Mapping early fate determination in Lgr5+ crypt stem cells using a novel Ki67-RFP allele

Abstract

Cycling Lgr5+ stem cells fuel the rapid turnover of the adult intestinal epithelium. The existence of quiescent Lgr5+ cells has been reported, while an alternative quiescent stem cell population is believed to reside at crypt position +4. Here, we generated a novel Ki67RFP knock-in allele that identifies dividing cells. Using Lgr5-GFP;Ki67RFP mice, we isolated crypt stem and progenitor cells with distinct Wnt signaling levels and cell cycle features and generated their molecular signature using microarrays. Stem cell potential of these populations was further characterized using the intestinal organoid culture. We found that Lgr5high stem cells are continuously in cell cycle, while a fraction of Lgr5low progenitors that reside predominantly at +4 position exit the cell cycle. Unlike fast dividing CBCs, Lgr5low Ki67- cells have lost their ability to initiate organoid cultures, are enriched in secretory differentiation factors, and resemble the Dll1 secretory precursors and the label-retaining cells of Winton and colleagues. Our findings support the cycling stem cell hypothesis and highlight the cell cycle heterogeneity of early progenitors during lineage commitment.

Keywords: Ki67; Lgr5; intestinal stem cells; quiescent stem cells; secretory lineage.

Figure 1. Characterization of the cell cycle of Lgr5-expressing cells

A, B Most of the Lgr5⁺ (Lgr5-GFP, green) cells have high levels of KI67 (red; white arrowheads) as seen on transverse sections. A small fraction of Lgr5⁺ stem cells do not express the KI67 protein (yellow arrowheads). Lysozyme (LYZ, blue)-expressing Paneth cells mark the Paneth cell zone and are negative for Lgr5 and KI67. Scale bar, 20 μm. Quantification of the results is shown in (B). C A cross section of an intestinal crypt showing Lgr5⁺ stem cells (Green), KI67 (Red), chromogranin A (CHGA, white) and the nucleus (DAPI, blue). Scale bar, 10 μm. D Quantification of the position of Lgr5⁺ and CHGA⁺ cells seen in (C). pos: position, total: sum of Lgr5⁺ (left) and CHGA⁺ (right) cells in each position. Each square shows the percentage of cells with a given antigen expression at the respective position among Lgr5⁺ (left) and CHGA (right)-expressing cells. Lgr5 expression is visualized using the endogenous Lgr5^GFP florescence.

Figure 2. Generation and characterization of the Ki67^RFP knock-in mouse

A The Ki67^RFP targeting construct. B, C Ki67^RFP expression can be visualized on live section from the small at low (B) and high (C) magnification. D RFP-expressing cells from Ki67^RFP-expressing small intestines can be identified and sorted using FACS. E, F Graph showing Hoechst 34580 staining on dissociated live intestinal crypt cells. The x-axis shows the dye intensity, which is directly correlated to the DNA content of the cells. Red and blue lines indicate RFP⁺ and RFP⁻ fractions, respectively. Quantification of results is shown in (F). All indicates the profile of total live crypt cells. G Quantitative PCR analysis of sorted Ki67^RFP+ and Ki67^RFP− population. Levels in sorted RFP⁺ cells is compared to RFP⁻ cells from the same mouse to indicate the fold change (n = 3). Results are normalized to expression levels in crypt, which is 1. H, I Sorted single cells were stained using anti-KI67 antibody. RFP⁺ cells express KI67 while most RFP⁻ cells lack KI67 expression. Quantification of the results is shown in (I). J, K Sorted RFP⁺ cells can initiate organoid cultures while RFP⁻ cells are less efficient. Quantification of the results is shown in (K). Data information: Error bars indicate the standard deviation. In (D), FL1 channel is used as a blank channel to visualize autofluorescence. Scale bars: 100 μm (B), 20 μm (C), 10 μm (H), 200 μm (J). In (B) and (C), the gamma settings were adjusted for better visualization of the dim RFP fluorescence.

Figure 3. Identification of CBCs with distinct KI67 expression

A Intestinal crypts from control (WT), Ki67^RFP, and Lgr5^GFPKi67^RFP mice used for the FACS sorting. Scale bar, 80 μm. The gamma settings were adjusted for better visualization of the dim RFP fluorescence. B We defined 4 populations of CBCs on FACS using different GFP and RFP expression levels: Lgr5^GFP high Ki67^RFP positive (L^highK⁺), Lgr5^GFP high Ki67^RFP negative (L^highK⁻), Lgr5^GFP low Ki67^RFP positive (L^lowK⁺), Lgr5^GFP low Ki67^RFP negative (L^lowK⁻). C, D Organoid cultures derived from sorted CBC populations (C) and their quantification (D). Error bars indicate the standard deviation. Scale bar, 80 μm.

