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Aneuploidy causes premature differentiation of neural and intestinal stem cells.

Gogendeau D, Siudeja K, Gambarotto D, Pennetier C, Bardin AJ, Basto R - Nat Commun (2015)

Bottom Line: We show that aneuploidy causes brain size reduction due to a decrease in the number of proliferative neural stem cells (NSCs), but not through apoptosis.Moreover, we show that this response to aneuploidy is also present in adult intestinal stem cells but not in the wing disc.Our work highlights a neural and intestine stem cell-specific response to aneuploidy, which prevents their proliferation and expansion.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, PSL Research University, CNRS UMR144, 12 rue Lhomond, Paris 75005, France.

ABSTRACT
Aneuploidy is associated with a variety of diseases such as cancer and microcephaly. Although many studies have addressed the consequences of a non-euploid genome in cells, little is known about their overall consequences in tissue and organism development. Here we use two different mutant conditions to address the consequences of aneuploidy during tissue development and homeostasis in Drosophila. We show that aneuploidy causes brain size reduction due to a decrease in the number of proliferative neural stem cells (NSCs), but not through apoptosis. Instead, aneuploid NSCs present an extended G1 phase, which leads to cell cycle exit and premature differentiation. Moreover, we show that this response to aneuploidy is also present in adult intestinal stem cells but not in the wing disc. Our work highlights a neural and intestine stem cell-specific response to aneuploidy, which prevents their proliferation and expansion.

