Dynamic Lkb1-TORC1 signaling as a possible mechanism for regulating the endoderm-intestine transition - PubMed (original) (raw)

Dynamic Lkb1-TORC1 signaling as a possible mechanism for regulating the endoderm-intestine transition

Kathryn E Marshall et al. Dev Dyn. 2010 Nov.

Erratum in

Dev Dyn. 2011 Feb;240(2):459

Abstract

The intestinal epithelium arises from undifferentiated endoderm via a developmental program known as the endoderm-intestine transition (EIT). Previously we found that the target of rapamycin complex 1 (TORC1) regulates intestinal growth and differentiation during the EIT in zebrafish. Here we address a possible role for the tumor-suppressor kinase Lkb1 in regulating TORC1 in this context. We find that TORC1 activity is transiently upregulated during the EIT in both zebrafish and mouse. Concomitantly, Lkb1 becomes transiently localized to the nucleus, suggesting that these two phenomena may be linked. Morpholino-mediated knockdown of lkb1 stimulated intestinal growth via upregulation of TORC1, and also induced precocious intestine-specific gene expression in the zebrafish gut epithelium. Knockdown of tsc2, which acts downstream of lkb1, likewise induced early expression of intestine-specific genes. These data suggest that programmed localization of Lkb1 could represent a novel mechanism for regulating the EIT during intestinal development in vertebrates.

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Figures

Figure 1. TORC1 is transiently upregulated during the EIT in zebrafish

(A) Zebrafish embryos were stained via whole mount immunofluorescence for P-RPS6ser240/244, followed by sectioning in JB-4 and counterstaining with DAPI. The white line delineates the gut epithelium (g). The number of immunopositive cells and staining increases from 48 to 72 hpf, then decreases upon cytodifferentiation of the epithelium. (B) To verify that the fluorescent signal is a specific readout of TORC1, we treated embryos with 100 μM rapamycin for 24 hours prior to fixation. This resulted in significant reduction of the immunofluorescence signal. (C) Mean fluorescence intensity (grayscale 0-4095) of the epithelium immunostained for P-RPS6ser240/244. Sections shown in panel A, as well as additional representative sections (total n=4 for each stage) were subjected to histometric analysis using the program Openlab to calculate the mean fluorescence intensity for the intestinal epithelium. For panel B, only the sections shown were subjected to analysis. P-values less than 0.05 are indicated by an asterisk.

Figure 2. TORC1 is transiently upregulated during the EIT in the mouse embryonic intestine

(A) Sections of embryonic mouse intestine were stained for P-RPS6ser240/244 and then counterstained with DAPI. Weak immunostaining is seen at e13.5 (pre-EIT), with marked increase in number and intensity of immunopositive cells at e14.5 (mid-EIT). At e15.5, shortly after the formation of a simple columnar epithelium, a few residual epithelial cells remain positive, but the signal is substantially decreased compared with e15.5. The white line delineates the epithelium from subjacent mesenchyme. (B) Quantification of fluorescence intensity in the epithelium shows a pattern in which mean intensity peaks at e14.5. Shown are representatives of n=4 sections from each stage that were analyzed. T-test showed significant differences (p<0.05) between e13.5 vs 14.5, and e14.5 vs 15.5).

Figure 3. Zebrafish lkb1 is widely expressed

a-f: Whole-mount in situ hybridization to zebrafish lkb1 shown in dorso-lateral view with the head to the left. Background staining was determined by omitting the RNA probe in panel f. g and h: Histological sections of embryos in panels c and d, respectively. Solid lines delineate gut tube from the yolk. Original magnification: a-f, 40×; g and h, 400×. Abbreviations: g, gut; L, liver; p, pancreas; y, yolk.

Figure 4. . Lkb1 displays dynamic subcellular localization during the EIT in zebrafish

(A) Lkb1 subcellular localization was monitored in a Tg[β_-actin: lkb1- mCherry_] zebrafish line at 48 hpf, 72 hpf*, 77 hpf, and 96 hpf. Frozen sections were counterstained with DAPI (blue). A white line outlines the gut tube at 48 hpf, and asterisks mark the gut lumen. Original magnification: 630×.

Figure 5. Lkb1 displays dynamic subcellular localization during the EIT in embryonic mouse intestine

(A) Confocal images of Lkb1 (red) counterstained with phalloidin (green) to reveal cell apices and DAPI (blue) to mark nuclei. Lkb1 becomes increasingly localized to nuclei as the EIT proceeds from primitive gut tube (e13.5) to transitional epithelium. At e14.5, a secondary lumen within the epithelium is marked by phalloidin (arrowhead). Upon formation of a simple columnar epithelium at 15.5, staining in the nuclei is relatively decreased, though still present. Original magnification, 630×. Insets in merged images show 3× digital magnification of area outlined by boxes, with a single cell boundary and nucleus outlined by green and white, respectively. (B) Histometric analysis of Lkb1 staining intensity within the epithelium reveals dynamic distribution of Lkb1 in nuclear and non-nuclear (i.e. cytoplasmic) cellular compartments during the EIT.

