In Vivo Analysis of Centromeric Proteins Reveals a Stem Cell-Specific Asymmetry and an Essential Role in Differentiated, Non-proliferating Cells  Ana García del Arco, Bruce A. Edgar, Sylvia Erhardt  Cell Reports  Volume 22, Issue 8, Pages 1982-1993 (February 2018) DOI: 10.1016/j.celrep.2018.01.079 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 22, 1982-1993DOI: (10.1016/j.celrep.2018.01.079) Copyright © 2018 The Authors Terms and Conditions

Figure 1 Endogenous Centromeric Proteins Are Differentially Distributed within Different Cell Types of the Midgut (A) Reads per kilobase million (RPKM) data of CENP-A, CAL1, and CENP-C expression in different cell types of the Drosophila midgut (source: http://flygutseq.buchonlab.com/). (B) Drosophila midgut expressing CENP-A-GFP under the control of CENP-A endogenous promoter. Centromeric enrichment of CENP-A-GFP in the so-called “small cells” Scale bar, 25 μm. (C) Midgut of w1118 flies stained for CENP-A (green), CENP-C (red), and Delta (white). ISCs are marked by yellow circles and shown in the zoomed images below. Scale bars, 5 μm and 2.5 μm. (D) Midgut of Su(H)-lacZ transgenic flies stained for CENP-C (white, left) and CAL1 (white, right). (E) Midgut of w1118 flies stained for CENP-A (green), CAL1 (red), and the EE marker Prospero (white). See also Figure S1. Cell Reports 2018 22, 1982-1993DOI: (10.1016/j.celrep.2018.01.079) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Pre-existing CENP-A Is Preferentially Retained by ISCs (A) A dual labeling transgene that allows to differentiate pre-existing and newly synthesized CENP-A protein. The Gal4-activatable construct expresses CENP-A-GFP. Upon heat shock the flies activate a flippase that removes CENP-A-GFP by recombination and starts expressing CENP-A-mKO. UAS, upstream activating sequence; FRT, FLP (flippase) recombination target, CENP-A; mKO, monomeric Kusabura-Orange; ub.GAL4, ubiquitous GAL4 driver; hs-FLP, heat shock-inducible FLP recombinase. (B) Heat shock regimen for the analysis of pre-existing (old) and newly synthesized CENP-A distribution. Adult female flies 7 days after eclosion were heat shocked and guts were analyzed at different time points. (C and D) Distribution of old CENP-A (green) and newly synthesized CENP-A (red) in fixed and stained female fly midguts at different recovery time points (C, 1 day; D, 5 days). No antibody was added to enhance GFP or mKO signal. Samples were stained with β-galactosidase to detect ISCs marked by Delta-lacZ (white signal). The arrows mark the ISCs. (C′ and D′) Zooms of the same guts as in (C) and (D), DAPI and β-galactosidase. (C″–D′″) Zooms of the same guts as in (C) and (D) showing GFP and mKO signal, respectively, arrows point to β-Gal-positive cells. Scale bars, 25 μm. The inlets on the right show the zoom of the cells within the yellow box. (E) Fixed and stained female fly gut showing the distribution of pre-existing (green) and newly synthesized CENP-A (red) at day 1 after heat shock. ISC are marked by Delta (β-Gal). Scale bar, 5 μm. (F) Quantification of GFP and mKO fluorescent intensity ratios at different time points between ISCs and EBs. CENP-A ISC/EB GFP ratio >1, EB/ISC mKO ratio >1, >25 pairs were analyzed; error bars represent SEM. (G) Percentage of ISC-EB pairs that distribute CENP-A-GFP and CENP-A-mKO asymmetrically and symmetrically (see also Figure S2). n = 3, >30 pairs counted per case in each experiment. (H) Distribution of old CENP-A (green) and newly synthesized CENP-A (red) in fixed female fly guts 20 days after heat shock. ISCs are marked by Delta (white, arrows). Scale bar, 25 μm. See also Figures S1–S6. Cell Reports 2018 22, 1982-1993DOI: (10.1016/j.celrep.2018.01.079) Copyright © 2018 The Authors Terms and Conditions

