Sphingolipid metabolism orchestrates the establishment of the adult hair follicle stem cell niche to control skin homeostasis

  1. Franziska Peters
  2. Susanne Brodesser
  3. Kai Kruse
  4. Hannes C.A. Drexler
  5. Jiali Hu
  6. Dominika Lukas
  7. Esther von Stebut
  8. Martin Krönke
  9. Carien M. Niessen
  10. Sara A. Wickström

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Bioactive sphingolipids serve as an essential building block of membranes, forming a selective barrier that ensures subcellular compartmentalization and facilitates cell type-specific intercellular communication through regulation of the plasma membrane receptor repertoire. How cell type-specific lipid compositions are achieved and what is their functional significance in tissue morphogenesis and maintenance has remained unclear. Here, we identify a stem-cell specific role for ceramide synthase 4 (CerS4) in orchestrating fate decisions in skin epidermis. Deletion of CerS4 in the epidermis prevents the effective development of the adult hair follicle bulge stem cell (HFSCs) niche due to altered differentiation trajectories of HFSC precursors towards upper hair follicle and inner bulge fates. Mechanistically, HFSC differentiation defects arise from an imbalance of key ceramides and their derivate sphingolipids in HFSCs associated with hyperactivity of canonical Wnt signaling. Impaired HFSC niche establishment leads to disruption of hair follicle architecture and hair follicle barrier function, ultimately triggering a T helper cell 2 - dominated immune infiltration closely resembling human atopic dermatitis. This work uncovers a fundamental role for a cell state-specific sphingolipid profile in epidermal stem cell homeostasis and the role of an intact stem cell niche in maintaining an intact skin barrier.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

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

    Deletion of CerS4 in the entire mouse epidermis throughout development via the K14-Cre results in enlarged sebaceous glands and perturbed HFSC molecular phenotype. There is low or no expression of CD34, a known marker of the HFSCs along with apparent reduction of several other HFSC markers and acquisition of a more differentiated cell phenotype in these cells. Interestingly, skin and hair follicles seem to remain normal otherwise up to advanced age, though this contradicts the notion that HFSC were indeed affected at the functional level. The data does not demonstrate 'gradual decline' in the HFSC compartment, as claimed by the authors, but rather seem to indicate that the adult HFSC compartment is not properly established in its molecular signatures. Organoid cultures document defects in HFSC, which included reduced proliferation in the CerS4 KO cells. Lipid composition in plasma membranes was also affected by the CerS4 KO. Associated with this, Wnt signal transduction is also affected according to experiments that enhance the strength of wnt signals via a specific small molecular agonist of the pathway. Finally, the authors discover a resemblance of the mouse KO immune-phenotype, with human atopic dermatitis. The study is likely of interest to a specialized readership in skin biology and dermatology and adds to previous studies on CerS4 in skin that erroneously placed its role in the sebaceous gland. [The authors here demonstrate that deletion of CerS4 in the sebaceous glands via SCD3-Cre led to no phenotype, contradicting the previous assessment that CerS4 is important in sebaceous glands.] The study would need to be corrected in a few of its interpretations regarding stem cells to better match the data, as indicated below. *

    We thank the reviewer for the constructive comments that will help us to improve the manuscript. In particular, it is clear that we have not been sufficiently clear in the data presentation.

    Firstly, contrary to what the reviewer states, the CerS4epi-/- mice have a very strong hair follicle phenotype that results in complete hair loss. Also the epidermis is not normal as an inflammatory phenotype develops later, after the hair follicle architecture and function has been disrupted. Thus, there are clear functional consequences to the hair follicle and epidermis that arise from the dysfunction of the HFSC compartment. We will edit the manuscript and add photodocumentation of the macroscopic phenotype to ensure clarity.

    We fully agree with the reviewer that the initial phenotype is inability to establish the adult hair follicle stem cell niche, as shown by the single cell sequencing data and as also stated in the manuscript title. We will further edit the manuscript to clarify this conclusion. Importantly, however, some hair follicle stem cells are generated but these become gradually depleted. So there is a dual phenotype: an inability to efficiently establish and maintain the hair follicle stem cell population. We will clarify this in the text.

    Finally, we want to emphasize that the main finding of this manuscript is that hair follicle stem cells contain a unique lipid profile and perturbing this profile by deleting CerS4 leads to profound defects in stem cell fate regulation through Wnt. This is a completely new finding that has implications far beyond dermatology.

    Major revisions: Fig1B - the data seems to simply shows that bulge cells express less or no CD34 and not that ' CerS4epi-/- mice showed reduced HFSC numbers'; the primary FACS data should be shown somewhere too.