Figure 6. Co-imaging of Lkb1 localization and TORC1 activation

Zebrafish expressing Lkb1-mCherry (red) at the indicated stages were immunostained for P-RPS6ser240/244 (green). The stages shown represent pre-, mid-, and early post-EIT. At 55 hpf, Lkb1 is diffusely present in the scant cytoplasm of intestinal epithelial cells, though it can be seen in the nuclei of various other cell types outside the intestine. Staining for P-RPS6ser240/244 is low relative to surrounding tissue. At 72 hpf, Lkb1-mCherry is seen in the nuclei of most of the intestinal epithelial cells, and P-RPS6ser240/244 immunostaining is increased relative to 55 hpf. At 84 hpf, nuclear localization of Lkb1-mCherry is sporadically seen in the epithelium, and most (though not all) of those cells stain positive for P-RPS6ser240/244. A white line encloses the gut epithelium. Original magnification, 200×. Insets, 600×. e, epithelium; m, mesenchyme.

Figure 7. Knockdown of lkb1 stimulates epithelial cell proliferation and growth

(A) Histological sections through the intestine of uninjected, spl morphant, and ATG morphant, embryos at 48 hpf, 72 hpf, and 96 hpf. At 48 hpf the gut lumen is outlined by solid black line. g, gut. Shown are representative sections that correspond to the median gut size as determined by morphometry and depicted in the bar graph. Sections were stained with H&E and original magnification was 400×. (B) Depiction of morphometric analysis for cell number and cell shape (height-to-width ratio) using 25 embryos per treatment. A-P positions were matched between embryos using standard anatomic landmarks (pancreatic islet, gall bladder, neural tube). (C) Analysis of proliferation rate by BrdU incorporation comparing uninjected (black squares) and spl morphant (white squares) at 48, 72, and 96 hpf. Significance was determined using Student's two-tail, unpaired t-test (n=25 embryos). Single asterisk, p<0.05; double asterisk, p<0.005.

Figure 8. Lkb1 regulates intestine size via TORC1

(A) Histological analysis of intestine at 96 hpf in lkb1 spl morphants in the presence and absence of rapamycin. Sections were stained with H&E. Original magnification: 400×. (B) Morphometric analysis of cell number showing no morphant phenotype in the presence of rapamycin (n=6 embryos).

Figure 9. Knockdown of lkb1 induces precocious expression of intestinal markers

(A) In situ hybridization to intestinal fatty acid binding protein (ifabp) at 48 hpf. Arrow indicates the gut tube, showing increased expression in the lkb1 morphant. Original magnification, 40×. (B) Relative mRNA expression of intestinal markers ifabp and iap at 48 hpf and 72 hpf, and slc10a2 at 96 hpf in uninjected, spl, ATG, 5 mm, and 25 N determined by qRT-PCR. Samples were run in triplicate and normalized to ef1α to control for total RNA. Significance was determined using Student's two-tail, unpaired t-test. (C) Rhodamine-conjugated wheat germ agglutinin (WGA) staining for goblet cells in the uninjected and lkb1 spl morphant at 96 hpf and 121 hpf. Very few goblet cells are visible in the control embryo, whereas the morphant displays a much brighter signal. At 121 hpf, the fluorescent signals are qualitatively similar between control and morphant. White line encircles the hindgut. Original magnification: 40×.

Figure 10. Tsc2 knockdown induces precocious expression of intestinal markers

(A) RT-PCR demonstrating knockdown of tsc2 in spl MO-injected embryos. (B) Western blot of extract from tsc2 spl morphants at 53 hpf for P-RPS6ser240/244, showing upregulation of TORC1 signaling. (C) qRT-PCR of tsc2 morphant RNA for markers of intestinal differentiation showing upregulation of ifabp and iap expression at 48 hpf and 72 hpf, and slc10a2 at 96 hpf. Samples were normalized to ef1α and significance was determined using Student's two-tail, unpaired t-test.

Figure 11. Suggested model for the role of Lkb1 in regulating the EIT

We hypothesize that the EIT requires the formation of a developmental intermediate characterized by the transient relocalization of Lkb1 from the cytoplasm to the nucleus. Consequently, TORC1 becomes upregulated, promoting epithelial growth and differentiation. After the intestine reaches a target size, Lkb1 then relocalizes to the cytoplasm, TOR activity decreases, and the cells become stabilized in a terminally differentiated state. The source and identity of the signals regulating Lkb1 in this context remain to be identified.

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Dynamic Lkb1-TORC1 signaling as a possible mechanism for regulating the endoderm-intestine transition - PubMed (original) (raw)