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Intestine stem cells undergo premature differentiation upon bub3 depletion.(a) Intestinal stem cell (ISC)s division scheme: ISC expresses Escargot (Esg) and Delta (Dl), divides asymmetrically to self-renew and to generate an enteroblast (Esg+) that generate enterocytes (ECs) (Pdm1+) that undergo polyploidization and enteroendocrine cells (EEs), diploid (Pros+). (b) Fluorescent in situ hybridization in Esg+ (green) clones. Wild-type ECs appear homogeneous in size and the FISH probes appear clustered in a large dot. bub3RNAi inset shows a nucleus with abnormal morphology, never seen in WT and with several unclustered signals that allow the distinction between ECs (>2 n, that would still be Esg+) and non-euploid Esg+ cells. Scale bar, 10 μm. (c) Fluorescent in situ hybridization quantification (n=99 and 246 WT and bub3RNAi Esg+ cells, respectively). Statistical significance determined using Fisher's exact test (Fet). *(P=0.012). (d) WT or bub3RNAi GFP+ MARCM clones 12 days after heat shock (AHS) (in yellow), Dl (cytoplasmic red) marks SCs and Pros (nuclear red) marks EEs. Scale bar, 20 μm. (e) Quantification of number of cells/clone in WT and bub3RNAi (n=31 and 45 clones, respectively). The line represents the mean and the error bars the s.d. Statistical significance determined using an unpaired t-test. ***(P<0.0001). (f) Quantification of Delta+ cells in WT and bub3RNAi (n=31 and 45 clones, respectively). The line represents the mean and the error bars the s.d. Statistical significance determined using unpaired t-test. ***(P<0.0001). (g) Percentage of WT and bub3RNAi (n=31 and 45 clones, respectively) without Dl+ cells. Statistical significance determined using Fet ***(P<0.0001). (h) Percentage of single cell clones in WT and bub3RNAi (n=32 and 55 clones, respectively). Statistical significance determined using Fet. **(P=0.029). (i) WT and bub3RNAi midguts expressing Esg GFP (green). DNA is in blue. Scale bars, 20 μm. (j) WT or bub3RNAi midguts basal side with Dl (labelling ISCs, in white) and GFP. Wild-type Esg+ cells remain basally located and some express vesicular Dl, ISC marker (yellow arrows). Scale bars, 20 μm. (k) Apical side of the same WT or bub3RNAi midguts than in (j) with GFP and Pdm1. Esg+, Pdm1+ cells are only observed in bub3RNAi midguts (magenta arrows). Scale bars, 20 μm. (l) Percentage of Esg+ cells with nuclear size >7 μm in WT (n=40) and bub3RNAi (n=54) midguts. Statistical significance determined using Fet ***(P<0.0001).
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f6: Intestine stem cells undergo premature differentiation upon bub3 depletion.(a) Intestinal stem cell (ISC)s division scheme: ISC expresses Escargot (Esg) and Delta (Dl), divides asymmetrically to self-renew and to generate an enteroblast (Esg+) that generate enterocytes (ECs) (Pdm1+) that undergo polyploidization and enteroendocrine cells (EEs), diploid (Pros+). (b) Fluorescent in situ hybridization in Esg+ (green) clones. Wild-type ECs appear homogeneous in size and the FISH probes appear clustered in a large dot. bub3RNAi inset shows a nucleus with abnormal morphology, never seen in WT and with several unclustered signals that allow the distinction between ECs (>2 n, that would still be Esg+) and non-euploid Esg+ cells. Scale bar, 10 μm. (c) Fluorescent in situ hybridization quantification (n=99 and 246 WT and bub3RNAi Esg+ cells, respectively). Statistical significance determined using Fisher's exact test (Fet). *(P=0.012). (d) WT or bub3RNAi GFP+ MARCM clones 12 days after heat shock (AHS) (in yellow), Dl (cytoplasmic red) marks SCs and Pros (nuclear red) marks EEs. Scale bar, 20 μm. (e) Quantification of number of cells/clone in WT and bub3RNAi (n=31 and 45 clones, respectively). The line represents the mean and the error bars the s.d. Statistical significance determined using an unpaired t-test. ***(P<0.0001). (f) Quantification of Delta+ cells in WT and bub3RNAi (n=31 and 45 clones, respectively). The line represents the mean and the error bars the s.d. Statistical significance determined using unpaired t-test. ***(P<0.0001). (g) Percentage of WT and bub3RNAi (n=31 and 45 clones, respectively) without Dl+ cells. Statistical significance determined using Fet ***(P<0.0001). (h) Percentage of single cell clones in WT and bub3RNAi (n=32 and 55 clones, respectively). Statistical significance determined using Fet. **(P=0.029). (i) WT and bub3RNAi midguts expressing Esg GFP (green). DNA is in blue. Scale bars, 20 μm. (j) WT or bub3RNAi midguts basal side with Dl (labelling ISCs, in white) and GFP. Wild-type Esg+ cells remain basally located and some express vesicular Dl, ISC marker (yellow arrows). Scale bars, 20 μm. (k) Apical side of the same WT or bub3RNAi midguts than in (j) with GFP and Pdm1. Esg+, Pdm1+ cells are only observed in bub3RNAi midguts (magenta arrows). Scale bars, 20 μm. (l) Percentage of Esg+ cells with nuclear size >7 μm in WT (n=40) and bub3RNAi (n=54) midguts. Statistical significance determined using Fet ***(P<0.0001).

Mentions: We then analyse adult ISCs using the Mosaic analysis with a repressible cell marker (MARCM) technique to induce expression of bub3RNAi. ISCs divide asymmetrically to self-renew and to generate one enteroblast (EB). EBs differentiate either into enteroendocrine cells (EEs) or enterocytes (ECs) that undergo polyploidization (Fig. 6a). First, to ascertain that bub3 RNAi triggers aneuploidy in ISCs, we used the temporal and regional gene expression targeting (TARGET) method (EsgGal4>GFP, tubGal80ts) to express bub3RNAi in adult ISCs and progenitor cells by shifting the temperature during 12 consecutive days to degrade the Gal4 inhibitor, tubGAL80. FISH revealed that 14.2% of bub3RNAi Esg+ cells were aneuploid with only 4.3% in WT (Fig. 6b,c).


