Hmga1 drives expansion of the ISC compartment

A prior gene expression profile study showed that Hmga1 is among the genes enriched in Lgr5+ ISCs (ref. 29). HMGA1 is also among the genes most highly expressed in diverse epithelial human cancers as compared to normal epithelium, including intestinal malignancies12,13,14,17,33. We therefore sought to elucidate the functional role of Hmga1 in ISCs, both in normal intestinal epithelial homeostasis and in intestinal neoplasia. To this end, we crossed our Hmga1 transgenic mice onto Lgr5-EGFP mice6, which mark Lgr5+ ISCs with enhanced green fluorescent protein (EGFP). The Hmga1 transgene is driven by the H-2Kb promoter and μ enhancer, which confer transgene expression in intestinal crypt basilar cells37, lymphoid cells35 and uterine tissue36. In both Hmga1 transgenic and wild-type (WT) mice, Hmga1 protein localizes to the nuclei of Lgr5+ ISCs (Fig. 1a–d). Interestingly, Lgr5+ ISCs extend further up the crypts in the Hmga1 transgenic mice compared to WT mice in all regions of the small intestine (duodenum, jejunum, ileum; Fig. 1a–f), consistent with the expansion in the ISC pool and enhanced self-renewal in vivo. The percentage of Lgr5+ ISCs per total crypt cells by fluorescent stain (Fig. 1f; **P<0.00001; Mann-Whitney test) and relative frequency of Lgr5+ISCs per crypt cell isolates by fluorescence-activated cell sorting (FACS) were significantly (P<0.05; two-tailed Student’s t-test) increased in the Hmga1 transgenic small intestine (Fig. 1g,h), further validating the expansion in ISCs. To determine whether Hmga1 gene expression accounts for the elevated protein levels in the Lgr5+ ISCs, we assessed Hmga1 mRNA in WT mice and found that it is increased by ∼4-fold in Lgr5+ ISCs isolated by FACS compared to Lgr5− cells (Fig. 1g,h). We also assessed Hmga1 mRNA in crypt cells from the transgenic model and found that Hmga1 expression is increased about twofold in both Lgr5+ ISCs and Lgr5− cells compared to the same cell populations in WT mice. In both WT and transgenic models, Hmga1 mRNA is enriched in the Lgr5+ ISC population (Fig. 1g,h).

Figure 1: Hmga1 localizes to ISCs and its overexpression expands the ISC compartment in transgenic mice. (a) ISCs from WT/Lgr5-EGFP+ mice stain green (upper panel) in small intestinal cross-sections. Hmga1 stains red and localizes to GFP+ ISCs (lower panel). (b) Intranuclear Hmga1 stains brown (immunohistochemistry (IHC)) in small intestinal cross-sections from WT/Lgr5-EGFP+ mice. (c) ISCs stain green in Hmga1/Lgr5-EGFP+ transgenic mice and extend further up the base of the crypt in Hmga1 transgenic mice compared to WT mice. (Cells with red nuclear staining outside of crypts are Hmga1 transgenic lymphocytes) (d) Intranuclear Hmga1 stains brown (IHC) in small intestinal cross-sections from Hmga1/Lgr5-EGFP+ mice. (e) Fluorescent staining identifies GFP+ ISCs at each region in the small intestine of WT and Hmga1 mice (duodenum, jejunum, ileum). (f) The percentage (mean±s.d.) of GFP+ ISCs per crypt in WT and Hmga1 transgenic mice are shown; **P<0.00001, Mann–Whitney test (n=50 crypts per region, 3 mice per genotype). Dot plot shows individual data points. (g) Hmga1 transgenic mice have higher GFP+ ISC frequency as assessed by flow cytometry; mean frequency±s.d. from two experiments are shown. (h) Hmga1 mRNA is enriched in ISCs isolated by flow cytometry for GFP+ cells using quantitative, real-time PCR (qPCR) in WT and Hmga1 transgenic mice. Hmga1 is higher in both GFP+ ISCs and GFP− crypt cells from the Hmga1 transgenic model compared to WT mice. Bars show mean relative Hmga1 expression±s.d. from three experiments performed in triplicate; Gapdh was used as a loading control; *P<0.05; two-tailed Student’s t-test. Scale bars, 20 μm. Full size image