    Outer bulge hair follicle stem cells are defined as a population of cells that expresses CD34 and integrin-a6. The quantifications in Fig 1B show the quantitative FACS analyses of the size of this population and indicate less CD34+/integrin-a6+ cells in CerS4epi-/- epidermis. The mean fluorescence intensity of CD34 and integrin-a6 was not reduced in these CerS4epi-/- stem cells. This FACS analysis therefore allows the conclusion that there are less CD34+/integrin-a6+ cells in CerS4epi-/- epidermis. We will include the original FACS plot data to support this notion and the quantifications.

    The conclusion that stemness is affected, and HFSCs lose their normal gene expression signature is at more convincing after looking at other HFSC markers down the road in the paper. However, in the absence of functional assays that would demonstrate stem cell function is lacking and seeing that hair follicles are maintained and grow in long-term, the notion that stem cells are lacking in these conditions is not supported by the data.

    We appreciate that the reviewer finds the marker gene analysis convincing. To assay stem cell functionality, we have used the organoid assays (spheroid formation is classical, widely used assay for stemness). Using these functional assays we observe impaired self-renewal of stem cells (Fig. 3D), enhanced differentiation (Fig. 3I), and altered Wnt responsiveness (Fig. 5 E, F), all indicative of stem cell dysfunction and explaining the in vivo phenotypes of altered stem cell differentiation and inability to establish and maintain the stem cell population.

    In the revised manuscript we will also include measurements of stem cell self-renewal in vivo using BrdU incorporation and provide more detailed description on the hair loss phenotype of the mice to further strengthen this conclusion.

    *The conclusion after figure 2: "Collectively, these data indicate that CerS4-deficiency triggers ... ... gradual depletion of the quiescent HFSC compartment." There is no data showing gradual depletion of the quiescent HFSC compartment. We would need to see a gradual activation of HFSCs with over proliferation to conclude this. There is some data albeit not always convincing (see NFAC1 staining in Fig. 5C) indicating loss of markers associated with quiescence but there is no data indicating 'gradual' loss of markers. *

    We agree with the reviewer that showing gradual activation of the HFSCs in vivo is important to conclude loss of quiescence. We will include in situ stainings of in vivo BrdU labeling and quantify proliferation in the hair follicle bulge stem cell region. Preliminary data of P47 mice already shows a clear increase in BrdU+ cell in the stem cell compartment in CerS4epi-/- skin . Further analysis at P21 will be carried out during the revision.

    *Minor revisions: *

    • Figure 1C legend - please spell out what are the abbreviations for the different subpopulations; please show these populations as % as opposed to absolute numbers. *

    We will edit the Figure 1C legend for clarity and express the populations as %.

    *Figure 1D - please make it clear in the cartoon what the different sub-populations listed are; *

    We will edit the cartoon for clarity.

    Is OB1 a CD34- HFSC population?

    The outer bulge 1 (OB1) is a population of cells that expresses hair follicle stem cell markers, including CD34. We will clarify this in the legend.

    Fig. 3B - the colors of the legend do not match the colors in the data so it is confusing as to which one is which!

    We will alter the colors to match the data and thank the reviewer for pointing this out.

    Fig 5C - the differences in NFATc1 are not visible in the images shown

    We apologize for the suboptimal quality of these images and will replace them with higher resolution images to more clearly demonstrate the difference.

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

    The manuscript authored by Peters et al. titled "Sphingolipid metabolism orchestrates the establishment of the adult hair follicle stem cell niche to control skin homeostasis" elucidates the critical role of ceramide synthase 4 (CerS4) in the epidermal stem cell niche, particularly in regulating hair follicle bulge stem cells (HFSCs). Using epidermal specific CerS4 knockout mice as an in vivo model and hair follicle organoid culture as an ex vivo model, the authors conducted a comprehensive analysis, which includes cutting edge approaches such as scRNA-seq, proteomics, and lipidomics. The results highlight CerS4's function in the establishment/maintenance of the HFSC niche, as absence of CerS4 changes HFSCs' number and differentiation state. Potential underlying mechanisms identified include altered membrane lipid profiles and Wnt signaling responsiveness. Possible link to a chronic inflammatory skin disease, atopic dermatitis, is also implicated. The data presented are generally of high quality, and the work is significant as it uncovers a new regulator of HFSC fate with mechanistic connection to lipid metabolism. *

    We thank the reviewer for the positive assessment of our work and finding it to be of high quality and significance. We further appreciate the constructive comments that will further help us to improve the manuscript.