Aneuploidy causes premature differentiation of neural and intestinal stem cells.

Gogendeau D, Siudeja K, Gambarotto D, Pennetier C, Bardin AJ, Basto R - Nat Commun (2015)

Intestine stem cells undergo premature differentiation upon bub3 depletion.(a) Intestinal stem cell (ISC)s division scheme: ISC expresses Escargot (Esg) and Delta (Dl), divides asymmetrically to self-renew and to generate an enteroblast (Esg+) that generate enterocytes (ECs) (Pdm1+) that undergo polyploidization and enteroendocrine cells (EEs), diploid (Pros+). (b) Fluorescent in situ hybridization in Esg+ (green) clones. Wild-type ECs appear homogeneous in size and the FISH probes appear clustered in a large dot. bub3RNAi inset shows a nucleus with abnormal morphology, never seen in WT and with several unclustered signals that allow the distinction between ECs (>2 n, that would still be Esg+) and non-euploid Esg+ cells. Scale bar, 10 μm. (c) Fluorescent in situ hybridization quantification (n=99 and 246 WT and bub3RNAi Esg+ cells, respectively). Statistical significance determined using Fisher's exact test (Fet). *(P=0.012). (d) WT or bub3RNAi GFP+ MARCM clones 12 days after heat shock (AHS) (in yellow), Dl (cytoplasmic red) marks SCs and Pros (nuclear red) marks EEs. Scale bar, 20 μm. (e) Quantification of number of cells/clone in WT and bub3RNAi (n=31 and 45 clones, respectively). The line represents the mean and the error bars the s.d. Statistical significance determined using an unpaired t-test. ***(P<0.0001). (f) Quantification of Delta+ cells in WT and bub3RNAi (n=31 and 45 clones, respectively). The line represents the mean and the error bars the s.d. Statistical significance determined using unpaired t-test. ***(P<0.0001). (g) Percentage of WT and bub3RNAi (n=31 and 45 clones, respectively) without Dl+ cells. Statistical significance determined using Fet ***(P<0.0001). (h) Percentage of single cell clones in WT and bub3RNAi (n=32 and 55 clones, respectively). Statistical significance determined using Fet. **(P=0.029). (i) WT and bub3RNAi midguts expressing Esg GFP (green). DNA is in blue. Scale bars, 20 μm. (j) WT or bub3RNAi midguts basal side with Dl (labelling ISCs, in white) and GFP. Wild-type Esg+ cells remain basally located and some express vesicular Dl, ISC marker (yellow arrows). Scale bars, 20 μm. (k) Apical side of the same WT or bub3RNAi midguts than in (j) with GFP and Pdm1. Esg+, Pdm1+ cells are only observed in bub3RNAi midguts (magenta arrows). Scale bars, 20 μm. (l) Percentage of Esg+ cells with nuclear size >7 μm in WT (n=40) and bub3RNAi (n=54) midguts. Statistical significance determined using Fet ***(P<0.0001).
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f6: Intestine stem cells undergo premature differentiation upon bub3 depletion.(a) Intestinal stem cell (ISC)s division scheme: ISC expresses Escargot (Esg) and Delta (Dl), divides asymmetrically to self-renew and to generate an enteroblast (Esg+) that generate enterocytes (ECs) (Pdm1+) that undergo polyploidization and enteroendocrine cells (EEs), diploid (Pros+). (b) Fluorescent in situ hybridization in Esg+ (green) clones. Wild-type ECs appear homogeneous in size and the FISH probes appear clustered in a large dot. bub3RNAi inset shows a nucleus with abnormal morphology, never seen in WT and with several unclustered signals that allow the distinction between ECs (>2 n, that would still be Esg+) and non-euploid Esg+ cells. Scale bar, 10 μm. (c) Fluorescent in situ hybridization quantification (n=99 and 246 WT and bub3RNAi Esg+ cells, respectively). Statistical significance determined using Fisher's exact test (Fet). *(P=0.012). (d) WT or bub3RNAi GFP+ MARCM clones 12 days after heat shock (AHS) (in yellow), Dl (cytoplasmic red) marks SCs and Pros (nuclear red) marks EEs. Scale bar, 20 μm. (e) Quantification of number of cells/clone in WT and bub3RNAi (n=31 and 45 clones, respectively). The line represents the mean and the error bars the s.d. Statistical significance determined using an unpaired t-test. ***(P<0.0001). (f) Quantification of Delta+ cells in WT and bub3RNAi (n=31 and 45 clones, respectively). The line represents the mean and the error bars the s.d. Statistical significance determined using unpaired t-test. ***(P<0.0001). (g) Percentage of WT and bub3RNAi (n=31 and 45 clones, respectively) without Dl+ cells. Statistical significance determined using Fet ***(P<0.0001). (h) Percentage of single cell clones in WT and bub3RNAi (n=32 and 55 clones, respectively). Statistical significance determined using Fet. **(P=0.029). (i) WT and bub3RNAi midguts expressing Esg GFP (green). DNA is in blue. Scale bars, 20 μm. (j) WT or bub3RNAi midguts basal side with Dl (labelling ISCs, in white) and GFP. Wild-type Esg+ cells remain basally located and some express vesicular Dl, ISC marker (yellow arrows). Scale bars, 20 μm. (k) Apical side of the same WT or bub3RNAi midguts than in (j) with GFP and Pdm1. Esg+, Pdm1+ cells are only observed in bub3RNAi midguts (magenta arrows). Scale bars, 20 μm. (l) Percentage of Esg+ cells with nuclear size >7 μm in WT (n=40) and bub3RNAi (n=54) midguts. Statistical significance determined using Fet ***(P<0.0001).
Mentions: We then analyse adult ISCs using the Mosaic analysis with a repressible cell marker (MARCM) technique to induce expression of bub3RNAi. ISCs divide asymmetrically to self-renew and to generate one enteroblast (EB). EBs differentiate either into enteroendocrine cells (EEs) or enterocytes (ECs) that undergo polyploidization (Fig. 6a). First, to ascertain that bub3 RNAi triggers aneuploidy in ISCs, we used the temporal and regional gene expression targeting (TARGET) method (EsgGal4>GFP, tubGal80ts) to express bub3RNAi in adult ISCs and progenitor cells by shifting the temperature during 12 consecutive days to degrade the Gal4 inhibitor, tubGAL80. FISH revealed that 14.2% of bub3RNAi Esg+ cells were aneuploid with only 4.3% in WT (Fig. 6b,c).

Bottom Line: We show that aneuploidy causes brain size reduction due to a decrease in the number of proliferative neural stem cells (NSCs), but not through apoptosis.Moreover, we show that this response to aneuploidy is also present in adult intestinal stem cells but not in the wing disc.Our work highlights a neural and intestine stem cell-specific response to aneuploidy, which prevents their proliferation and expansion.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, PSL Research University, CNRS UMR144, 12 rue Lhomond, Paris 75005, France.

ABSTRACT
Aneuploidy is associated with a variety of diseases such as cancer and microcephaly. Although many studies have addressed the consequences of a non-euploid genome in cells, little is known about their overall consequences in tissue and organism development. Here we use two different mutant conditions to address the consequences of aneuploidy during tissue development and homeostasis in Drosophila. We show that aneuploidy causes brain size reduction due to a decrease in the number of proliferative neural stem cells (NSCs), but not through apoptosis. Instead, aneuploid NSCs present an extended G1 phase, which leads to cell cycle exit and premature differentiation. Moreover, we show that this response to aneuploidy is also present in adult intestinal stem cells but not in the wing disc. Our work highlights a neural and intestine stem cell-specific response to aneuploidy, which prevents their proliferation and expansion.

Show MeSH
Related in: MedlinePlus