ISCs are regulated by factors from the stromal compartment in addition to intestinal epithelial cells1,2,3,4,5,6,7,8,9,38,39,40. To define the role of Hmga1 in ISCs within the epithelial compartment, we used organoids, an in vitro intestinal crypt cell culture model1,2,3,4,41. Organoid buds are a surrogate for ISC function because they comprise crypt-like structures with ISCs on the tips; differentiated epithelial cells extend towards the luminal centres of the organoids. We derived organoids from small intestinal epithelial crypt cells isolated from transgenic or WT mice and compared bud formation and projected surface area. Prior to culture, an equal number of crypts from WT or Hmga1 transgenic mice of similar sizes were isolated by passage through a 70 μM filter41. Similar to the histologic results in the transgenic mouse intestine, we found a marked increase in projected surface area per organoid (Image-Pro Plus Version 6) and bud number per organoid in those derived from the Hmga1 transgenic mice as compared to WT controls (Fig. 2a–c; Supplementary Fig. 1a,b). There were more Hmga1 organoids with >3 buds as compared to WT organoids by day 6 in culture (Fig. 2b). The majority of WT organoids had ⩽1 buds per organoid, while the majority of Hmga1 organoids had ≥2 buds at day 6 (Fig. 2b). Next, we isolated small crypts by passage through a 40 μM filter40,41 and performed time-lapsed confocal microscopy42,43,44,45 to ascertain the rate of self-renewal in Lgr5+ ISCs and bud development from day 0 to day 6 in 3D organoid culture (Fig. 2d–f). A representative image of crypt cells from days 0 and 6 from WT mice or Hmga1 transgenics, respectively, are shown (Fig. 2d). We discovered a marked increase in the rate of self-renewal at day 6 in the Lgr5+ ISCs from the Hmga1 transgenics compared to the WT mice based on the projected surface area of Lgr5+ cells (μManager software; Stanford Photonics; Fig. 2e,f). Similar to our prior results using standard microscopy, we also found more buds and greater projected surface area of Lgr5+ cells in the Hmga1 organoids versus the WT organoids by confocal microscopy (Fig. 2f).

Figure 2: Hmga1 enhances ISC function in gut organoid cultures. (a) Typical 3D organoids cultured from crypts are shown from WT or Hmga1 transgenic mice. (b) Bud numbers were ascertained in 3D organoids from isolated crypts (n>100 organoids per mouse; 3 mice per genotype). Bars show mean percentage of organoids±s.d. with different bud numbers. (Yellow≥3, grey=2, pink=1, blue=0). The mean percentage of organoids with ≥ 3 buds was increased in Hmga1 organoids compared to WT organoids. *P<0.05; two-tailed Student’s t-test. (c) Organoid projected surface area (PSA) is shown (mean±s.d.) from WT or Hmga1 mice (n>20 organoids/genotype). **P<0.01; two-tailed Student’s t-test. (d) Representative confocal imaging of crypt cells (day 0) and organoids (day 6) with GFP+ staining for Lgr5+ ISCs, Phalloidin (F-actin; red) for cell clusters and organoid structure, and Hoechst (blue) for nuclei are shown. (e) Relative expansion rate of ISCs was calculated by the ratio of the PSA of GFP+ ISCs on day 6 (n≥42 organoids per group) over the PSA on day 0 (n≥72 crypt cell clusters/group). **P<0.01; Mann–Whitney test. (f) ISC PSA (y axis) and bud number (x axis) at days 0 and 6 are shown. **P<0.01, *P<0.05; Mann–Whitney test. (g) Lgr5-GFP+ ISCs were purified by flow cytometry and cultured in matrigel. (h) Colony (organoid)-forming efficiency (mean±s.d.) was calculated from purified GFP+ ISCs isolated as single cells (n=3,000 cells per group) from WT and Hmga1 mice in three experiments; **P<0.01; two-tailed Student’s t-test. (i) Re-plating efficiency (mean±s.d.; n=3,000 cells per group) was assessed after dissociating colonies from purified GFP+ ISCs isolated as single cells from WT and Hmga1 mice using flow cytometry. **P<0.01; two-tailed Student’s t-test. Scale bars, 50 μm. Full size image

Previous work from our group demonstrated that forced expression of Hmga1 prevents differentiation in human embryonic stem cells16. We therefore reasoned that Hmga1 expression would predominate at the crypt-like regions enriched for ISCs at the bud tips and decrease in regions of differentiated cells at the base of the buds. To test this, we assessed Hmga1 protein levels throughout the organoids and found that Hmga1 is enriched at the bud tips where ISCs predominate, and undetectable in the differentiated cells, suggesting that differentiation is permitted in cells with lower levels of Hmga1 (Supplementary Fig. 2). Together, these results suggest that Hmga1 is a key factor for ISC maintenance and function.

Because we could not exclude the possibility that the enhanced stem cell function observed in the Hmga1 organoids resulted from in vivo exposure to lymphoid or other cells with transgenic Hmga1 expression and downstream factors, we engineered WT organoids to overexpress Hmga1. To this end, we transduced organoids via lentivirus expressing Hmga1 and GFP (FUGW-Hmga1) and compared this to control organoids transduced with lentivirus expressing GFP alone (FUGW). The WT organoids engineered to overexpress Hmga1 exhibited a similar phenotype to the organoids derived from the Hmga1 mice with increased bud formation (Supplementary Fig. 3), demonstrating that this phenotype was dependent on Hmga1.

