Peer review of: Erythroid differentiation regulator-1 induced by microbiota in early life drives intestinal (2020)

Reviewers' 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. 
3. The authors should perform qPCR of Notch target genes, such as Hes1, to test whether Erdr1 
activates Notch signaling in addition to Wnt signaling pathways. 
4. The authors should examine if Erdr1 regulates YAP-1 expression, given that YAP-1 is important for 
intestinal regeneration. 

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 time-dependent 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-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. 
Figure 3: Authors should show pictures of entire culture well on Figure3c. Organoid experiment 
showed short term culture result, how about long term culture? 
Figure 5: Did you check the expression level of Edr1 in SKCO15 cells? 
Figure 6: Add 2D functional assay by using Edr1KO organoid. 
Figure 7,8 : Authors did not show the adding Erd1 directly act on EC cells but not other immune cells. 
Previous report showed downreguation of Erd1 increased the number of Th17 pathogenic cells in EAE 
model. Authors need to eliminate the possibility of affecting other immune cells, by using RAGKO mice 
or with anti CD4/ anti NK1.1 antibody.