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. 2015 Jun 15;142(12):2147-62.
doi: 10.1242/dev.121046. Epub 2015 May 26.

Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche

Soumyashree Das  1 , Shiyan Yu  1 , Ryotaro Sakamori  1 , Pavan Vedula  1 , Qiang Feng  1 , Juan Flores  1 , Andrew Hoffman  2 , Jiang Fu  3 , Ewa Stypulkowski  1 , Alexis Rodriguez  1 , Radek Dobrowolski  1 , Akihiro Harada  4 , Wei Hsu  3 , Edward M Bonder  1 , Michael P Verzi  5 , Nan Gao  6
Affiliations

Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche

Soumyashree Das et al. Development. .

Abstract

Communication between stem and niche supporting cells maintains the homeostasis of adult tissues. Wnt signaling is a crucial regulator of the stem cell niche, but the mechanism that governs Wnt ligand delivery in this compartment has not been fully investigated. We identified that Wnt secretion is partly dependent on Rab8a-mediated anterograde transport of Gpr177 (wntless), a Wnt-specific transmembrane transporter. Gpr177 binds to Rab8a, depletion of which compromises Gpr177 traffic, thereby weakening the secretion of multiple Wnts. Analyses of generic Wnt/β-catenin targets in Rab8a knockout mouse intestinal crypts indicate reduced signaling activities; maturation of Paneth cells - a Wnt-dependent cell type - is severely affected. Rab8a knockout crypts show an expansion of Lgr5(+) and Hopx(+) cells in vivo. However, in vitro, the knockout enteroids exhibit significantly weakened growth that can be partly restored by exogenous Wnts or Gsk3β inhibitors. Immunogold labeling and surface protein isolation identified decreased plasma membrane localization of Gpr177 in Rab8a knockout Paneth cells and fibroblasts. Upon stimulation by exogenous Wnts, Rab8a-deficient cells show ligand-induced Lrp6 phosphorylation and transcriptional reporter activation. Rab8a thus controls Wnt delivery in producing cells and is crucial for Paneth cell maturation. Our data highlight the profound tissue plasticity that occurs in response to stress induced by depletion of a stem cell niche signal.

Keywords: Gpr177; Intestinal stem cell; Paneth cell; Rab8a; Wnt secretion; Wntless.

