Mac-/Lactosylceramide regulates intestinal homeostasis and secretory cell fate commitment by facilitating Notch signaling
- National Institute of Biological Sciences, China
- Academy for Advanced Interdisciplinary Studies, Peking University, China
- School of Life Sciences, Tsinghua University, China
- School of Basic Medicine, Nanchang Medical College, China
- Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, China
Figures
Figure 1 with 1 supplement
A mosaic genetic screen identifies GlcT as a tumor suppressor in adult Drosophila midgut.
(A) Diagram showing the lineage hierarchy of intestinal stem cells (ISCs) in the Drosophila intestine. EB, enteroblast; EEP, enteroendocrine progenitor; EC, enterocyte; EE, enteroendocrine cell. (B–D) Mosaic Analysis with a Repressible Cell Marker (MARCM) clones (green) induced for 5 days with Pros (red) staining (B), together with quantification of cell number (C, n=6) and the percentage of Pros+ EE cells per clone (D, n=6) in EA30, E230, and GlcT null allele (GlcTΔ8) mutant. (E, F) Overexpression of GlcT in EA30 or E230 mutant clones with Pros staining, along with quantification of the percentage of Pros+ EE cells per clone (F, n=4). (G–J) esg >GlcT IR in adult fly gut for 14 days. PH3 staining (G) and Pros staining (I) with quantification of positively stained cells (H, n=11; J, n=12). Error bars represent mean ± SEM, with p-values indicated in the figure (two-tailed Student’s t-test). Scale bars: 25 μm.
Figure 1—figure supplement 1
Cross-scheme for the mosaic genetic screen on the 2R chromosome.
(A) A schematic illustrating the principles of FLPase-induced mitotic recombination for generating homozygous mutant cells in heterozygous animals. (B) The cross-scheme and workflow for the forward genetic mosaic screen conducted in this study. EMS, ethyl methanesulfonate.
Figure 2
Genetic analysis of the components of the glycosphingolipid (GSL) synthesis pathway reveals specific tumor-suppressive activity for egh, in addition to GlcT.
(A) Diagram illustrating a portion of the GSL metabolic pathway synthesized from ceramide, highlighting the key enzymes involved at each step. (B–D) GlcTΔ8 clones induced for 7 days without or with co-expression of p35 or CDase, stained with anti-DCP-1 or anti-Pros (B). Quantification of the percentage of Pros+ cells in clones of the indicated genotypes (C, D, n=5). (E–G) Mosaic Analysis with a Repressible Cell Marker (MARCM) clones induced in several mutants of key enzymes in the GSL synthesis pathway. Pros staining in egh mutant clones (eghA, eghB, egh7), brn228, β4GalNAcTA-IR, and α4GT1-IR clones (E). Quantification of cell number (F, n=7) and the percentage of Pros+ cells (G, n=7) in clones of the indicated genotypes. Error bars represent mean ± SEM, with p-values indicated (two-tailed Student’s t-test). Scale bars: 25 μm.
Figure 3
The EE tumor phenotype is a result of MacCer deficiency.
(A–C) LacCer feeding after Mosaic Analysis with a Repressible Cell Marker (MARCM) clone induction. Anti-Pros staining of anterior midgut (AM) and posterior midgut (PM) in GlcTΔ8 clones (A). Quantification of cell number (B, n=7) and the percentage of Pros+ cells (C, n=7) in clones of the indicated genotypes. Error bars represent mean ± SEM, with p-values indicated (two-tailed Student’s t-test). Scale bars: 25 μm.
Figure 4 with 1 supplement
Loss of GlcT leads to reduced activation of Notch signaling in intestinal stem cell (ISC) progenies.
(A, B) Expression of NRE-lacZ, the Notch activity reporter, in GlcTΔ8 clones (A) and quantification of LacZ fluorescence intensity for signal positive cell (B, n=3). (C) Tk and AstC staining in GlcTΔ8 clones in the posterior (R5) region of the Drosophila midgut. (D) Pros staining in Nintra-overexpressing GlcTΔ8 clones. Error bars represent mean ± SEM, with p-values indicated (two-tailed Student’s t-test). Scale bars: 25 μm.