To determine whether the enhanced stem cell function in organoid-forming ability and bud development resulted from cell-autonomous properties of the Hmga1 ISCs or from ISC interactions with other crypt epithelial cells, we purified Lgr5+ ISCs isolated as single cells from transgenic and WT mice using flow cytometry and compared their efficiency in organoid formation and re-plating assays. The purified Hmga1 ISCs generated organoids at a fivefold greater efficiency as compared to WT ISCs (Fig. 2g,h). Moreover, re-plating efficiency was also enhanced fivefold in the Hmga1 ISCs (Fig. 2i). Interestingly, the Hmga1 organoids generated from purified Lgr5+ ISCs adopt a cyst-like, spherical structure similar to the morphology observed in organoids treated with Wnt or engineered to overexpress Ascl2, a transcription factor important in maintaining stem cell identity in ISCs7. To determine whether Hmga1 organoids remained in a cyst-like structure because they were comprised predominantly of undifferentiated Lgr5+ stem cells, we stained for the ISC marker, Lgr5-GFP+. Strikingly, most of the cells within the Hmga1 organoids generated from purified Hmga1 Lgr5-GFP+ ISCs maintain this stem cell marker (Lgr5-GFP+; Supplementary Fig. 4), in contrast to WT organoids, which only stain for Lgr5-GFP+ at the tips of the buds (Fig. 2d). Together, these studies demonstrate that Hmga1 maintains undifferentiated Lgr5+ ISCs and enhances self-renewal in this model.

Next, we sought to determine whether Hmga1 is required for self-renewal and ISC function. First, we silenced Hmga1 in WT crypt cells using lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting Hmga1 (shHmga1) and compared this to WT crypt cells transduced with a control lentiviral vector in organoid cultures (Fig. 3a). Crypt cells were incubated with Wnt3a to enhance transduction efficiency as previously reported2,3. Hmga1 expression was repressed in the organoids transduced to express the shHmga1 (Fig. 3b). Strikingly, the crypt cells with Hmga1 silencing formed very few organoids with smaller projected surface areas and decreased bud number, while those transduced with control vector organized into typical 3D structures and generated new buds (Fig. 3a–c; Supplementary Fig. 5a). To rule out any potential nonspecific toxicity from the shHmga1 vector, we also tested an inducible, shRNA lentiviral vector targeting Hmga1 (inducible-shHmga1), which is linked to a gene encoding red fluorescent protein (RFP); both RFP and shHmga1 are induced by doxycycline. Crypt cells transduced with the lentivirus, but not induced to express the shHmga1 vector, organized into 3D organoids with buds similar to what we observed in WT crypt cultures (Fig. 3d; top panels). As expected, the uninduced organoids (cultured without doxycycline) do not express RFP. In contrast, those organoids in which the shHmga1 vector was induced by doxycycline express RFP and exhibit an impaired capability to proliferate and generate 3D organoids; the projected organoid surface area was decreased compared to uninduced controls (Fig. 3d,e). Bud formation was also disrupted (Supplementary Fig. 5b). Hmga1 gene silencing in organoids transduced with the inducible shHmga1 was confirmed by quantitative, reverse transcription PCR (qPCR; Fig. 3e). To further validate these findings, we transduced organoids derived from the Hmga1 mice with the shHmga1 vector and found that silencing Hmga1 also disrupted their ability to form organoids and generate buds (Supplementary Fig. 5c). These studies indicate that Hmga1 is a key factor involved in self-renewal, bud formation and organization into 3D organoid structures.

Figure 3: Hmga1 deficiency disrupts ISC function in 3D organoids. (a) Representative images of organoid formation after silencing Hmga1 by delivery of lentiviral vector expressing shRNA targeting Hmga1 (knockdown or KD) or control lentivirus (Cont) are shown. Scale bars, 50 μm. (b) Relative Hmga1 expression (mean±s.d.) was assessed from two experiments performed in triplicate (via qPCR) in organoids transduced with lentivirus (Cont versus KD) at day 10. Gapdh was used to control for loading. **P<0.01; two-tailed Student’s t-test. (c) PSA (mean±s.d.) was assessed in organoids transduced with lentivirus (Cont versus KD) at the indicated time points (n=20 organoids per group). *P<0.05, **P<0.01; two-tailed Student’s t-test. (d) Representative image of organoid formation±knockdown of Hmga1 via an inducible lentiviral vector expressing red fluorescent protein (RFP) and shRNA targeting Hmga1 are shown. Doxcyline (0.5 μg ml−1) was used to induce RFP and shRNA. Scale bars, 50 μm. (e) Relative Hmga1 expression (mean±s.d.) in organoids with or without induction of lentivirus shRNA targeting Hmga1 was assessed from two experiments performed in triplicate (via qPCR); Gapdh was used to control for loading. *P<0.05, **P<0.01; two-tailed Student’s t-test. (f) PSA (mean±s.d.) of organoids with or without induction of Hmga1 knockdown at the indicated time points are shown (n=20 organoids per group from two different virus transduction experiments). **P<0.01; Mann–Whitney test. Full size image

