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. 2012 Oct 5;11(4):529-40.
doi: 10.1016/j.stem.2012.06.017.

The Par complex and integrins direct asymmetric cell division in adult intestinal stem cells

Spyros Goulas  1 , Ryan Conder, Juergen A Knoblich
Affiliations

The Par complex and integrins direct asymmetric cell division in adult intestinal stem cells

Spyros Goulas et al. Cell Stem Cell. .

Abstract

The adult Drosophila midgut is maintained by intestinal stem cells (ISCs) that generate both self-renewing and differentiating daughter cells. How this asymmetry is generated is currently unclear. Here, we demonstrate that asymmetric ISC division is established by a unique combination of extracellular and intracellular polarity mechanisms. We show that Integrin-dependent adhesion to the basement membrane induces cell-intrinsic polarity and results in the asymmetric segregation of the Par proteins Par-3, Par-6, and aPKC into the apical daughter cell. Cell-specific knockdown and overexpression experiments suggest that increased activity of aPKC enhances Delta/Notch signaling in one of the two daughter cells to induce terminal differentiation. Perturbing this mechanism or altering the orientation of ISC division results in the formation of intestinal tumors. Our data indicate that mechanisms for intrinsically asymmetric cell division can be adapted to allow for the flexibility in lineage decisions that is required in adult stem cells.