Figure 4. Comparison of CBC populations

1. Heatmap generated using the differentially expressed genes between CBC populations calculated as significantly different (ANOVA test, P < 0.01). Red indicates higher expression.

2. Two-group plotters comparing Ki67⁺Lgr5^high to Ki67⁺Lgr5^low (left) and Ki67⁻Lgr5^high to Ki67⁻Lgr5^low (right).

3. Quantification of the number of genes that are significantly different (ANOVA, P < 0.01) between groups and is expressed at least twofold (black bars) of 1.5-fold higher (gray bars) in one of the groups.

4. Gene set enrichment analysis (GSEA) reveals high similarity between the top 200 stem cell genes (Munoz et al, 2012) and 58 genes upregulated upon deletion of APC in the small intestine (Sansom et al, 2004) to the genes enriched in Ki67⁺Lgr5^high versus Ki67⁺Lgr5^low and Ki67⁻Lgr5^high versus Ki67⁻Lgr5^low. Results are displayed in log2.

Figure 5. The cell cycle dynamics of CBC populations

1. Heatmap displaying genes with the combined GO term “cell cycle” that are differentially expressed between CBC populations (ANOVA test, P < 0.01). Red indicates higher expression.

2. qPCR analysis for the expression of cell cycle indicators mKi67 and Ccnb2 in CBC populations, crypt, and the villus. Results are normalized to the expression level in the crypt, which is displayed as 0. Error bars indicate the standard deviation.

3. Quantification of the number of genes that are significantly different (ANOVA, P < 0.01) between groups and is expressed at least twofold (black bars) of 1.5-fold higher (gray bars) in one of the groups.

4. Two-group plotters comparing Lgr5^highKi67⁺ to Lgr5^highKi67⁻ (left) and Lgr5^lowKi67⁺ to Lgr5^lowKi67⁻ (right).

Figure 6. Lgr5^lowKi67⁻ population corresponds to the secretory precursors

1. Heatmap displaying genes with the combined GO terms “transcription factor” and “transcriptional regulatory activity” that are differentially expressed between CBC populations (ANOVA test, P < 0.01). Red indicates higher expression.

2. Plots displaying the distribution of the proposed quiescent stem cell markers to CBC populations. The numbers on the y-axis show the normalized expression values on microarrays in log2. Each dot represents a separate sample.

3. Gene set enrichment analysis (GSEA) analysis. Genes significantly enriched in Lgr5^lowKi67⁻ population was used to generate a molecular signature, which was compared to published data sets including the Dll1⁺ (van Es et al, 2012), Paneth (PC; van Es et al, 2012), enteroendocrine (EEC; van Es et al, 2012) and label-retaining cell (LRC; Buczacki et al, 2013) expression profiles. NES: Normalized enrichment score.

Figure 7. Stem and progenitor cell compartmentalization in the small intestine

Paneth cells mark the stem cell zone where dividing Lgr5^high stem cells reside. In the “zone of differentiation”, progenitors are specified to the absorptive or the secretory lineages (Bjerknes & Cheng, 1981). The “+4” markers are shared between both Lgr5^high stem cells and Lgr5^low progenitors independent of their cell cycle status. Lgr5^lowKi67⁻ intermediate secretory precursors arise from Lgr5^high stem cells and overlap with Dll1⁺ cells, the common precursors of the Paneth, goblet, and the enteroendocrine cells (van Es et al, 2012). The label-retaining cells are the longest-lived secretory precursors that differentiate into Paneth and enteroendocrine cells (Buczacki et al, 2013). Both LRCs and Dll1⁺ can re-enter the cell cycle to generate multiple secretory cells, displaying limited proliferation potential (secretory precursors, red nucleus). Partially overlapping secretory precursors likely represent different maturational stages. Lgr5^lowKi67⁺ cells rapidly proliferate and generate enterocytes through Lgr5⁻Ki67⁺ and/or Lgr5⁻Ki67⁻ transient amplifying progenitors. Dark and light green indicate high and low Lgr5 expression, respectively. Red nucleus: mitotically active state; black nucleus: G₀ state.