Hmga1 induces self-renewal through Wnt/β-catenin signalling

When organoids from Hmga1 mice were incubated with Wnt3a to enhance lentiviral transduction, we observed a striking phenotype whereby the Hmga1 organoids formed very large, cyst-like, spherical organoids comprised predominantly of Lgr5+ ISCs, in contrast to WT organoids, which generated smaller cysts at a decreased frequency (Fig. 4a; Supplementary Fig. 6). These findings suggested that the Hmga1 organoids are more sensitive to Wnt signalling. Wnt/Tcf4/β-catenin signalling is an evolutionarily conserved pathway important for self-renewal in epithelial crypt ISCs and many other tissue-specific adult stem cells38. Moreover, hyperactive Wnt signalling leads to malignant transformation in intestinal epithelium38. To begin to define the role of Hmga1 in Wnt/Tcf4/β-catenin signalling, we assessed immunostaining for β-catenin as a surrogate for canonical Wnt signalling. β-catenin was markedly increased in the Hmga1 transgenic intestinal epithelium and concentrated at the base of the crypts (Fig. 4b). A similar increase was also observed in WT organoids transduced to overexpress Hmga1 (Fig. 4c). Since Hmga1 functions as an architectural transcription factor that alters gene expression, we hypothesized that Hmga1 could upregulate expression of factors that enhance Wnt signalling. To test this, we first compared expression of genes encoding Wnt agonist receptors that function in intestinal epithelium, including Lgr5, Frizzled (Fzd)5, Fzd7, low-density lipoprotein receptor-related protein 5 (Lrp5) and Lrp6 in purified Lgr5+ ISCs isolated from WT or transgenic mice. We found an increase in expression of all Wnt agonist receptors tested (Lgr5, Fzd5/7, Lrp5/6) in the Hmga1 ISCs by >2 to 4-fold compared to control ISCs (Fig. 4d). Together, these results indicate that Hmga1 amplifies Wnt signalling by upregulating genes encoding Wnt agonist receptors in ISCs, including the ISC marker, Lgr5, in our model.

Figure 4: Hmga1 amplifies Wnt signalling in ISCs. (a) Representative organoids after exposure to Wnt3a (10 ng μl−1) are shown. Hmga1 organoids form large, cyst-like spheres (standard (top) and fluorescent (bottom) microscopy). Scale bars, 50 μm. (b) β-Catenin IHC staining is shown in small intestines from WT and Hmga1 mice. Scale bars, 20 μm. (c) β-Catenin IHC staining is shown in organoids engineered to express control lentivirus (FUGW; left) or Hmga1 (FUGW-Hmga1; right). Scale bar, 20 μm. (d) Relative expression (mean±s.d.) of genes encoding Wnt agonist receptors was ascertained by qPCR in GFP+ ISCs from WT (blue) or Hmga1 transgenic mice (orange) in two experiments performed in triplicate. Gapdh was used to control for loading. **P<0.01; two-tailed Student’s t-test. (e) Relative expression (mean±s.d.) of genes encoding Wnt/Tcf4/β-catenin transcriptional targets was ascertained by qPCR in GFP+ ISCs from WT or Hmga1 transgenic mice in two experiments performed in triplicate. Gapdh was used to control for loading. **P<0.01; two-tailed Student’s t-test. (f) HEK 293 (left) and Caco2 (right) cells infected with the synthetic Wnt reporter construct containing seven optimal Tcf4/β-catenin-binding sites showed activation in cells overexpressing Hmga1. Purified Wnt3A (100 ng ml−1) protein was used as a positive control. Bars show mean luciferase activity±s.d. from two experiments performed in triplicate. **P<0.01; two-tailed Student’s t-test. (g) Representative image of GFP+ ISCs from WT or Hmga1 mice are shown in 3D culture with Wnt porcupine inhibitors IWP-2 (0.5 μM) and C59 (0.5 μM). Scale bars, 50 μm. (h) Relative colony-forming efficiency (mean±s.d.) from three experiments is shown with GFP+ ISCs from WT or Hmga1 mice in 3D culture with the Wnt porcupine inhibitors (IWP-2, C59). *P<0.05; two-tailed Student’s t-test. Full size image

Once β-catenin is released from an inhibitory complex following Wnt signalling, it binds to DNA together with its partner, Tcf4, to induce Wnt pathway genes. In human embryonic stem cells, HMGA1 induces expression of c-MYC (ref. 16), a WNT/TCF4/β-catenin gene target, suggesting that Hmga1 could cooperate with Wnt/Tcf4/β-catenin to regulate the Wnt stem cell program. We therefore assessed expression of Wnt/Tcf4/β-catenin target genes, including Axin2, Ascl2, β-catenin, Cd44, c-Myc, Ephb2, Ets, Tcf4 and Prom-1. Expression of all of these genes, excluding Prom-1, was increased by >2 to 6-fold in Hmga1 ISCs versus WT control ISCs (Fig. 4e). Together, these results indicate that Hmga1 not only activates Wnt target genes together with Tcf4/β-catenin, but also amplifies Wnt signals by inducing expression of Wnt agonist receptor genes.

