Erythroid differentiation regulator-1 induced by microbiota in early life drives intestinal stem cell proliferation and regeneration

Gut microbiota and their metabolites are instrumental in regulating intestinal homeostasis. However, early-life microbiota associated influences on intestinal development remain incompletely understood. Here we demonstrate that co-housing of germ-free (GF) mice with specific-pathogen free (SPF) mice at weaning (exGF) results in altered intestinal gene expression. Our results reveal that one highly differentially expressed gene, erythroid differentiation regulator-1 (Erdr1), is induced during development in SPF but not GF or exGF mice and localizes to Lgr5+ stem cells and transit amplifying (TA) cells. Erdr1 functions to induce Wnt signaling in epithelial cells, increase Lgr5+ stem cell expansion, and promote intestinal organoid growth. Additionally, Erdr1 accelerates scratch-wound closure in vitro, increases Lgr5+ intestinal stem cell regeneration following radiation-induced injury in vivo, and enhances recovery from dextran sodium sulfate (DSS)-induced colonic damage. Collectively, our findings indicate that early-life microbiota controls Erdr1-mediated intestinal epithelial proliferation and regeneration in response to mucosal damage.

cells during regeneration. Additionally, apoptosis should be analyzed (e.g., cleaved caspase-3). These additional experiments are required because it is unclear whether Erdr1 affects the proliferation or protects from IR injury of ISCs and TA cells. This is also due to the co-treatment of Erdr1 with IR, which makes it difficult to assess the effects of Erdr1 on intestinal regeneration (radioprotection, prevention, or increased regeneration?), which might also be addressed by pre-treatment of Erdr1 followed by IR. 6. Fig. 7. Did GF or ExoGF mice treated with IR display the severe impairment in intestinal regeneration with lethal phenotype? If they did, a gain-of-function approach for rescue (Erdr1 administration) might further address the crucial roles of Erdr1 in intestinal regeneration. Conversely, as shown in Fig. 8, anti-Erdr1 ab can be used for a loss-of-function approach to corroborate this claim. 7. How does Erdr1 activate Wnt/beta-catenin signaling?
Minor comments 1. 'Intestinal epithelial cells are highly sensitive to DNA damage caused by radiation': This is inaccurate. Only proliferating cells (Lgr5+ ISCs and TA) are sensitive to radiation (Suh et al., Cell Reports 2017 PMCID: PMC5138641). 2. The recommendation is to revise the references (#37, 38). For example, Kuo lab at Stanford or Capecchi's Bmi1+ ISCs. 3. Fig. 2. DAPI labeling is missing in images. 4. Fig. 4. Which mice were used to isolated IECs/organoids? Assumed exGF or GF? 5. Fig. 5. What is the expression level of Erdr1 in IEC cell lines? 6. Fig. 5. Erdr1-activated beta-catenin target genes. Please include beta-catenin IF images upon Erdr1 treatment. 7. Two IEC cell lines were used but for specific assays for each. Any justification? 8. Fig. 6. Why does Mode-K cell express the endogenous Erdr1 although this cell line is not Lgr5+ ISCs or TA cells? 9. Fig. 7. Are the results from GF/ExoGF mice or SPF mice?
Reviewer #3 (Remarks to the Author): Abo et al showed Edr1 expressed Intestinal stem cells and TA cells in SPF mice, but not GF, eGF mice. Edr1 expression increased total number of stem cells and Edr1 is beneficial for the recovery tissue damage. Mouse model was elegant but there are issues to be solved.
First, Authors showed Edr1 was expressed SPF mice, however, previous report SPF CD4 T cell reduced the expression level of Erd1 compared to GF condition (Weis AM et al. Gut microbe 2018.). Authors need to compare isolated EC cells between GF, eGF and SPF condition. If possible, to analyse Edr1 expression of CD4 or NK cells in each condition is helpful to clarify their findings. Second, authors demonstrated microbe is essential to increase Erd1 expression in SPF mice, but exGF mice did not increase Erd1 expression. Other papers demonstrated Erd1 expression is decreased by TLR2-Myd88 pathway (Soto Ret al PNAS 2017). Authors should show expression of Erd1 in intestinal epithelial cells is regulated by TLR-Myd88 or not. Also, recently Nabhani ZA showed microbe-IFNg signaling at weaning age is essential for the low susceptibility (Nabhani ZA et al. Immunity 2019). Authors need to address the reason exGF mice express less Edr1 under the microbe rich condition. Is specific microbe or cytokine essential for the upregulation of Edr1 in stem cells? Third, authors did not show the precise mechanism of Edr1 control the number of stem cells. Previous report showed Erd1 protein induce apoptosis by increased Fas/ Caspase8 and 3 on T cells (Weis AM et al.). Erd1 signal is Fas dependent manner. Authors data is completely opposite result from precious report. These results might be Erd1 unknown receptor did not express the intestinal stem cells compared to the T/ NK cells. The receptor signal is further study but at least, authors need to address apoptosis factor in vitro and vivo. Also Edr1KO organoid assay should be important to understand for Erd1-Wnt signal.
Overall, the finding that microbe regulate Erd1 expression of intestinal stem/TA cells is potentially interesting, however current results cannot satisfy the readers. Authors need to address new insight/ pathway that is not preciously reported.
Minor: Figure 2 : Authors need to show the Erd1 staining was fine by staining CD4 T cells in GF mice. See the major comments.