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Figures

Fig. 1.
Fig. 1.
RAB8A intersects GPR177 traffic. (A) Flag-GPR177 was immunoprecipitated (IP) from lysates of a stable human HeLa cell line in the presence of 1% Triton X-100. Precipitates were blotted (IB) for various vesicular markers. (B) Flag-GPR177Δ44 lacking the C-terminal tail failed to co-immunoprecipitate with RAB8A. (C) GST pull-down showed binding of Flag-GPR177 to GST-RAB8A, but not to GST, GST-CDC42 or GST-JFC-D1. Data are representative of three independent experiments. (D) Live cell imaging detected GPR177-mCherry in EGFP-RAB8A vesicles in HeLa cells. The line scan histogram (along the dotted line in the merge) shows colocalization of the two fluorescent signals (arrows). (E) Single vesicle tracks of GPR177-mCherry vesicles in Rab8a+/+ (top) and Rab8a−/− (bottom) MEFs (t=10 s; n=8 vesicles in each cell). Arrows indicate the start and end of an individual vesicle track. See supplementary material Fig. S1B, Movies 1 and 2. (F) Instantaneous vesicle speed distribution (nm/s), with a bin size of 70 nm/s. The speed of Gpr177 vesicles in Rab8a+/+ cells peaks at 130-140 nm/s (green dashed line), agreeing with myosin V-powered movement (bar), which was reduced in Rab8a−/− MEFs (red dashed curve). 1717 steps for wild type and 995 steps for Rab8a−/− were analyzed. Scale bars: 10 μm.
Fig. 2.
Fig. 2.
Rab8a deletion reduces Wnt secretion. (A) Concentrated conditioned media or cell lysates, in increasing amounts, from Rab8a+/+ and Rab8a−/− MEFs were analyzed by western blot. Rab8a−/− MEFs showed less secreted, but more intracellular, Wnt5a/b. The ratio of secreted to intracellular Wnt5a/b was deduced from corresponding samples (n=3, bar chart). Histone H3 was used to detect medium contamination by cell lysates and to normalize corresponding Wnt5a/b bands. (B) WNT5A expression constructs or empty vectors were transiently transfected together with Topflash reporter and Renilla luciferase plasmids into Rab8a+/+ and Rab8a−/− MEFs, followed by dual-luciferase assays. Rab8a+/+ and Rab8a−/− MEFs showed 14.6-fold and 3.9-fold inductions of Topflash activities, respectively, by transfected WNT5A as compared with empty vector-transfected counterparts. **P<0.01. (C) Rab8a−/− MEFs secrete less Wnt3a-Gluc. Wnt3a-Gluc was transiently transfected into wild-type, Rab8a−/−, Rab8b knockdown (KD), Rab8a−/−/Rab8b KD and Gpr177−/− MEFs, with firefly luciferase serving as control for transfection efficiency. Secretion of Wnt3a-Gluc was eliminated by 5 mM C59 or Gpr177 depletion. **P<0.01, ***P<0.001; n.s., not significant. (D) Rab8a−/− MEFs showed insignificant changes in the secretion of Shh-Renilla, Met-Luc or the biosynthetic cargo alkaline phosphatase (AP). (E) Surface protein biotinylation and isolation detected decreased Gpr177 in Rab8a−/− MEFs. Consistent results were obtained in two independent experiments. (F) Rab8a−/− MEFs showed similar levels of cell surface Fzd (1-10) and Lrp6 compared with Rab8a+/+ MEFs. Note that Wnt3a stimulated surface Lrp6 phosphorylation (Ser1490) in Rab8a+/+ and Rab8a−/− MEFs. (G) Topflash assays showed that Rab8a−/− MEFs, with a significantly lower basal Wnt signaling activity (##P<0.01, compared with vehicle-treated Rab8a+/+ MEFs), responded strongly to Wnt3a stimulation. **P<0.01, ***P<0.001, compared with vehicle-treated cells.
Fig. 3.
Fig. 3.
Rab8a deletion impairs canonical Wnt signaling in intestines. (A) Quantitative RT-PCR showed reduced Tcf1, Olfm4, Axin2 and Ascl2 expression in Rab8a−/− intestines. (B) Western blots showed reduced Tcf1, Tcf4, Sox9 and β-catenin levels in Rab8a−/− intestines. (C) Immunohistochemistry for β-catenin showed a reduced number of crypts with nuclear β-catenin+ cells (arrows). Note that only crypts with detectable Paneth cells were scored for their positive or negative inclusion of nuclear β-catenin (n=50 for each genotype). (D) β-Gal staining of mouse small intestines showed a significant reduction of Axin2 reporter activity in crypts in Axin2lacZ/+;Rab8a−/− mice. Thirty continuous crypts were analyzed in each section of independent wild-type and knockout mice. (E) β-Gal-stained Axin2lacZ/+ and Axin2lacZ/+;Rab8a−/− intestinal organoids showed significantly reduced bud number and size in the absence of Rab8a. Images were taken at day 10 after crypt plating. Arrow points to a small bud. Twenty organoids of each genotype were quantified for β-gal-stained bud areas (circled in red). *P<0.05, **P<0.01, ***P<0.001. Scale bars: 10 μm in C,D; 15 μm in E.
Fig. 4.
Fig. 4.