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Figures

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Graphical abstract
Figure 1
Figure 1
Adult Drosophila ISCs Divide Asymmetrically and Segregate Members of the Par Complex into the Apical Daughter Cell during ISC Division (A) Schematic diagram of the Drosophila intestinal epithelium. EC, enterocyte; ISC, intestinal stem cell; BM, basement membrane; SM, surrounding musculature. (B) Transverse view of Delta-positive ISC (red) and its immediate progeny, the EB (green), expressing Su(H)GBE Gal4 > UASCD8::GFP. (C and D) Quantification of the percentage of cell doublet populations in terms of cell fate (C, n = 137) and the number of Delta-positive ISCs within 4–5 day clones (D, n = 33). (E–G) Members of the Par complex are expressed and polarized apically. Endogenous Baz (E, red) apically localizes with respect to the basement membrane (E, Vkg::GFP, green) within small ISC-like diploid cells. Par-6::GFP (F, green) is expressed in Delta-positive ISCs (arrowheads, red) and EBs (asterisks), but not Pros-positive ee cells (white). Par-6::GFP (G, green) is polarized in Delta-positive ISCs (red) along the apicobasal axis at interphase. (H–J) Localization of Par-6::GFP in mitotic ISCs. In metaphase (H), apical asymmetric localization of Par-6::GFP is maintained, and in telophase (I), it is segregated into the apical daughter cell. Par-6::GFP is mislocalized/symmetric (J) in a small fraction of ISC divisions. (K–M′) Localization of other members of the Par complex. Endogenous Baz (K, green) is apical in interphase ISCs (red) and is apically asymmetric in telophase (L). Endogenous aPKC (M) is asymmetric in telophase ISCs. All images were taken as confocal Z-stacks and were processed as maximum projections. Scale bars = 10 μm (E and F) and 5 μm (G–M). See also Figure S1.
Figure 2
Figure 2
The Par Complex Is Required for Differentiation of EBs and the Kinase Activity of aPKC Is Sufficient to Induce Differentiation in ISCs (A–H) RNAi knockdown of Par-6 (B and F), Baz (C and G), and aPKC (D and H [KK line, VDRC]) results in the ectopic clusters of undifferentiated ISCs and EBs (green) to varying extents in comparison to wild-type (A and E). Arrowheads (B) show mitotic ISCs (red). Lineage staining using Delta (red) to label ISCs and Pros (white) to label ee cells shows an increase in Delta upon knockdown of Par complex members (F–H) in comparison to wild-type (E). ee lineages (E–H, white) are largely unaffected but occasionally also form clusters (G, asterisk). (I–L) RNAi knockdown in the ISCs, specifically by Delta Gal4, of Par6 (J), Baz (K), or aPKC (L) leads to increased Delta-positive cells (red) in comparison to wild-type (I). (M and N) Quantification of Par complex RNAi knockdown using esg Gal4 (M, n = 4 guts/genotype) and Delta Gal4 (N, n = 12 guts/genotype). (O–S) Clonal analysis using the Act-FLP out-Gal4 system comparing aPKC-CAAX overexpression to that of wild-type clones. Histogram representing the percentage of clones with respect to clonal size, 4–5 days after clonal induction (O) is shown. Nuclear-LacZ (Nls-LacZ, green) positively labels mitotic clones in wild-type (P), and aPKC-CAAX overexpression (Q) with Pros (red) marks ee cells. Schematic diagram of ISC lineage identity in wild-type and aPKC-CAAX backgrounds (R and S) is shown. Statistical significance was determined by Student’s t test (∗∗∗p < 0.001). All images were taken as confocal Z-stacks and were processed as maximum projections. Scale bar = 10 μm (A–L) and 5 μm (P and Q). See also Figure S2.
Figure 3
Figure 3
aPKC Kinase Activity Acts on Notch Signaling and Notch Is Epistatic to the Par Complex (A–C) Notch activity within the intestinal epithelium increases upon the overexpression of aPKC-CAAX. The subset of Su(H)GBE-LacZ-positive cells (red) within esg-positive cells (green) increases upon the aPKC-CAAX expression (B) in comparison to wild-type (A). Histogram of the percentage of Su(H)GBE-LacZ-positive cells within esg-positive cells (C) is shown. (D–G″) Notch is epistatic to aPKC. In comparison to the internal control (D), aPKC IR (E, 1B1 aPKC IR) results in an increase in esg-positive (green) and Delta-positive (red) cells, whereas overexpression of Nintra leads to the depletion of esg-positive and Delta-positive cells (F). Overexpression of Nintra rescues the aPKC IR phenotype, reducing the number of esg-positive cells (G). These cells are not Delta-positive (G), indicating that they are EBs. Error bars, standard deviation; n = 12 guts in wild-type, n = 11 guts in aPKC-CAAX overexpression, n = 10 guts in aPKC-CAAXKD. Statistical significance was determined by Student’s t test (∗p < 0.05, ∗∗∗p < 0.001). All images were taken as confocal Z-stacks and were processed as maximum projections. Scale bar = 10 μm. See also Figure S3.
Figure 4
Figure 4
Integrins Regulate ISC Proliferation and Self-Renewal (A and A′) Integrin localization in intestinal epithelium. βPS-Integrin (red) is in contact with the ISCs (A, green). (B and C) ISC divisions increase in Integrin knockdown. Single ISCs (green) dividing (pH3, red) are compared with multiple dividing ISCs in mew knoc′kdown (C). (D–G) Integrin knockdown in the ISCs and EBs leads to ectopic esg-positive cell clusters. Clusters of esg-positive (green) and Delta-positive (red) cells form in mew (E), mys (F), and if (G) RNAi knockdowns in comparison to wild-type (D). (H–K) Integrin RNAi phenotype with Delta Gal4 driver. Confocal Z projections of Delta Gal4 driver line alone (H) and Delta Gal4 driving mew (I), mys (J), and if knockdown (K) in ISCs marked by Delta (red) is shown. (L–N) Quantification of mitotic cells upon Integrin knockdown with the esg Gal4 driver line (L, n = 15 guts/genotype) and the quantification of the increase in Delta-positive cells in Integrin knockdown with the esg Gal4 (M, n = 15 guts/genotype) and Delta Gal4 (N, n = 10 guts/genotype) drivers. Error bars, standard deviation. Statistical significance was determined by Student’s t test (∗∗∗p < 0.001). Scale bar = 10 μm. See also Figure S4.
Figure 5
Figure 5
Integrins Regulate Spindle Orientation and Localization of Asymmetric Proteins (A and B) 3D reconstruction of confocal projections of mitotic esg-positive ISCs (green) labeled by pH3 (red) in wild-type (A) and mew (B) knockdown cells. Basement membrane is labeled by Laminin (red). (C–E) Quantification of angles of spindle orientation away from the basement membrane upon knockdown of mew (n = 14), mys (n = 6), if (n = 12), and apc (n = 7) (C) cells. Overexpression of TorD/βcyt (n = 14) and FAK56wt (n = 9) dominant-negative adhesion constructs in shown in (D) and knockdowns of Par complex members Par-6 (n = 21), Baz (n = 7), and aPKC (n = 8) is shown in (E). Statistical significance was determined by Wilcoxon rank sum test. Scale bars = 5 μm. See also Figure S5.
Figure 6
Figure 6
Integrins Regulate the Localization and Are Genetically Upstream of the Par Complex (A–G) Asymmetric localization of the Par complex is affected upon Integrin knockdown. 3D reconstruction of mitotic (red) Baz::GFP (green) ISCs in wild-type (A) and Integrin knockdown (B) is shown. Statistical quantification of the percentage of asymmetric/symmetric Baz::GFP between wild-type (n = 24 cells) and Integrin knockdown (C, mew IR, n = 24 cells, if IR, n = 33 cells) is shown. Endogenous Baz localization (red) is also perturbed upon knockdown of if, leading to apical symmetric (E) or general mislocalization (F) (if IR, n = 20 cells metaphase/two-cell stage) in comparison to the wild-type (D, n = 15 cells metaphase/two-cell stage) in mitotic (red) ISCs (green). Histogram (G) of the localization of endogenous Baz between wild-type and if RNAi is shown. (H–K″) The Par complex is epistatic to integrins. RNAi knockdown of if results in supernumerary Delta-positive ISCs (I) whereas aPKC-CAAX overexpression leads to the loss of ISCs (J) in comparison to the internal control (H). The phenotypic severity observed in if RNAi is rescued upon the overexpression of aPKC-CAAX (K). Images (D–F and H–K) were taken as confocal Z-stacks and were processed as maximum projections. Scale bars = 5 μm (A, B, and D–F) and 10 μm (H–K).
Figure 7
Figure 7
The Par Complex and Integrins Direct Asymmetric Cell Division in ISCs (A and B) Adult Drosophila ISCs divide asymmetrically through Integrin-dependent asymmetric localization the Par complex during mitosis. Different fates might arise either because the two daughter cells reside in different microenvironments and receive different signals (A) or because levels of aPKC activity are higher in one of the two daughter cells (B).
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