Reviewer #1 (Remarks to the Author)
The manuscript by Abo and colleagues entitled "Erythroid differentiation regulator-1 is induced by microbiota in early life and drives intestinal stem cell proliferation and regeneration" provides new data showing that Erdr1 is expressed in the intestinal stem/TA compartment in mice in early life in SPF conditions, but not in germ-free conditions. Interestingly, Erdr1 is not induced to normal levels even in ex-germ-free mice. Data is presented showing that Erdr1 induced Wnt signaling, increased Lgr5+ ISCs and organoid growth. Notably, Erdr1 accelerated wound closure in scratch-wound assays in vitro and increased Lgr5+ ISC regeneration following radiation and promoted recovery of mice from DSS-induced colonic injury. Alternatively, treatment of mice with anti-Erdr1 antibody was shown to delay recovery from DSS-induced colonic injury.
Overall, this is a timely and important area of investigation. The manuscript is nicely written and notably thorough in experimentation with compelling in vitro and in vivo data. Addressing the following points will further strengthen this manuscript: 1. The authors should confirm that recombinant Erdr1 has the expected properties using SDS-PAGE and/or mass spec.
2. The authors should show data using organoids generated from TLR4-deficient mice to verify that effects are not influenced by LPS.

Reviewer #2 (Remarks to the Author)
Denning and colleagues suggested that erythroid differentiation regulator-1 (Erdr1) is transactivated by microbiota at the early stage of development and positively modulates intestinal stem cell proliferation and regeneration. Overall, the authors' finding is very interesting in regards to its novelty, the crucial roles of microbiota in physiology and pathology of the intestine. However, such interest is dampened by the lack of mechanisms including how Erdr1 expression is induced by microbiota or microbial metabolites and how Erdr1 activates Lgr5+ intestinal stem cells (ISCs) possibly via Wnt/beta-catenin signaling.
Major comments 1. The recommendation is to include RNA-seq results as a supplementary table.
2. It may need justification for why Erdr1 was chosen among others.
3. Fig. 1. Epigenetic regulation of Erdr1. This needs more discussion. Also, additional experiments using organoids treated with 5'-Aza-deoxycytidine or HDAC inhibitor may address some part of the potential mechanism of epigenetic regulation of Erdr1.
4. Wound healing assays (Fig. 6) seems somewhat irrelevant to the main topic, i.e., ISCs and intestinal regeneration given that Erdr1 expression is specifically enriched in ISCs and TA cells. Any justification? 5. Fig. 7. To claim the proliferative/regenerative effects of Erdr1 on Lgr5+ ISCs and TA cells upon ionizing radiation (IR), authors should show the Ki67 images in a timedependent manner (after IR). Also, BrdU incorporation or migration assays will better address the effects of Erdr1 on ISCs and TA cells during regeneration. Additionally, apoptosis should be analyzed (e.g., cleaved caspase-3). These additional experiments are required because it is unclear whether Erdr1 affects the proliferation or protects from IR injury of ISCs and TA cells. This is also due to the co-treatment of Erdr1 with IR, which makes it difficult to assess the effects of Erdr1 on intestinal regeneration (radioprotection, prevention, or increased regeneration?), which might also be addressed by pre-treatment of Erdr1 followed by IR. 6. Fig. 7. Did GF or ExoGF mice treated with IR display the severe impairment in intestinal regeneration with lethal phenotype? If they did, a gain-of-function approach for rescue (Erdr1 administration) might further address the crucial roles of Erdr1 in intestinal regeneration. Conversely, as shown in Fig. 8, anti-Erdr1 ab can be used for a loss-offunction approach to corroborate this claim. epithelial cells were cultured for 8 days before infecting SV40 large T antigen. During this 8-day culture, proliferating cells were selected. While clearly not prototypic ISCs or TA cells, whether these intestinal epithelial cells maintained any lineage relationship with ISCs or TA cells remains unclear.
Author's response to Reviewer 2, Comment 9: We thank Reviewer 2 for this comment. We got results of Figure 7 from SPF mice. We have now clarified this in the results section and figure legend.

Reviewer #3 (Remarks to the Author)
Abo et al showed Edr1 expressed Intestinal stem cells and TA cells in SPF mice, but not GF, eGF mice. Edr1 expression increased total number of stem cells and Edr1 is beneficial for the recovery tissue damage. Mouse model was elegant but there are issues to be solved.
First, Authors showed Edr1 was expressed SPF mice, however, previous report SPF CD4 T cell reduced the expression level of Erd1 compared to GF condition (Weis AM et al. Gut microbe 2018.). Authors need to compare isolated EC cells between GF, eGF and SPF condition. If possible, to analyse Edr1 expression of CD4 or NK cells in each condition is helpful to clarify their findings.
Second, authors demonstrated microbe is essential to increase Erd1 expression in SPF mice, but exGF mice did not increase Erd1 expression. Other papers demonstrated Erd1 expression is decreased by TLR2-Myd88 pathway (Soto Ret al PNAS 2017). Authors should show expression of Erd1 in intestinal epithelial cells is regulated by TLR-Myd88 or not. Also, recently Nabhani ZA showed microbe-IFNg signaling at weaning age is essential for the low susceptibility (Nabhani ZA et al. Immunity 2019). Authors need to address the reason exGF mice express less Edr1 under the microbe rich condition. Is specific microbe or cytokine essential for the upregulation of Edr1 in stem cells? Third, authors did not show the precise mechanism of Edr1 control the number of stem cells. Previous report showed Erd1 protein induce apoptosis by increased Fas/ Caspase8 and 3 on T cells (Weis AM et al.). Erd1 signal is Fas dependent manner. Authors data is completely opposite result from precious report. These results might be Erd1 unknown receptor did not express the intestinal stem cells compared to the T/ NK cells. The receptor signal is further study but at least, authors need to address apoptosis factor in vitro and vivo. Also Edr1KO organoid assay should be important to understand for Erd1-Wnt signal.
Overall, the finding that microbe regulate Erd1 expression of intestinal stem/TA cells is potentially interesting, however current results cannot satisfy the readers. Authors need to address new insight/ pathway that is not preciously reported.
Minor: Figure 2 : Authors need to show the Erd1 staining was fine by staining CD4 T cells in GF mice. See the major comments.