    *However, some issues were identified, most of them having to do with in vivo characterization and data interpretation:

    *Major:

    1. The in vivo HFSC phenotype can be better characterized. "Collective, these data show that CerS4 in HFSCs is essential to establish the adult stem cell compartment and to assure lineage fidelity." - this statement premature based on order of the data shown. Also the trajectory difference shown in Figure 2A is not striking. Subclustering out the relative cell subsets and redo the analysis might help to tease out the difference. Additional experiments such as lineage tracing would be useful to support the notion that there is lineage fidelity issue in the mutant - though it is understood that this is quite involved and may lie outside the scope of the current study. Are bulge cells in the mutant proliferative? - the authors should consider in vivo Edu labelling experiment or the like to assess the quiescence/proliferation of the bulge cells. Finally, analyzing hair follicles at earlier stages might help to clarify when and where the bulge and sebaceous gland changes start - is possible that aberrant divergence of bulge/sebaceous fates occur prior to the establishment of a stable bulge fate? *

    We thank the reviewer for suggesting additional analyses of the single cell sequencing data. We have performed subclustering of the relevant populations for the trajectory analyses to more clearly demonstrate the altered lineage trajectories. We will include these new analyses in the manuscript. Importantly, the in vitro organoids show abnormal differentiation (Fig. 3H, 3I, Supplementary Fig. 2G), closely resembling the in vivo phenotypes, thereby strengthening the conclusion of cell-autonomously altered lineage trajectories of the hair follicle stem cells.

    We will further preform in vivo BrdU labeling as suggested. These experiments have already been initiated and preliminary data show increased proliferation in the bulge stem cell region of CerS4epi-/- mice in older mice (P47). The data will be included to emphasize long term loss of quiescence in the stem cell compartment in CerS4epi-/- mice.

    Understanding the early development of bulge and sebaceous fates is indeed an interesting question. This will be addressed by detailed analyses of stem cell fate at early stages (P17-P21) using key markers of stem cell state and sebaceous linages (CD34, Krt15, Lhx2, Nfatc1, SCD1 and FASN).

    Finally, we have initiated lineage tracing experiments using the stem cell-specific Lgr5-Cre to conclusively demonstrate that Cers4-deletion leads to altered routing of hair follicle stem cells into upper hair follicle and sebaceous gland fates. This notion is supported by the preliminary analyses of these experiments. We will finalize these analyses and include them in the manuscript.

    *2. The exclusion of IFE contribution is not backed up by data. Figure 6D - model emphasizes HFSC involvement in atopic dermatitis, but this could be due to epidermal barrier defect. Barrier defect could already be present even though IFE morphology appears normal. Maybe TEWL is measured at the time of analysis and shows no change - if so, this data should be included. HFSC changes might contribute but the involvement of IFE cannot be excluded. The conclusion that "CerS4 expression was restricted to the hair follicle" is not supported by data. IFE expression is apparent in Figure S1C. Along this line, there is also an apparent expansion of IFE basal II in the mutant (Figure 1C). *

    We acknowledge that we have not been clear enough with the evidence that allowed us to exclude the involvement of an IFE-mediated barrier defect in the early skin inflammation phenotype. To address a potential barrier defect early on, we have performed careful analysis of TEWL. In Peters at al., 2020 we demonstrate no changes in TEWL at P0, a reduced TEWL at P21 and an increased TEWL in adult CerS4epi-/- mice starting only at P33. The reduction of the TEWL in adolescent CerS4epi-/- mice (P21) is likely linked to an increased production of sebaceous lipids lubricating the skin surface at this time point (Peters et al., 2020). Thus, defects in the hair follicle stem cell compartment, present at adolescence (P21) arise prior to defects in the adult (P33) IFE barrier function. We will clarify this in the manuscript.

    Cers4 expression is overall low in skin, as is typical for enzymes. In situ stainings of Cers4mRNA (Fig.S1C) indeed show a sparse signal also in the IFE. This signal is also detected in CerS4-/- sections, although the KO skin cannot be conclusively used to control background as these mice were generated by deletion of exon 3 only, and Cers4 RNAscope probes might detect remnant Cers4 RNA in these mice. Importantly, our data on FACS sorted basal cells of the IFE shows no substantial Cers4 mRNA expression in IFE progenitors (Fig. S1D) and no mRNA is detected in the IFE in the single cell sequencing. Thus, while we cannot fully exclude low levels of CerS4 expression in the IFE, the levels are substantially lower than in the HFSC and SG compartments, and the phenotype, including the slight expansion of the IFE basal II population, is very minor compared to the hair follicle phenotype. However, to avoid overinterpreting our data, we will carefully edit the conclusions to be less strong on the involvement of the IFE. Furthermore, we will perform hair follicle stem cell lineage tracing experiments as outlined in resspose to the previous point to strengthen the conclusion on the hair follicle stem cell-autonomous phenotype.

    3. Figure 1 - single cell analysis was done using only 2 pairs of mice, and data in E lack statistical assessment. At the very least, data for individual pairs should be shown in supplemental data to ensure that changes are consistent in both mutant mice rather than being dominated by dramatic alteration in only 1 mutant mouse.

    We naturally have rigorously analyzed the replicates to ensure that the phenotype is consistently present in both. We will include the separate analysis of the mice to document this and include statistical analysis.