Rab8a deficiency-impaired organoid growth is restored by Wnt3a. (A) Crypts (n=100) of each genotype were seeded in triplicate and the number of surviving organoids then counted daily. Growth of 80% of Rab8a−/− intestinal organoids was arrested within the first 2 days. Rab8aΔIEC did not show any significant improvement in organoid survival. Data were collected from three independent experiments. (B,C) After seeding, Rab8+/+ and Rab8a−/− intestinal organoids (n=100 for each genotype) were immediately supplemented with Wnt3a or the Gsk3β inhibitor CHIR at the indicated concentrations. Media containing these supplements were replenished daily. Rab8a−/− intestinal organoids showed similar morphological and proliferative features as Rab8+/+ organoids upon Wnt3a or CHIR treatment. (D) In the above experiments, surviving organoids were counted daily during 1 week after seeding. Percentages of surviving organoids are plotted. Note that Wnt3a and CHIR significantly increased the number of surviving Rab8a−/− organoids. Scale bars: 15 μm.
Fig. 5.
Fig. 5.
Rab8a deletion impairs Paneth cell maturation. (A) Lysozyme staining showed reduced numbers of lysozyme+ Paneth cells in Rab8a−/− crypts. ***P<0.001. (B) Quantitative RT-PCR showed no significant change in the Paneth cell gene expression signature in Rab8a−/− intestinal tissues. (C) Lysozyme and EdU (1 h) staining of Rab8a−/− organoids detected lysozyme+ Paneth cells in proliferating buds of surviving organoids. (D) TEM showed that the residual Paneth cells in Rab8a−/− crypts exhibit a reduction in the typical number and size of granules. Arrow points to a granule in a Rab8a−/− crypt. (E) An expansion of smooth ER was observed in all remaining Rab8a−/− Paneth cells. The thickness of stacked ER cisternae was significantly expanded in Rab8a−/− (834±46 nm) as compared with Rab8a+/+ (333±7 nm) Paneth cells. Scale bars: 10 µm in A; 15 µm in C; 2 µm in D; 1 µm in E.
Fig. 6.
Fig. 6.
Rab8a deletion affects Gpr177 transport to the plasma membrane but not ER export. (A) Quantification of Gpr177+ immunogold particle distributions in the ER, non-ER Golgi/vesicle and plasma membrane of Rab8a+/+ and Rab8a−/− Paneth cells (particles counted from 12 Rab8a+/+ and nine Rab8a−/− Paneth cells from three Rab8a+/+ and two Rab8a−/− mice, respectively). (B) The majority of Gpr177+ immunogold particles (arrows) were detected in the ER of Rab8a+/+ and Rab8a−/− Paneth cells. (C,D) Representative micrographs showing Gpr177+ immunogold particles in Golgi and lysosomes. Gold particles were detected in lysosomes (open arrowheads) in Rab8a−/− (D) but not in Rab8a+/+ (C). (E) Gpr177+ immunogold particles were frequently detected at apical or basolateral plasma membranes in wild-type Paneth cells. A significant reduction in plasma membrane-localized Gpr177+ particles was detected in Rab8a−/− Paneth cells per unit length of plasma membrane. Data were collected for a total length of 312 µm plasma membrane in 12 Rab8a+/+ Paneth cells and 328 µm plasma membrane in 14 Rab8a−/− Paneth cells from three Rab8a+/+ and two Rab8a−/− mice, respectively. Arrowheads point to plasma membranes between a stem cell and a Paneth cell. lu, lumen. *P<0.05. Scale bars: 500 nm.
Fig. 7.
Fig. 7.
Adaptive changes in Rab8a−/− crypts. (A) EdU labeling (1 and 3 h, red) of mouse small intestine showed a significant increase in proliferative Lgr5+ cells in Rab8a−/−;Lgr5EGFP−IRES−CreERT2 mice. The total number of Lgr5+ cells was also increased in Rab8a−/− crypts. Around 45 crypts that contained Lgr5+ cells were analyzed in each tissue section of independent Lgr5EGFP−IRES−CreERT2 or Rab8a−/−;Lgr5EGFP−IRES−CreERT2 mice. (B) EdU labeling of the colon showed an increase in proliferative Lgr5+ cells in Rab8a−/−;Lgr5EGFP−IRES−CreERT2 mice. (C) Co-staining of BrdU (1 h, brown) and β-gal (blue) showed increased BrdU+ cells in Axin2lacZ/+;Rab8a−/− intestines, despite decreased Axin2 reporter activity in the same crypts. One hundred continuous crypts were analyzed in each section of independent Rab8a+/+ and Rab8a−/− mice. (D) Rab8a−/− intestines contained more cells with the strongest level of Hopx immunoreactivity (arrows). Arrowheads point to cells with moderately higher immunoreactivity than in wild type. One hundred continuous crypts were analyzed in each section of independent Rab8a+/+ and Rab8a−/− mice. (E) Quantitative RT-PCR detected increased Wnt3a levels in Rab8a−/− intestines (n=3 for each genotype). (F,G) RNA in situ hybridization to detect Wnt3 (F) or Wnt3a (G), showing ectopic activation of Wnt3a in Rab8a−/− crypts, whereas Wnt3 was largely unaffected. *P<0.05, ***P<0.001. Scale bars: 10 µm.
Fig. 8.
Fig. 8.
Rab8a facilitates anterograde transport of Gpr177-Wnt. Rab8a facilitates the transport of post-Golgi Gpr177-Wnt vesicles to the plasma membrane for secretion. Loss of Rab8a attenuates Gpr177 exocytotic traffic and, via an unknown mechanism (red dotted arrows), may reroute Gpr177 into endolysosomes. TGN, trans-Golgi network.
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