Major comments
Reviewer 3, Comment 1: Authors showed Edr1 was expressed SPF mice, however, previous report SPF CD4 T cell reduced the expression level of Erd1 compared to GF condition (Weis AM et al. Gut microbe 2018.). Authors need to compare isolated EC cells between GF, eGF and SPF condition. If possible, to analyse Edr1 expression of CD4 or NK cells in each condition is helpful to clarify their findings.
Author's response to Reviewer 3, Comment 1: We thank Reviewer 3 for this comment. In Figure 2a and new Supplementary Figure 2 our data clearly demonstrate Erdr1 expression predominantly in the crypt region and TA zone of SPF, but not GF or exGF mouse intestine. It is important to clarify that these data are not inconsistent with those of Soto et al, PNAS, 2017, since that report did not compare the expression of Erdr1 in gut CD4 T cells from SPF and GF mice, but rather splenic CD4 T cells from SPF and GF mice. Further, we observed Erdr1 staining in the lamina propria region of the small intestine from SPF mice which is consistent with expression of Erdr1 by other cells types perhaps including CD4 T cells, as reported by Soto et al, PNAS, 2017 in the spleen. Interestingly, we did not observe detectable Erdr1 expression in the intestines of GF or exGF mice, so we did not pursue co-staining for specific cell types as that data would also be negative for Erdr1 expression. Collectively, these data suggest that microbiotadependent regulation of Erdr1 expression may differ between the gut and periphery.

Reviewer 3, Comment 2: Authors demonstrated microbe is essential to increase Erd1 expression in SPF mice, but exGF mice did not increase Erd1 expression. Other papers demonstrated Erd1 expression is decreased by TLR2-Myd88 pathway (Soto Ret al PNAS 2017). Authors should show expression of Erd1 in intestinal epithelial cells is regulated by TLR-Myd88 or not. Also, recently Nabhani ZA showed microbe-IFNg signaling at weaning age is essential for the low susceptibility (Nabhani ZA et al. Immunity 2019). Authors need to address the reason exGF mice express less Edr1 under the microbe rich condition. Is specific microbe or cytokine essential for the upregulation of Edr1 in stem cells?
Author's response to Reviewer 3, Comment 2: We thank for Reviewer 3 for this important comment. To address this comment, we performed qPCR analysis using total SI and LI tissue from Myd88 KO mice. Knock out of Myd88 did not result in altered Erdr1 expression (Supplementary Figure 1). These data suggest that Erdr1 expression in the mouse intestine is independent of Myd88 signaling.
Further, the reason why exGF mice still express less Erdr1 even after being colonized under SPF conditions is because they have missed a critical window for microbiota exposure in early life (prior to weaning) and explainable, at least in part, by epigenetic changes in the Erdr1 promoter. As shown in Figure 1d and 3b, microbiota exposure prior to weaning promotes histone H3 acetylation on the Erdr1 promoter region and this epigenetic change is associated with increased Erdr1 expression. Since exGF mice were not exposed to microbiota (and their associated metabolites) before weaning, histone H3 acetylation on the Erdr1 promoter and Erdr1 expression was dramatically reduced. These finding indicates unknown mediators include metabolites produced by microbiota induce Erdr1. Study to find specific mediator which upregulates Erdr1 is outside the scope of this study. We have now included these data in Supplementary Figure. 1.
While it is certainly of interest to define the specific microbe(s)/metabolite(s) and/or cytokine(s)/factor(s) that regulate Erdr1 expression in ISCs, we hope the reviewer can appreciate that doing so is a massive undertaking that would last numerous years and is well beyond the scope of this initial study (currently 8 main figures and 16 supplementary figures) defining novel expression patterns of Erdr1 in the gut, regulation of Erdr1 by the microbiota, and functions of Erdr1 in intestinal regeneration and colitis.