    *Minor:

    1. CerS4SCD3-/+ nomenclature is mis-leading.*

    We will edit this for clarity

      1. Figure 2- "Furthermore, we observed expansion of the inner bulge identity marker Krt6 protein expression into outer bulge stem cells and along the infundibulum in CerS4epi-/- hair follicles, whereas in control mice Krt6 was restricted to the inner bulge (Fig. 2C)." - Krt6 staining is presented in Fig 2D, not 2C. *

    We thank the reviewer for pointing out this mistake that will correct.

      1. Figure 3C - size of the organoids should be quantified with statistics. The images shown do not support the statement that "Strikingly, CerS4epi-/- organoids showed altered morphology characterized by smaller size and loss of cohesion of peripheral cells from the organoid clusters (Fig. 3C), ...".*

    We will include quantifications.

      1. Section titled "CerS4 regulates HFSC differentiation in a stem cell autonomous manner": "CD34- integrin- a6+ cells, which based on extensive transcriptome and marker expression analyses represent a mixture of HFSCs, hair follicle outer root sheath (ORS) cells and inner bulge cells (collectively termed non-HFSCs)." - shouldn't the CD34- integrin- a6+ population also contain IFE stem/progenitor cells? Are hair follicles micro-dissected out for FACS? *

    The hair follicles are not micro-dissected out for FACS, and the entire basal cell population is initially isolated. However the organoid culture conditions speficically promote the expansion of the hair follicle linage, whereas cells of the IFE are not expanded and long term maintained as extensively documented in previous publications using this organoid system (see for example Kim et al., Cell Metabolism 2020; Chacon-Martinez EMBOJ 2016).

    *5. Figure 5D - please provide the working concentration of Chir99021. *

    We will provide the working concentration.

    *6. Figure 5F - explain what arrows mean in legends. *

    We will define the arrows.

      1. Figure 6A - no significant changes in Th2 and ILC2 were observed at a 95% confidence interval. Increasing mouse number will help to increase statistical power*.

    We agree with the reviewer and acknowledge that this experiment was unfortunately underpowered. We will repeat it with a larger cohort.

      1. Additional Wnt target genes such as Axin 2 should be looked at.*

    We thank the reviewer for this suggestion, we will include analyses of additional Wnt target genes.

      1. The increased BMP signaling and decreased Nfatc1 expression are seemingly contradictory.*

    We apologize for the lack of clarity here. Single cell sequencing showed increased BMP-signaling of outer bulge cell cells to inner bulge cells on mRNA level (Figure S4A). No alteration in BMP signaling was detected within the outer bulge stem cell compartment (Figure 5A). Nfatc1 protein expression was analyzed in the upper bugle (Figure 5C). The data indicate no differential gene expression of ligand receptor pairs mediating BMP-signaling within the outer bulge. A decrease in Nfact1 protein expression (Fig. 5C) together with an increased proliferation (see above) and loss of label retention (Peters et al., 2015) indicates loss of quiescence in this compartment. This data does not contradict an increased BMP signaling in the inner bulge (Figure S4A). An increase in BMP signaling in the inner bulge is in line with reduced inner bulge cell cluster detected in CerS4epi-/- skin via single cell sequencing, likely contributing to the hair loss observed. We will edit this paragraph to make this more clear.

      1. Paragraph starting with "It is interesting to note that ceramide availability was shown to regulate Wnt signaling in Drosophila through strong effects on recycling endocytosis of the receptors (Pepperl et al., 2013)." Is redundant in the manuscript.*

    We apologize for the accidental duplication of this paragraph and thank the reviewer for noticing this mistake.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    The authors created CerS4 mutant mice to test the role of sphingolipids in hair follicle stem cells (HFSCs) and the hair cycle. This work extends previous studies that show that loss of this enzyme leads to defects in the hair cycle and eventually hair loss. In this study the authors look early on in the course of the deletion in an attempt to understand why loss of this enzyme leads to the phenotype described previously. They use single cell profiling, proteomics, and in situ imaging to pinpoint issues in the stem cell niche that drive phenotypes and propose novel interactions between sphingolipid metabolism, Wnt signaling, and inflammation in regulation of HFSC homeostasis. The data are nicely presented, and the text is well written. The conclusions are clearly defined.*

    We thank the reviewer for the positive assessment of our work and finding it well written and presented. We further appreciate the constructive comments that will further help us to improve the manuscript.

    *While the data are clearly presented, there are numerous issues that are confusing to this reviewer. In addition, some of the phenotypes described are subtle, and thus do not make a convincing case.