Figure 3 The Key Centromere Proteins CAL1, CENP-A, and CENP-C Are Required for Proliferation of Progenitor Cells and Regenerative Response (A) ISCs division scheme: ISC divides into an asymmetric lineage of a self-renewable stem cell or an EB that generate either enterocytes (ECs) or enteroendocrine cells (EEs). ISCs and EBs (crossed out in red) express escargot (esg) and were targeted by esg RNAi. (B and C) 5-day-old clones generated by the esg F/O system were marked with GFP (green), nuclei were visualized by DAPI (blue) staining. (B) Control adult midgut expressing UAS::GFP alone, (C) knockdown of CAL1. Scale bar, 25 μm. n = 3, >10 midguts were analyzed. (D and E) Esg F/O midguts expressing UAS::GFP alone (control, D) or CAL1RNAi (E) 2 days after P. entomophilae infection. Guts were stained for pH3 (red, arrows). Scale bar, 25 μm. (F) The sizes of the esg F/O clones were quantified by counting cell numbers per clone after 1, 3, 5, and 7 days of clone induction. (G) Kaplan-Meier survival curve of flies of the indicated genotype with (dotted line) and without (straight line) P. entomophilae infection, n = 50 for each genotype under normal conditions, n = 20 for each genotype with P.e infection. (H) ISCs division scheme. ISCs (crossed out in red) express escargot (esg) but lack Su(H) expression. (I and J) Knockdown of CAL1 in ISCs using the esgts, Su(H)-Gal80 system. ISCs were marked by GFP (green). Samples were stained for PH3 (red) and DAPI (blue). (I) Control adult midgut, (J) CAL1-depleted midgut after 5 days induction at 29°C. Decrease in the number of GFP-positive cells were observed in CAL1-depleted midguts. (K) Quantification of PH3-positive cells in adult midguts of the indicated genotype. (L) Quantification of GFP-positive cells in adult midguts of the indicated genotype. Statistical significance was determined by Student’s t test (∗∗∗∗p < 0.0001). n = 3, > 10 midguts were analyzed. Error bars in each graph represent SEM. See also Figure S7. Cell Reports 2018 22, 1982-1993DOI: (10.1016/j.celrep.2018.01.079) Copyright © 2018 The Authors Terms and Conditions

Figure 4 The Loading Factor of CENP-A Is Required in Post-mitotic Cells of the Midgut (A) ISC lineages in the adult midgut. EBs (crossed out in red) are characterized by the expression of Su(H). (A′ and A″) Knockdown of CAL1 specifically in EBs using the Su(H)ts system. EBs were marked by GFP (green). Samples were stained for PH3 (red) and DAPI (blue). (A′) Control midgut, (A″) CAL1-depleted midgut after 3 days induction at 29°C. Scale bar, 25 μm. (B) Quantification of GFP-positive cells in midguts of the indicated genotype. n = 3, >10 midguts were analyzed. (C) ISCs lineages in the midgut. ECs (crossed out in red) are characterized by the expression of Myo1A. (C′–C″) Knockdown of CAL1 specifically in ECs using the Myo1Ats system. ECs were marked by GFP (green). Samples were stained for PH3 (red) and DAPI (blue). (C′) Control midgut, (C″) CAL1-depleted midgut after 3 days induction at 29°C. Decrease in the number of GFP-positive cells were observed in CAL1-depleted midguts. Scale bar, 25 μm. (D) Quantification of PH3-positive cells in midguts of the indicated genotype. Increase in the number of dividing ISCs (PH3-positive cells) were observed in midguts whose ECs were deficient for CAL1. Statistical significance was determined by Student’s t test (∗∗∗∗p < 0.0001). Error bars in each graph represent SEM. (E) Kaplan-Meier survival curve of flies with (dotted line) and without (solid line) P.e. infection, n = 25 for each genotype under normal conditions, n = 20 for each genotype under P.e infection. (F) Immunostaining of CAL1 in control adult midguts (top) and CAL1-depleted midguts (bottom) after 5 days induction at 29°C. (G) DAPI-stained regions mark the measured nuclear area. See also Figures S8–S10. Cell Reports 2018 22, 1982-1993DOI: (10.1016/j.celrep.2018.01.079) Copyright © 2018 The Authors Terms and Conditions

Figure 5 CAL1 Depletion Impairs Endoreduplication in Salivary Glands (A–C′) Knockdown of CAL1 in salivary glands using the ptc-Gal4 driver. (A and A′) Salivary gland cells were stained with DAPI. (A) Control third instar salivary gland. (A′) CAL1-depleted salivary glands. (B and B′) Polytene chromosomes stained with HP1 (green). (B) Control polytene chromosome, (B′) polytene chromosome from CAL1-depleted salivary gland. (C and C′) Salivary glands stained for DNA (blue) and incorporated EdU (red). (C) Control and (C′) CAL1-depleted salivary glands were dissected and labeled with EdU for 5 hr. Scale bars, 25 μm. (D) Quantification of mean nuclear area of the indicated genotype. n = 3, >10 salivary glands pairs were analyzed. (E) Quantification of EdU incorporation. (F) Adult offspring frequencies of control genotypes versus CAL1-depleted salivary glands. Statistical significance was determined by Student’s t test (∗∗∗∗p < 0.0001). Error bars in each graph represent SEM. Cell Reports 2018 22, 1982-1993DOI: (10.1016/j.celrep.2018.01.079) Copyright © 2018 The Authors Terms and Conditions