelial signaling and prevent mesenchymal condensation.”

    Whole kidney qPCR results are not enough to support the claim of incomplete deletion of Fgf8 in mouse models. Protein staining or mRNA detection in section is required to support the claim. In addition, clear explanation is required on how the phenotypes of Fgf8 KO mice are associated the function of Fgf8 for NPC condensation.

    The qPCR results of the figure 3I and J have been carried out on the nephrospehere assays. In figure 3I, the nephrosphere assay which consist of culturing the kidney mesenchyme with ectopic FGF8 ligand for 24 hours. This showed that the NPCs markers were sustained due to FGF8 ligand. This is further confirmed with the staining of PAX2 a marker for progenitors and early nephron precursors which stained the aggregated NPC cells expressing SIX2 marker as in Dapkunas et al. 2019 (Figure 3E-G).

    I can not understand the last sentence well "Further work is required to reveal how Fgf8 along with its receptors and inhibiting factors orchestrates NPC condensation, its ......"

    We have modified the text. We have written “It is known that FGF8 ligand interacts with several FGF receptors and such interaction can also be modulated by heparan sulfate proteoglycan which will consequently regulate the gradient of FGF8 concentration (Harish et al. 2022 bioRXiv). Towards this goal a detailed ligand/receptor interactions in \textit{in vivo} is required to fully understand how FGF8 imparts its function.”

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Major concerns:

    • Parametric tests are not appropriate for a small sample size; non-parametric tests should be applied to examine if data are robust. Specifically, in Figures 3F and 3G, where n=3.

    *We would like to thank the reviewer as we did not write the correct statistical test that we have carried out. It is the 2-way Anova Sidak multiple comparison that is a non-parametric test. We have corrected the legend of the figure 3. *

    • In Figure 2E it is unclear how many kidneys were analyzed since the graph shows an n=5 per genotype while the figure legend indicates an n=6. Similarly, please indicate the number of independent biological samples analyzed in panel 2G rather than only the total number of cells, and specify the statistical test used for data analysis.

    We would like to thank the reviewer for catching our written mistake for the figure 2E. We have analysed 5 kidneys and we have corrected the legend. We have added the number of independent biological samples analyzed in the figure 2G and the statistical test (Wilcoxon signed-rank test) used in the legend.

    • Can the authors clarify how the experiment described in Figure 3E was performed? It is unclear if NPCs were treated with FGF8 ligand (as indicated in the chart legend) or with an anti-FGF8b antibody (as described in the figure legend). Moreover, the authors stated that "the loss of Six2 expression as a result of the absence of FGF8 was not completely due to cells death as more live cells were observed", however, the number of live cells seems similar between control and FGF8-treated nephrospheres. Can the authors comment on that?

    *The figure was mislabeled the NPC were treated with the ectopic FGF8 ligand (mistake has been corrected in manuscript). *

    To detect the number of cells (dead/live), the flow cytometry was utilized on a full cultured kidney for 24 hours with or without ectopic FGF8 ligand. This method requires several washing steps which can remove the dead cells during the procedure. However, we are planning to repeat the same experiment using the nephrosphere assay where the nephrosphere will be cultured with or without ectopic FGF8 ligand for 24 hours before being sorted to check the live and dead cells.

    • The authors argued that "the NPCs, the Six2+ cells, accumulate around the UB tip and that NPC induction is interrupted failing the PTAs formation". Please include quantification of Lhx1-positive structures to assess the number of PTA structures in wildtype, as well as, in Pax8Cre;Fgf8n/c and Wnt4Cre;Fgf8n/c mutant kidneys.

    As we never have worked with Lhx1 antibody before, we are optimizing the protocol for the staining of the Lhx1 antibody combined with Troma (epithelial marker) and Six2 (NPC population marker) antibodies to highlight the NPC population from the ureteric bud in the WT kidney slides. We have few sample slides for Pax8Cre;Fgf8n/c and Wnt4Cre;Fgf8n/c mutant kidneys and we would like a working protocol before any staining.

    • It is unclear if both male and female offspring were collected. If so, did the authors observe sex-related differences in outcomes?

    *We have use male and female embryos. We did not genotype for the sex of the embryos in any of the experiments. In such way the sample collection was unbiased regarding the sex of the embryos. *

    Minor concerns:

    • Abbreviations should be defined at first mention in the text (e.g. "MET" in the second paragraph of the Introduction) and in each figure/table legend (e.g. UB, CM, tNPCs, PTA in Figure 1).

    We appreciate the suggestion and include all the abbreviation in the text and in the legend of Fig1.

    • In Figure 7, please label the structures (kidney; ureters, and bladder) in the urogenital system of control and mutant mice to facilitate the reader's understanding.

    We appreciate the suggestion and have labelled the structures in the figure 7.

    • For consistency, please include an inset showing a higher magnification image for Figure 8C. We followed the reviewer’s suggestion and have included a higher magnification image for the figure 8C.
    • Please revise the following sentence for clarity: "Primary antibody incubation duration and temperature was."

    We clarified the sentence and have added the temperature and the time for the staining for each staining in the table 2.

    • The legend of Supplementary Figure 1 needs to be improved, as it does not contain all information required to fully understand the presented results.