    1, In figure 1C, the cell proportion analysis suggests there are no OBII or SG in WT. I am not sure how this could be possible. In addition, there appears to be almost no sebaceous cells in either, but the mutant supposedly has much larger sebaceous glands (in Fig 2). In Fig S1I, there is no change in bulge cells? In Fig 1B, there is less HFSCs in the mutant than in the WT, but in 1C, there is more OBI in the mutant. The results in Fig 1B and C are confusing. Also, the schematic in Fig 1 is hard to read, the authors should color code the text with the image.*

    It is important to emphasize that the single cell RNA sequencing was carried out at P19 when the bulge stem cell compartment only starts to be established. This explains why only few bulge stem cells are detected at this point. Nevertheless, the OBII and SG cluster is visible also in the wt in Figure 1D. We will include subclustering of the relevant subpopulations to make these populations more clearly visible also in the wt. We will also edit the labels for clarity.

    Mature sebocytes are very large cells, and inherent to the single cell sequencing workflows these large cells are excluded from the sequencing libraries. Importantly, we do not detect a change in bulge stem cells in a mouse line in which CerS4 was specifically deleted only in sebocytes (Figure S1I). This analysis was carried out to exclude a sebocyte intrinsic effect on the hair follicle stem cell state and fate. The data does not contradict Fig. 1, as data in Fig. 1 was generated using a different mouse line in which CerS4 is deleted in the entire epidermal stem cell population using K14Cre. We will edit the manuscript to make this more clear.

    Data presented in Fig 1B and C focus on two different aspects. Fig 1B shows the inefficient establishment and maintenance of CD34+/integrin-a6+ bulge hair follicle stem cells. The quantification is based on FACS analyses of cells expressing these cell surface molecules/stem cell markers. Fig 1C shows the quantification of the various cell states based on single cell RNA expression and subsequent clustering of the control and CerS4epi-/- epidermal cells together. The “outer bulge” cluster was annotated based on these cells expressing hair follicle stem cell markers. While the CerS4epi-/- epidermis shows increased number of cells in this cluster, the expression of all key stem cell genes (CD34, Sox9, Krt15, Lhx2) is reduced in CerS4epi-/- outer bulge 1 compartment compared to control. Thus, while this “outer bulge” population is expanded in the KO, the stem cell properties of this population are clearly attenuated, as defined by decreased expression of key stem cell transcription factors and increased expression of differentiation genes. We will clarify this in the revised version of the manuscript and also rename this cluster “outer bulge-like” to highlight that these cells are not necessarily bona fide stem cells and might not express high levels of CD34+/integrin-a6+ protein.

    *2, In Figure 5, the signaling chart shows a strong upregulation of non-canonical Wnt signaling in the mutant bulge. Canonical Wnt signaling appears to be unchanged between wt and ko. Thus, it is not clear why the authors came to the conclusion that Wnt signaling is induced in the mutant. They further show expression of Lef1 and Nfatc1, but these are not typical markers used to denote canonical wnt activation, as implied. In fact, the data in Fig S4B suggest the induction of Lef1 and Tcf4 is actually very subtle. Instead, the authors should use nuclear b-catenin or transcriptional targets such as Axin or CyclinD. The authors should in fact explore the observation of Wnt5, as that appears to be the most dramatic change. In addition, the authors should use an ontological analysis with the single cell data from the tissue in the same manner that they did for organoids to take another look at molecular consequences of loss of CerS4. *

    We agree with the reviewer that further analysis of canonical and non-canonical Wnt signaling will strengthen this conclusion. In our experience, nuclear b-catenin is very difficult to detect in the skin even when Wnt is highly active, but we will investigate Axin2 and CyclinD1 expression. We will also investigate Wnt5a signaling by analyzing its expression as well as its downstream target genes. We will further perform additional ontological analyses from the single cell sequencing data to strengthen the conclusions on the signaling alterations.

    3, The authors suggest that much of the phenotype is due to inflammation. In Fig 6A, they showed analysis of CD45 cells in the skin. However, the only change was a very subtle change in Th2 cells, while no other CD45+ cells were altered.*

    We agree with the reviewer and acknowledge that this experiment was unfortunately underpowered. We will repeat these analyses with a larger cohort.

    4, The authors showed upregulation of Immune response in Fig 6C, but then in Fig S2, the genes downregulated are also related to immune response...how do the authors reconcile this?

    We apologize for this confusion. Importantly, keratinocyte-intrinsic downregulation of homeostatic immune modulating activity is a key driver of allergic disorders, like atopic dermatitis. This barrier intrinsic immune modulation is distinct from immune cell-mediated inflammation. There is a strong overlap of genes constituting the term “Inflammatory abnormality of the skin” (Human phenotype ontology terms) Fig 6C and “Immune system process” (GOBP terms) Fig S2F. To name some, i.e. Adam-, ALOX-, ASXL- family members are annotated by both terms. Mutations in these genes are known to cause skin diseases associated with immune dysregulation but are likewise known to regulate immune responses.