Figure 4—figure supplement 1
The EGFR/Ras/MAPK and JAK/STAT signaling activities in GlcT mutant clones.
GlcT mutant clones stained with pERK (red, upper panels), a direct indicator of EGFR/Ras/MAPK signaling activity, and STAT92E (red, lower panels), whose nuclear localization is indicative of JAK/STAT signaling activity. Note that pERK was not significantly increased in GlcT mutant cells compared to normal cells outside of the clones, and STAT92E activity was also not obviously altered in GlcT mutant cells compared to normal cells outside the clones. Scale bar: 25 μm.
Figure 5
GlcT regulates the endocytic trafficking of Delta.
(A, B) Pros staining in flies carrying GlcT-IR driven by Dlts and NREts, respectively (A), and quantification of the percentage of Pros+ cells (B, n=4). (C, D) Dl staining in an antibody uptake assay performed in GlcTΔ8 flies after 3-hour clone induction (C) and quantification of internalized antibody-Dl area inside and outside of the clones (D, n=5). (E, F) Dl staining in esg >GlcTIR flies co-stained with Rab5-GFP, Rab7-GFP, or Rab11-GFP (E); yellow arrows indicate co-localization of Dl and GFP. Quantification of Dl and GFP co-localization (F, n=6). Error bars represent mean ± SEM, with p-values indicated (two-tailed Student’s t-test). Scale bars: 25 μm (10 μm in E).
Figure 6
GlcT shows tissue specificity in regulating Notch signaling activity.
(A) Cut staining in wing disc of GlcTΔ8 clones. (B) EYA staining in follicle cell clones (non-green areas) of GlcTΔ8 flies. (C) Dl staining in germ cell clones (non-green area) of GlcTΔ8 mutant clone, yellow arrows indicate punctate Dl accumulation. Scale bars: 25 μm.
Figure 7 with 1 supplement
Transient loss of Ugcg in mouse small intestine causes reduced number of intestinal stem cells (ISCs) and increased number of goblet cells.
(A, B) Olfm4 staining in the duodenum and ileum of VillinCreERT2; Ugcgflox/flox mice 48 hours after tamoxifen injection (A, n=3) and quantification of Olfm4 fluorescence intensity (B). (C, D) Muc2 staining of goblet cells in the duodenum and ileum of Ugcg-CKO mice (C) and quantification of Muc2+ goblet cells per villus (D, n=3). (E) Schematic model illustrating the role of MacCer in regulating the Notch signaling pathway. Error bars represent mean ± SEM, with p-values indicated (two-tailed Student’s t-test). Scale bars: 100 μm.
Figure 7—figure supplement 1
The Crypt-Villus morphology in Ugcg CKO mice.
Small intestine (duodenum) from control and VillincreERT2, Ugcgflox/flox mice stained with K20, which marks enterocytes and goblet cells, and Ki67, which marks proliferative cells. Note that the mutant intestine has shortened villi and elongated crypts. Dotted lines indicate the boundary between the crypt and the villus. Samples were collected at 48 hours after tamoxifen injection. Scale bar: 100 μm.
Author response image 1
To investigate whether MacCer modifies Dl by Western blot.
(A) Four lanes were loaded: the first two contained 20 μL of membrane extract (lane 1: GlcT-IR, lane 2: control), while the last two contained 10 μL of membrane extract. (B) Full blot images are shown under both long and shortexposure conditions.
Additional files
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Supplementary file 1
Summary of tumor suppressive gene loci identified in the screen.
- https://cdn.elifesciences.org/articles/106184/elife-106184-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/106184/elife-106184-mdarchecklist1-v1.docx
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Source data 1
Raw data of the experiments presented in this article.
- https://cdn.elifesciences.org/articles/106184/elife-106184-data1-v1.xlsx
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Mac-/Lactosylceramide regulates intestinal homeostasis and secretory cell fate commitment by facilitating Notch signaling
eLife 14:RP106184.
https://doi.org/10.7554/eLife.106184.3