To better define the role of Hmga1 in regulating Wnt signalling, we cultured Hmga1 organoids in the absence of the Wnt receptor agonist, R-spondin 1 (R-spo1), which is essential for organoid formation in this 3D culture system. R-spo1 is secreted by intestinal stromal cells and binds to the Lgr5 receptor to activate Wnt signalling39,40. We discovered a striking difference in the response of Hmga1 organoids to the absence of R-spo1 as compared to the WT controls: Hmga1 organoids continued to survive and proliferate for over 2 weeks, while the WT organoids lost their 3D structural organization and ultimately died by day 5 (Supplementary Fig. 7). By 3 weeks, however, the Hmga1 organoids also stopped proliferating and lost their 3D organization, suggesting that Hmga1 overexpression partially rescues loss of Wnt signalling via R-spo1, but does not completely recapitulate or bypass Wnt.

To further investigate the link between HMGA1 and Wnt, we determined whether cells overexpressing HMGA1 have enhanced Wnt reporter activity using an established Wnt reporter construct that includes seven canonical Tcf4/β-catenin-binding sites in tandem upstream of a luciferase reporter gene46. Both HEK 293 human embryonal kidney cells and Caco-2 colon cancer cells were transduced with the Wnt reporter lentiviral vector, which includes a puromycin resistance gene. After selection for stable integration of the Wnt reporter construct by puromycin, the cultured cells were transduced with either control retrovirus or retrovirus expressing human HMGA1. In both cell types, HMGA1 activated Wnt reporter activity, while retroviral delivery of the control virus failed to do so (Fig. 4f). These experiments further support the role of HMGA1 in activating Wnt signalling.

To further test the link between Hmga1 and Wnt, we treated crypt cells with the Wnt inhibitor, C59, which blocks Wnt-mediated transcription and cell proliferation by inhibiting porcupine (PORCN), a protein required for Wnt palmitoylation, secretion and biological activity. Crypt cells isolated from Hmga1 or WT small intestine were selected for small crypt size and cell number by passage through a 40 μM filter. The Hmga1 crypts generated organoids and buds in C59 (0.25 μM) with a greater projected surface area than WT crypt cells cultured with C59 (Supplementary Fig. 8). In addition, the mean projected surface area of WT organoids cultured in C59 was reduced by days 6 and 10 compared to WT organoids cultured under control conditions with vehicle alone. In contrast, the Hmga1 crypt cells were relatively impervious to C59; by day 6, there was no difference in projected surface area of Hmga1 organoids compared to those cultured with vehicle alone, although the projected surface area decreased by day 10 when cultured with C59 compared to those cultured with vehicle control. This blunted response of Hmga1 organoids to Wnt inhibition could result from the ability of Hmga1 to amplify Wnt signalling, thereby mitigating the effects of Wnt inhibition. Alternatively, or in addition to amplifying Wnt signalling, the Hmga1 organoids could dampen the effects of Wnt inhibition by providing a buffer as a result of an increased size and cell number. Finally, an increase in the number of Paneth cells, which secrete Wnt agonists, could mitigate the effects of Wnt inhibition. To distinguish between these possibilities, we isolated single, purified Lgr5+ ISCs from transgenic or WT mice and cultured them in the presence of two different Wnt PORCN inhibitors, C59 and IWP-2. Strikingly, the Hmga1 ISCs proliferated and formed 3D, cyst-shaped organoids for up to 10 days, albeit at a slower rate between days 6–10 compared to purified Hmga1 ISCs cultured without inhibitor (Fig. 4g,h). In contrast, the WT ISCs generated very few organoids when cultured with C59. Moreover, no organoids formed from WT ISCs cultured in IWP-2 (Fig. 4g,h). Together, these findings demonstrate that Hmga1 ISCs have a blunted response to Wnt inhibitors, which is consistent with their ability to amplify Wnt signals.