    Data in Figure 6C shows enrichment of this term from both up and downregulated proteins in the CerS4epi-/- condition compared to control, indicating that proteins involved in “Inflammatory abnormality of the skin” are dysregulated in CerS4epi-/- organoids. Data in Figure S2F shows the downregulation of these proteins in CerS4epi-/- organoids compared to control. We will clarify this in the text and figure legends.

    5, The author propose that the phenotype in CerS4 null mice is due to disruption of the stem cell Niche. However, the authors have not shown evidence for such an effect through any in situ analysis. The single cell approaches are valuable, but in that case the niche is dissociated. The organoid work is also nice, but not exactly a stem cell niche either. The authors should instead test their hypothesis through an in situ analysis.

    We have used the term niche to describe the cellular interactions between stem cells and the other niche resident cells such as the Krt6+ inner bulge cells that have been analyzed here. We will edit the conclusions for clarity. We will further include additional immunofluorescence analyses of the bulge compartment in situ, as suggested (including markers for quiescence and activation).

  2. Review Commons

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

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    The authors created CerS4 mutant mice to test the role of sphingolipids in hair follicle stem cells (HFSCs) and the hair cycle. This work extends previous studies that show that loss of this enzyme leads to defects in the hair cycle and eventually hair loss. In this study the authors look early on in the course of the deletion in an attempt to understand why loss of this enzyme leads to the phenotype described previously. They use single cell profiling, proteomics, and in situ imaging to pinpoint issues in the stem cell niche that drive phenotypes and propose novel interactions between sphingolipid metabolism, Wnt signaling, and inflammation in regulation of HFSC homeostasis. The data are nicely presented, and the text is well written. The conclusions are clearly defined.

    While the data are clearly presented, there are numerous issues that are confusing to this reviewer. In addition, some of the phenotypes described are subtle, and thus do not make a convincing case.

    1. In figure 1C, the cell proportion analysis suggests there are no OBII or SG in WT. I am not sure how this could be possible. In addition, there appears to be almost no sebaceous cells in either, but the mutant supposedly has much larger sebaceous glands (in Fig 2). In Fig S1I, there is no change in bulge cells? In Fig 1B, there is less HFSCs in the mutant than in the WT, but in 1C, there is more OBI in the mutant. The results in Fig 1B and C are confusing. Also, the schematic in Fig 1 is hard to read, the authors should color code the text with the image.
    2. In Figure 5, the signaling chart shows a strong upregulation of non-canonical Wnt signaling in the mutant bulge. Canonical Wnt signaling appears to be unchanged between wt and ko. Thus, it is not clear why the authors came to the conclusion that Wnt signaling is induced in the mutant. They further show expression of Lef1 and Nfatc1, but these are not typical markers used to denote canonical wnt activation, as implied. In fact, the data in Fig S4B suggest the induction of Lef1 and Tcf4 is actually very subtle. Instead, the authors should use nuclear b-catenin or transcriptional targets such as Axin or CyclinD. The authors should in fact explore the observation of Wnt5, as that appears to be the most dramatic change. In addition, the authors should use an ontological analysis with the single cell data from the tissue in the same manner that they did for organoids to take another look at molecular consequences of loss of CerS4.
    3. The authors suggest that much of the phenotype is due to inflammation. In Fig 6A, they showed analysis of CD45 cells in the skin. However, the only change was a very subtle change in Th2 cells, while no other CD45+ cells were altered.
    4. The authors showed upregulation of Immune response in Fig 6C, but then in Fig S2, the genes downregulated are also related to immune response...how do the authors reconcile this?
    5. The author propose that the phenotype in CerS4 null mice is due to disruption of the stem cell Niche. However, the authors have not shown evidence for such an effect through any in situ analysis. The single cell approaches are valuable, but in that case the niche is dissociated. The organoid work is also nice, but not exactly a stem cell niche either. The authors should instead test their hypothesis through an in situ analysis.

    Significance

    The authors created CerS4 mutant mice to test the role of sphingolipids in hair follicle stem cells (HFSCs) and the hair cycle. This work extends previous studies that show that loss of this enzyme leads to defects in the hair cycle and eventually hair loss. In this study the authors look early on in the course of the deletion in an attempt to understand why loss of this enzyme leads to the phenotype described previously. They use single cell profiling, proteomics, and in situ imaging to pinpoint issues in the stem cell niche that drive phenotypes and propose novel interactions between sphingolipid metabolism, Wnt signaling, and inflammation in regulation of HFSC homeostasis. The data are nicely presented, and the text is well written. The conclusions are clearly defined.