    We have rewritten the legend of the supplementary figure 1 as the reviewer suggested.

    • In Supplementary Figure 2, please include the phospho-GSK3β relative expression normalized to GSK3β expression.

    We have calculated the ratio of phospho Gsk3 to GSK3 and no statistical difference was found.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    Several minor comments regarding typos and simple errors have already been incorporated in the transferred manuscript. The changes are highlighted in blue in the revised submission.

    *We have addressed all the minor comments that the reviewers have kindly highlighted to us. We feel these were straightforward to do and feasible in a short time, so do not require a detailed listed plan. *

    As mentioned above we are planning to stain the samples WT, Pax8Cre;Fgf8n/c and Wnt4Cre;Fgf8n/c mutant kidneys with Lhx1 antibody counterstained with Troma and Six2 markers and quantify the number of observed PTA structures

    Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

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    Referee #2

    Evidence, reproducibility and clarity

    This is a well-written and organized manuscript that investigated the role of FGF8 in chemokinesis and condensation of nephron progenitor cells to the ureteric bud during metanephric kidney development. The results described in this present study are scientifically relevant, and the figures clearly support the content and authors' conclusions. However, there are some major and minor concerns that should be addressed by the authors.

    Major concerns:

    • Parametric tests are not appropriate for a small sample size; non-parametric tests should be applied to examine if data are robust. Specifically, in Figures 3F and 3G, where n=3.
    • In Figure 2E it is unclear how many kidneys were analyzed since the graph shows an n=5 per genotype while the figure legend indicates an n=6. Similarly, please indicate the number of independent biological samples analyzed in panel 2G rather than only the total number of cells, and specify the statistical test used for data analysis.
    • Can the authors clarify how the experiment described in Figure 3E was performed? It is unclear if NPCs were treated with FGF8 ligand (as indicated in the chart legend) or with an anti-FGF8b antibody (as described in the figure legend). Moreover, the authors stated that "the loss of Six2 expression as a result of the absence of FGF8 was not completely due to cells death as more live cells were observed", however, the number of live cells seems similar between control and FGF8-treated nephrospheres. Can the authors comment on that?
    • The authors argued that "the NPCs, the Six2+ cells, accumulate around the UB tip and that NPC induction is interrupted failing the PTAs formation". Please include quantification of Lhx1-positive structures to assess the number of PTA structures in wildtype, as well as, in Pax8Cre;Fgf8n/c and Wnt4Cre;Fgf8n/c mutant kidneys.
    • It is unclear if both male and female offspring were collected. If so, did the authors observe sex-related differences in outcomes?

    Minor concerns:

    • Abbreviations should be defined at first mention in the text (e.g. "MET" in the second paragraph of the Introduction) and in each figure/table legend (e.g. UB, CM, tNPCs, PTA in Figure 1).
    • In Figure 7, please label the structures (kidney; ureters, and bladder) in the urogenital system of control and mutant mice to facilitate the reader's understanding.
    • For consistency, please include an inset showing a higher magnification image for Figure 8C.
    • Please revise the following sentence for clarity: "Primary antibody incubation duration and temperature was."
    • The legend of Supplementary Figure 1 needs to be improved, as it does not contain all information required to fully understand the presented results.
    • In Supplementary Figure 2, please include the phospho-GSK3β relative expression normalized to GSK3β expression.

    Significance

    This study provides provide conceptual and methodological insights relevant to the field and it will be of considerable interest to the readers.

    My field of expertise: kidney development and disease (mouse models, kidney explants, cell culture). I do not have sufficient expertise to evaluate the 2D simulations of NPC condensation to the ureteric epithelium.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    NPC condensation is essential for nephron formation, but underlying regulation mechanisms remain elusive. Previous studies have demonstrated the important role of Fgf8 for the survival of NPCs. In this manuscript, Sharma et al reveal a novel function of Fgf8. By using mouse models, quantitative imaging assays, and data-driven computational modeling, they demonstrated the crucial role of Fgf8 signaling for the coordination of NPCs behaviors to the UB, especially for NPC condensation. Generally speaking, the manuscript was well organized and written. The experiments and analysis were well done. However, I have following concerns:

    1. Fig 3, I don't understand why the control lost Six2 expression (Fig 3A). Fig 3E is not consistent withFig 3d, which showed that the treatment of Fgf8 antibody significantly decreases the number of live cells. Lastly, the 3D culture matrix experiments did not provide evidence on the role of Fgf8 for NPC condensation.
    2. At the end of "A model based on Fgf8-induced motility leads to robust condensation of NPC", there is not a conclusive sentence.
    3. Whole kidney qPCR results are not enough to support the claim of incomplete deletion of Fgf8 in mouse models. Protein staining or mRNA detection in section is required to support the claim. In addition, clear explanation is required on how the phenotypes of Fgf8 KO mice are associated the function of Fgf8 for NPC condensation.
    4. I can not understand the last sentence well "Further work is required to reveal how Fgf8 along with its receptors and inhibiting factors orchestrates NPC condensation, its ......"

    Significance

    It is novel to study the role of Fgf8 for NPC condensation as a chemokine.

  5. Version published to 10.1101/2022.02.07.478973v1 on bioRxiv