Hmga1 expands the Paneth cell niche and upregulates Sox9

Paneth cells are terminally differentiated epithelial cells derived from ISCs and located at the base of intestinal crypts47,48,49,50. They support ISC survival by secreting Wnt3a and other factors, thus providing an epithelial niche for ISCs. To determine whether Hmga1 alters the Paneth cell niche in our transgenic models, we stained for lysozyme using alkaline phosphatase, which marks Paneth cells. There was a marked increase in lysozyme stain (Fig. 5a), which was confirmed quantitatively by immunohistochemistry (IHC) pixels (Fig. 5b). To determine whether the increase in lysozyme stain corresponds to an increase in Paneth cell number, Paneth cells were enumerated in mouse intestinal tissue after controlling for the region of the intestine (Fig. 5c; n=100 crypts per group from duodenal epithelium). There was a marked increase in Paneth cell number per crypt, which paralleled the increase in lysozyme stain by IHC. Next, we co-stained the duodenal epithelium with EpCAM to demarcate cell borders, DAPI to indicate individual nuclei, and lysozyme such that the frequency of cells staining positive for lysozyme could be enumerated within the crypts (Fig. 5d). There was also a marked increase in Paneth cell frequency (Fig. 5e; n=50 crypts per group from duodenal epithelium), consistent with an expansion in the Paneth cell niche in the Hmga1 transgenic duodenal epithelium as compared to WT control duodenal epithelium. To further confirm the increase in Paneth cells by Hmga1, we assessed Paneth cell number in Hmga1 organoids using two different approaches. First, Paneth cells have a distinctive granular appearance on phase contrast microscopy. We therefore compared the mean number of granular cells per bud in WT and Hmga1 organoids after controlling for bud size by dividing the Paneth cell number by the projected surface area per bud (Fig. 5f,g; n=35 buds per group). Similar to the Hmga1 mouse intestinal tissue, there was a significant (P<0.00001; Mann–Whitney test) increase in Paneth cell number per bud projected surface area in the Hmga1 organoids (Fig. 5g). Second, we assessed Paneth cell number by staining with lysozyme, which appeared to be increased in organoids transduced to express Hmga1 compared to control organoids (Fig. 5h). To quantitatively assess the frequency of Paneth cells per total bud cell number, we co-stained with EpCAM, DAPI and lysozyme. There was an increase in Paneth cell frequency using this approach, similar to our results with intestinal tissue and granular cell number (Fig. 5h,i). Finally, to further corroborate this result, we assessed expression of a transcript that is highly specific for Paneth cells, Defcr-rs51, and found that it is increased in the Hmga1 organoids compared to control organoids (Fig. 5j). Together, these results indicate that Hmga1 induces Paneth cell niche formation and expansion, which could help to promote ISC maintenance and expansion.

Figure 5: Hmga1 expands the Paneth cell niche. (a) IHC for lysozyme is shown from small intestines of WT and Hmga1 transgenic mice. Scale bar, 20 μm. (b) Lysozyme IHC pixels (mean±s.d.; imagine pro-plus 6.0 software) is shown in small intestinal cross-sections (n=78 crypts per group; 3 mice per genotype). **P<0.01; Mann–Whitney test. (c) Paneth cell number (mean±s.d.) per crypt (n=100 crypts per group; 3 mice per genotype). **P<0.00001; Mann-Whitney test. (d) Immunofluorescent co-staining for lysozyme, Ep-CAM and DAPI is shown from small intestines of WT and Hmga1 transgenic mice. Scale bars, 20 μm. (e) Paneth cell frequency (mean±s.d.) was obtained by dividing the Paneth cell number by total cell number per crypt in WT and Hmga1 intestine (n=50 crypts per group; 3 mice per genotype). **P<0.00001; Mann–Whitney test. (f) Granular cells in WT and Hmga1 organoid buds are shown via phase contrast microscopy. Scale bars, 50 μm. (g) Granular cells per bud PSA (mean±s.d.) from WT and Hmga1 organoids are shown (n=35 buds per group). **P<0.00001; Mann–Whitney test. (h) IHC for lysozyme (top) and immunofluorescent co-staining for lysozyme, Ep-CAM and DAPI (bottom) are shown in organoids from WT or Hmga1 mice. Scale bars, 50 μm. (i) Paneth cell frequency per bud (mean±s.d.) was calculated by dividing the Paneth cell number by the total bud cell number (n=30 organoids per group). **P<0.00001; Mann–Whitney test. (j) Relative expression (mean±s.d.) of Defcr-rs, the Paneth cell-specific transcript, is shown from two experiments performed in triplicate from organoids from WT and Hmga1 mice. Gapdh was used to control for loading. **P<0.01; two-tailed Student’s t-test. Full size image