  3. Review Commons

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

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The manuscript authored by Peters et al. titled "Sphingolipid metabolism orchestrates the establishment of the adult hair follicle stem cell niche to control skin homeostasis" elucidates the critical role of ceramide synthase 4 (CerS4) in the epidermal stem cell niche, particularly in regulating hair follicle bulge stem cells (HFSCs). Using epidermal specific CerS4 knockout mice as an in vivo model and hair follicle organoid culture as an ex vivo model, the authors conducted a comprehensive analysis, which includes cutting edge approaches such as scRNA-seq, proteomics, and lipidomics. The results highlight CerS4's function in the establishment/maintenance of the HFSC niche, as absence of CerS4 changes HFSCs' number and differentiation state. Potential underlying mechanisms identified include altered membrane lipid profiles and Wnt signaling responsiveness. Possible link to a chronic inflammatory skin disease, atopic dermatitis, is also implicated. The data presented are generally of high quality, and the work is significant as it uncovers a new regulator of HFSC fate with mechanistic connection to lipid metabolism.

    However, some issues were identified, most of them having to do with in vivo characterization and data interpretation:

    Major:

    1. The in vivo HFSC phenotype can be better characterized. "Collective, these data show that CerS4 in HFSCs is essential to establish the adult stem cell compartment and to assure lineage fidelity." - this statement premature based on order of the data shown. Also the trajectory difference shown in Figure 2A is not striking. Subclustering out the relative cell subsets and redo the analysis might help to tease out the difference. Additional experiments such as lineage tracing would be useful to support the notion that there is lineage fidelity issue in the mutant - though it is understood that this is quite involved and may lie outside the scope of the current study. Are bulge cells in the mutant proliferative? - the authors should consider in vivo Edu labelling experiment or the like to assess the quiescence/proliferation of the bulge cells. Finally, analyzing hair follicles at earlier stages might help to clarify when and where the bulge and sebaceous gland changes start - is possible that aberrant divergence of bulge/sebaceous fates occur prior to the establishment of a stable bulge fate?
    2. The exclusion of IFE contribution is not backed up by data. Figure 6D - model emphasizes HFSC involvement in atopic dermatitis, but this could be due to epidermal barrier defect. Barrier defect could already be present even though IFE morphology appears normal. Maybe TEWL is measured at the time of analysis and shows no change - if so, this data should be included. HFSC changes might contribute but the involvement of IFE cannot be excluded. The conclusion that "CerS4 expression was restricted to the hair follicle" is not supported by data. IFE expression is apparent in Figure S1C. Along this line, there is also an apparent expansion of IFE basal II in the mutant (Figure 1C).
    3. Figure 1 - single cell analysis was done using only 2 pairs of mice, and data in E lack statistical assessment. At the very least, data for individual pairs should be shown in supplemental data to ensure that changes are consistent in both mutant mice rather than being dominated by dramatic alteration in only 1 mutant mouse.

    Minor:

    1. CerS4SCD3-/+ nomenclature is mis-leading.
    2. Figure 2- "Furthermore, we observed expansion of the inner bulge identity marker Krt6 protein expression into outer bulge stem cells and along the infundibulum in CerS4epi-/- hair follicles, whereas in control mice Krt6 was restricted to the inner bulge (Fig. 2C)." - Krt6 staining is presented in Fig 2D, not 2C.
    3. Figure 3C - size of the organoids should be quantified with statistics. The images shown do not support the statement that "Strikingly, CerS4epi-/- organoids showed altered morphology characterized by smaller size and loss of cohesion of peripheral cells from the organoid clusters (Fig. 3C), ...".
    4. Section titled "CerS4 regulates HFSC differentiation in a stem cell autonomous manner": "CD34- integrin- a6+ cells, which based on extensive transcriptome and marker expression analyses represent a mixture of HFSCs, hair follicle outer root sheath (ORS) cells and inner bulge cells (collectively termed non-HFSCs)." - shouldn't the CD34- integrin- a6+ population also contain IFE stem/progenitor cells? Are hair follicles micro-dissected out for FACS?
    5. Figurer 5D - please provide the working concentration of Chir99021. Figure 5F - explain what arrows mean in legends.
    6. Figure 6A - no significant changes in Th2 and ILC2 were observed at a 95% confidence interval. Increasing mouse number will help to increase statistical power.
    7. Additional Wnt target genes such as Axin 2 should be looked at.
    8. The increased BMP signaling and decreased Nfatc1 expression are seemingly contradictory.
    9. Paragraph starting with "It is interesting to note that ceramide availability was shown to regulate Wnt signaling in Drosophila through strong effects on recycling endocytosis of the receptors (Pepperl et al., 2013)." Is redundant in the manuscript.