Reviewer 3, Comment 3: Authors did not show the precise mechanism of Edr1 control the number of stem cells. Previous report showed Erd1 protein induce apoptosis by increased Fas/ Caspase8 and 3 on T cells (Weis AM et al.). Erd1 signal is Fas dependent manner. Authors data is completely opposite result from precious report. These results might be Erd1 unknown receptor did not express the intestinal stem cells compared to the T/ NK cells. The receptor signal is further study but at least, authors need to address apoptosis factor in vitro and vivo. Also Edr1KO organoid assay should be important to understand for Erd1-Wnt signal.
Author's response to Reviewer 3, Comment 3: We thank for Reviewer 3 for this comment. We appreciate that previous papers have reported that Erdr1 induces apoptosis. Erdr1 has also been reported to be a survival factor during condition of cellular stress (Dormer et al, 2004). In addition, Erdr1-expressing stroma can promote cancer cell survival in vitro and cancer cell invasion in vivo (Mango et al, 2014). These papers and our finding strongly indicate that Erdr1 play unique roles depending on the cell type, condition and tissue. We have now included a discussion to highlight the important and expanding biological functions of Erdr1.
In addition, we performed Annexin V/PI staining using organoids. Erdr1 induced no change of apoptotic cells (Supplementary Figure 6). Also, at day 1 post radiation, Erdr1 treated mice showed the same level of apoptosis as compared to control mice (Supplementary Figure 14). These in vitro and in vivo data indicate Erdr1 does not induce apoptosis on IECs.
Lastly, we concur with Reviewer 3 that the study of Erdr1 receptor is well beyond the scope of the current study.

Reviewer 3, Comment 1: Figure 2: Authors need to show the Erd1 staining was fine by staining CD4 T cells in GF mice. See the major comments.
Author's response to Reviewer 3, Comment 1: Please see response above to Reviewer 3, Comment 1. Our Erdr1 staining is very specific and robust in the intestine. ISCs and TA zone cells express robust levels of Erdr1 in the intestine of SPF mice and cells in the lamina propria (perhaps CD4 T cells) also express Erdr1 albeit at what appears to be lower levels. Author's response to Reviewer 3, Comment 2: To avoid any concerns of nonrepresentative regions of the well being used to generate organoid data, we selected 4 random regions and used those same regions for calculating data for all treatment groups.
Our data demonstrating that Erdr1 enhances organoid efficiency, budding, and surface area is consistent whether we add Erdr1 during the first week of organoid generation (short-term) or after passage during week 2 or week 3 (long-term).

Reviewer 3, Comment 3: Figure 5: Did you check the expression level of Erdr1 in SKCO15 cells?
Author's response to Reviewer 3, Comment 3: We thank for Reviewer 3 for this comment. We performed qPCR for Erdr1 mRNA in Mode-K, HT-29 and SKCO15 cell lines. Mode-K cells express Erdr1 mRNA (Supplementary Figure 11), however, the expression in HT-29 and SKCO15 was undetectable despite using the same primers validated to work in human cells (Soto et al, PNAS 2017). Figure 6: Add 2D functional assay by using Erdr1KO organoid.

Reviewer 3, Comment 4:
Author's response to Reviewer 3, Comment 4: We thank for Reviewer 3 for this comment. Erdr1 knockout mice are not available since the previous report failed to generate whole body KO and flox mice (Soto et al, PNAS, 2017). Author's response to Reviewer 3, Comment 5: We thank for Reviewer 3 for this comment and would like to emphasize that our manuscript is focused on defining novel expression patterns of Erdr1 in the gut, regulation of Erdr1 by the microbiota, and functions of Erdr1 in intestinal regeneration and colitis. We have indeed demonstrated that Erdr1 has effects on intestinal epithelial cell lines (mouse and human; no immune cells present) and organoids (mouse and human; no immune cells present). These effects of Erdr1 are most definitely not due to effects on immune cells. We cannot and do not exclude a possible role for Erdr1 on other cell types beyond intestinal epithelial cells in vivo. We hope the reviewer can appreciate that requiring that we investigate the direct versus indirect effects of Erdr1 in vivo on numerous cells types is a massive undertaking that would last numerous years and is well beyond the scope of this initial study (currently 8 main figures and 16 supplementary figures).