Because terminal differentiation to a Paneth cell requires Sox9 (refs 47, 48, 49), we hypothesized that Hmga1 fosters Paneth cell expansion by upregulating Sox9 expression. HMGA1 induces the SOX family member, SOX2, in human embryonic stem cells16; further, both human and mouse SOX9/Sox9 have similar AT-rich regions and predicted Hmga1 DNA-binding sites in the 5′ untranslated region. Sox9 is also a β-catenin/Tcf4 target gene. We found that Hmga1 upregulates Sox9 expression in the Lgr5+ ISCs as compared to WT Lgr5+ ISCs (Fig. 6a). To determine whether Hmga1 directly induces Sox9 gene expression, we performed chromatin immunoprecipitation (ChIP) in intestinal crypt cells from WT mice. We discovered that Hmga1 binds directly to the Sox9 promoter at two conserved sites (Fig. 6b; Supplementary Fig. 9a,b). Enrichment for Hmga1 binding was greatest at the proximal site (denoted site 1) and significant, albeit lower, at the second most proximal site (site 2; P<0.01 by two-tailed Student’s t-test). Hmga1 binding was not enriched at the distal site (site 3). For a positive control, we used a histone H3 antibody, which showed significant enrichment (P<0.01 by two-tailed Student’s t-test) at all promoters tested. For a negative control, we used the IgG antibody, which had minimal binding to chromatin at all promoter regions tested. As a second negative control, we used the murine Hprt gene; this promoter region was previously shown to be negative for Hmga1 occupancy. We confirmed that Hmga1 does not occupy the Hprt promoter (Fig. 6b). Next, we performed transfection experiments to determine whether Hmga1 transactivates the Sox9 promoter reporter construct containing the Hmga1-binding site 1, which had the greatest enrichment for Hmga1 binding (Supplementary Fig. 9a–c). As a control, we included a Sox9 promoter reporter construct lacking this Hmga1-binding site (Supplementary Fig. 9b,c). We found that Hmga1 induced the Sox9 promoter construct that included site 1, while there was no induction of the promoter construct lacking this site (Supplementary Fig. 9d). As an additional control, we included a dominant-negative HMGA1 construct with mutations in the second AT-hook DNA-binding domain, which no longer binds to DNA16. The dominant-negative mutant also failed to transactivate either Sox9 promoter construct (Supplementary Fig. 9d). To further validate this relationship between Hmga1 and Sox9, we assessed Sox9 mRNA in organoid cells transduced to overexpress Hmga1 and found that Sox9 was induced by Hmga1 (Supplementary Fig. 9e). In contrast, we also found that Sox9 was repressed in organoids with Hmga1 knockdown following transduction with shRNA targeting Hmga1 compared to controls (Supplementary Fig. 9f).

Figure 6: Hmga1 directly induces Sox9 expression. (a) Relative Sox9 mRNA (mean±s.d.) by qPCR in GFP+ ISCs from WT and Hmga1 mice is shown from two experiments performed in triplicate. Gapdh was used to control for loading. **P<0.01; Student’s t-test. (b) Hmga1 binding to the Sox9 promoter by ChIP is shown from mouse crypt cells. IgG antibody and the Hprt promoter region were used as negative controls; histone H3 was a positive control. Bars show mean enrichment±s.d. from two experiments performed in triplicate; **P<0.01; two-tailed Student’s t-test. (c) Sox9 IHC staining (brown) is shown in intestinal cross-sections from WT and Hmga1 transgenic mice. Scale bars, 20 μm. (d) Western blots for Sox9, Hmga1, and Gapdh (loading control) were performed from freshly isolated crypt cells from WT and Hmga1 transgenic mice. Western blots were done three times; a representative blot is shown. Size markers (kDa) are indicated. (e) Densitometry analysis±s.d. was performed on repeat western blots. *P<0.05; two-tailed Student’s t-test. (f) Sox9 and Hmga1 IHC staining (brown) is shown in adjacent sections of small intestine from WT and Hmga1 mice. Arrows show representative staining for Hmga1 (H) or Sox9 (S); triangles show Hmga1 transgenic lymphocytes which have high levels of Hmga1 (but not Sox9). Scale bars, 50 μm. (g) Mean number of cells±s.d. staining positive for Hmga1, Sox9 or both were identified by IHC in adjacent cross-section on slides of small intestinal crypts from WT and Hmga1 mice (n≥21 crypts per group.) **P<0.01 by Mann–Whitney test. Full size image

To determine whether cells with high levels of Hmga1 protein also have high levels of Sox9 protein, we assessed Sox9 protein and found that it is increased in the crypts of the Hmga1 transgenic mice compared to WT controls by IHC (Fig. 6c). To quantitatively assess Hmga1 and Sox9 proteins, we performed western blot analysis of crypt cells from WT and transgenic mice; the Hmga1 transgenic crypt cells had an increase in both Hmga1 and Sox9 protein compared to WT crypt cells (Fig. 6d,e). Next, we compared adjacent sections stained for Hmga1 or Sox9 and found that the majority of cells with detectable Hmga1 at the base of the crypts also have detectable Sox9 protein in both WT and transgenic mice (Fig. 6f,g). In WT mice, >70% of cells that stained positive for Hmga1 also stained positive for Sox9. In the transgenic mice, which have more cells with Hmga1 staining and higher levels of Hmga1, we found that >95% of cells that stained positive for Hmga1 also stained positive for Sox9 (Fig. 6f,g).