    Significance

    The work uncovers a new regulator of HFSC fate with mechanistic connection to lipid metabolism and development signaling. The same group previously reported epidermal and hair cycling phenotypes of the same mutant mice, but this work now identifies a specific defect in HFSCs and present evidence for cellular, molecular and biochemical changes. Linking stem cell regulation to lipid metabolism is conceptually novel, and should have a broad audience. However, the study does have some limitations, such as lack of definitive evidence that CerS4 function in HFSCs is responsible for all the defects reported here, and that lipid alterations have a causal relationship with altered Wnt signaling.

    My expertise is in skin biology, stem cell control, and developmental signaling.

  4. Review Commons

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

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Deletion of CerS4 in the entire mouse epidermis throughout development via the K14-Cre results in enlarged sebaceous glands and perturbed HFSC molecular phenotype. There is low or no expression of CD34, a known marker of the HFSCs along with apparent reduction of several other HFSC markers and acquisition of a more differentiated cell phenotype in these cells. Interestingly, skin and hair follicles seem to remain normal otherwise up to advanced age, though this contradicts the notion that HFSC were indeed affected at the functional level. The data does not demonstrate 'gradual decline' in the HFSC compartment, as claimed by the authors, but rather seem to indicate that the adult HFSC compartment is not properly established in its molecular signatures. Organoid cultures document defects in HFSC, which included reduced proliferation in the CerS4 KO cells. Lipid composition in plasma membranes was also affected by the CerS4 KO. Associated with this, Wnt signal transduction is also affected according to experiments that enhance the strength of wnt signals via a specific small molecular agonist of the pathway. Finally, the authors discover a resemblance of the mouse KO immune-phenotype, with human atopic dermatitis. The study is likely of interest to a specialized readership in skin biology and dermatology and adds to previous studies on CerS4 in skin that erroneously placed its role in the sebaceous gland. [The authors here demonstrate that deletion of CerS4 in the sebaceous glands via SCD3-Cre led to no phenotype, contradicting the previous assessment that CerS4 is important in sebaceous glands.] The study would need to be corrected in a few of its interpretations regarding stem cells to better match the data, as indicated below.

    Major revisions:

    Fig1B - the data seems to simply shows that bulge cells express less or no CD34 and not that ' CerS4epi-/- mice showed reduced HFSC numbers'; the primary FACS data should be shown somewhere too. The conclusion that stemness is affected, and HFSCs lose their normal gene expression signature is at more convincing after looking at other HFSC markers down the road in the paper. However, in the absence of functional assays that would demonstrate stem cell function is lacking and seeing that hair follicles are maintained and grow in long-term, the notion that stem cells are lacking in these conditions is not supported by the data.

    The conclusion after figure 2: "Collectively, these data indicate that CerS4-deficiency triggers ... ... gradual depletion of the quiescent HFSC compartment." There is no data showing gradual depletion of the quiescent HFSC compartment. We would need to see a gradual activation of HFSCs with over proliferation to conclude this. There is some data albeit not always convincing (see NFAC1 staining in Fig. 5C) indicating loss of markers associated with quiescence but there is no data indicating 'gradual' loss of markers.

    Minor revisions:

    Figure 1C legend - please spell out what are the abbreviations for the different subpopulations; please show these populations as % as opposed to absolute numbers.

    Figure 1D - please make it clear in the cartoon what the different sub-populations listed are;

    Is OB1 a CD34- HFSC population?

    Fig. 3B - the colors of the legend do not match the colors in the data so it is confusing as to which one is which!

    Fig 5C - the differences in NFATc1 are not visible in the images shown

    Significance

    Deletion of CerS4 in the entire mouse epidermis throughout development via the K14-Cre results in enlarged sebaceous glands and perturbed HFSC molecular phenotype. There is low or no expression of CD34, a known marker of the HFSCs along with apparent reduction of several other HFSC markers and acquisition of a more differentiated cell phenotype in these cells. Interestingly, skin and hair follicles seem to remain normal otherwise up to advanced age, though this contradicts the notion that HFSC were indeed affected at the functional level. The data does not demonstrate 'gradual decline' in the HFSC compartment, as claimed by the authors, but rather seem to indicate that the adult HFSC compartment is not properly established in its molecular signatures. Organoid cultures document defects in HFSC, which included reduced proliferation in the CerS4 KO cells. Lipid composition in plasma membranes was also affected by the CerS4 KO. Associated with this, Wnt signal transduction is also affected according to experiments that enhance the strength of wnt signals via a specific small molecular agonist of the pathway. Finally, the authors discover a resemblance of the mouse KO immune-phenotype, with human atopic dermatitis. The study is likely of interest to a specialized readership in skin biology and dermatology and adds to previous studies on CerS4 in skin that erroneously placed its role in the sebaceous gland. [The authors here demonstrate that deletion of CerS4 in the sebaceous glands via SCD3-Cre led to no phenotype, contradicting the previous assessment that CerS4 is important in sebaceous glands.]

  5. Version published to 10.1101/2024.01.09.574628v1 on bioRxiv