While Sox9 is required for Paneth cell differentiation47,48,49,50,51, it is not known whether increased levels of Sox9 lead to expansion in Paneth cells. To test this, we transduced WT organoids with a lentivirus expressing Sox9 following induction by doxycycline (Fig. 7). Immunofluorescent staining showed increased Sox9 protein in the doxycycline-induced organoids compared to uninduced control organoids cultured for the same period of time (Fig. 7a). Sox9 protein (by western blot analysis) and mRNA (by qPCR) were both increased relative to uninduced organoids (Fig. 7b,c). Consistent with the observation that Sox9 decreases proliferation in vivo in intestinal epithelium49, we observed that the organoid growth (estimated by projected surface area) was decreased in organoids with Sox9 induction compared to uninduced organoids (Fig. 7d,e). We also found that the projected bud surface area was decreased with Sox9 induction (Fig. 7f). Next, we compared Paneth cell frequency in Sox9-induced and uninduced organoids using two different approaches. First, we used phase contrast microscopy to identify Paneth cells morphologically by the distinctive granules, which are characteristic of these cells. The relative Paneth cell number per bud-projected surface area was increased in the Sox9-induced organoids compared to uninduced control organoids (Fig. 7g,h). Second, we used lysozyme stain to identify Paneth cells, together with EpCAM stain (to delineate cell membranes)50 and DAPI (to demarcate nuclei). There was also an increase in the frequency of Paneth cells per total bud cell number in Sox9-induced organoids (n>50 buds per condition from >50 organoids per condition; Fig. 7i,j). To further substantiate these results, we assessed expression of a transcript that is highly specific for Paneth cells, Defcr-rs51, and showed that it is also increased in the organoids overexpressing Sox9 (Fig. 7k). These results suggest that Hmga1-mediated induction of Sox9 helps to expand the Paneth cell niche, although there are likely to be other factors that contribute to Paneth cell differentiation. Together, our results demonstrate that Hmga1 drives self-renewal and expansion of ISCs in addition to helping to establish an epithelial stem cell niche through Paneth cell differentiation.

Figure 7: Paneth cell number increases in organoids overexpressing Sox9. (a) Immunofluorescent stain for Sox9 (red), DAPI (blue) and EpCAM (green) is shown in organoids transduced with lentivirus expressing doxycycline-dependent inducible Sox9 (Sox9-Fuw). Sox9 (Sox9-Fuw Dox(+)) was induced by doxycycline (1 μg ml−1) for 5 days; controls were cultured for 5 days without doxycycline. (b) Representative western blot for Sox9 in control and induced organoids is shown. Densitometry±s.d. was performed on three repeat western blots. **P<0.01; two-tailed t-test. (c) Sox9 mRNA (mean±s.d.) was assessed by qPCR from two experiments performed in triplicate in control and induced organoids. Gapdh was used to control for loading. **P<0.01; two-tailed Student’s t-test. (d) Typical organoids from uninduced controls (left) and induced (right) cultures are shown. (e) PSA±s.d. of organoids (n=52 per group) are shown. **P<0.01; two-tailed Student’s t-test. (f) PSA±s.d. of buds (n=52 per group) are shown. **P<0.01; two-tailed Student’s t-test. (g) Paneth cells identified as granular cells using phase contrast microscopy within individual buds are shown. (h) Granular cell frequency (mean±s.d.) was estimated by dividing the total granular cell number by the total bud PSA (n=52 per group). **P<0.01; Mann–Whitney test. (i) Paneth cells were identified based on lysozyme stain (red) in cells delineated by EpCAM (green) for cell membrane and (DAPI) for nuclei. (j) Paneth cell frequency (mean±s.d.) was ascertained by dividing the number of lysozyme staining cells (red) delineated by EpCAM (green) by the total cell number (DAPI) per bud (n=35 per group). **P<0.01; Mann–Whitney test. (k) Relative expression of Defc-rs (mean±s.d.) was compared in uninduced control and Sox9-induced organoids by qPCR from two experiments performed in triplicate. Gapdh was used to control for loading. **P<0.01; two-tailed Student’s t-test. Scale bars, 50 μm. Full size image

HMGA1 and SOX9 in colonic epithelium and colorectal cancer

To determine whether our findings in mouse intestine are relevant to humans, we assessed expression of HMGA1 and SOX9 in human intestinal epithelium. Using the Cancer Genome Atlas (TCGA), we found that HMGA1 and SOX9 are positively correlated in normal colonic epithelium (P=0.008, r=0.52; Fig. 8a). We also stained human small intestine, and found that HMGA1 localizes to the columnar basal cells within the crypts where ISCs are located (Supplementary Fig. 10). Because HMGA1 is overexpressed in diverse epithelial cancers and correlates with cancer stem cell properties in experimental models, we also sought to determine whether HMGA1 and SOX9 are co-regulated in colorectal cancer. Strikingly, both HMGA1 and SOX9 are markedly upregulated in human colorectal cancer (P<0.0000001), although their expression was not correlated in this setting (Fig. 8a). Interestingly, the relative expression and amplitude of HMGA1 mRNA levels are greater than that of SOX9 (Fig. 8a). These data support a model whereby HMGA1 and SOX9 are crucial for normal ISC function, and both become upregulated in carcinogenesis (Fig. 8b,c). These findings also suggest that upregulation of both HMGA1 and SOX9 beyond a threshold may be necessary for early reprogramming of an epithelial cell to a neoplastic cell, while further increases in HMGA1 could drive tumour progression.