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. 2018 Apr;75(7):1285-1301.
doi: 10.1007/s00018-017-2697-4. Epub 2017 Nov 2.

SUMOylation Controls Stem Cell Proliferation and Regional Cell Death Through Hedgehog Signaling in Planarians

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Free PMC article

SUMOylation Controls Stem Cell Proliferation and Regional Cell Death Through Hedgehog Signaling in Planarians

Manish Thiruvalluvan et al. Cell Mol Life Sci. .
Free PMC article

Abstract

Mechanisms underlying anteroposterior body axis differences during adult tissue maintenance and regeneration are poorly understood. Here, we identify that post-translational modifications through the SUMO (Small Ubiquitin-like Modifier) machinery are evolutionarily conserved in the Lophotrocozoan Schmidtea mediterranea. Disruption of SUMOylation in adult animals by RNA-interference of the only SUMO E2 conjugating enzyme Ubc9 leads to a systemic increase in DNA damage and a remarkable regional defect characterized by increased cell death and loss of the posterior half of the body. We identified that Ubc9 is mainly expressed in planarian stem cells (neoblasts) but it is also transcribed in differentiated cells including neurons. Regeneration in Ubc9(RNAi) animals is impaired and associated with low neoblast proliferation. We present evidence indicating that Ubc9-induced regional cell death is preceded by alterations in transcription and spatial expression of repressors and activators of the Hedgehog signaling pathway. Our results demonstrate that SUMOylation acts as a regional-specific cue to regulate cell fate during tissue renewal and regeneration.

Keywords: Genomic instability; Patched; Rad51; Regeneration; Ubc9.

Figures

Fig 1.
Fig 1.. The SUMOylation pathway is conserved in the planarian Schmidtea mediterranea
(A) A general outline of the SUMOylation pathway with the names of enzymes involved and a brief description of major steps in the process. (B) A list of SUMO pathway components found in humans and their homologs in yeast and S. mediterranea. (C) Protein alignment of planarian UBC9 and homologs found in humans (NP_003336.1), mice (EDL06246.1), Drosophila (NP_476978.1), C. elegans (NP_001023158.1) and Xenopus (NP_001080758.1). Human and planarian UBC9 protein sequences are 73% identical.
Fig 2.
Fig 2.. Ubc9 is required for regional tissue maintenance
(A) RNAi schedule based on feeding with bacterially expressed dsRNA to knockdown Ubc9 expression. All controls were fed either Unc22 or gfp. Black bars represent feeding days and red represents fixation. (B) Representative images of control and Ubc9(RNAi) animals 25 dpf. The arrows indicate different abnormalities including dark tail, lesions, and tail loss. n=75 total animals used in three biological replicates. Scale bar = 200 μm. Note the scale bar on the right applies for the two pictures on the right portion of the figure. (C) Histogram illustrates the progression of the Ubc9 phenotype based on macroscopic abnormalities observed in “B”. (D) Change in surface area over the 25-day RNAi time course in controls and Ubc9(RNAi) animals. The experiment involves 15 animals per time point and three biological replicates. (E) Levels of Ubc9 expression measured with qPCR. The results show that the RNAi knockdown protocol is effective at reducing transcript levels of Ubc9 in both anterior and posterior regions. Data obtained from triplicates per experiment of at least two biological replicates. **** p<0.0001; two-way ANOVA.
Fig 3.
Fig 3.. Ubc9 is necessary for stem cell function and proper cell cycle transition
(A) Whole mount in situ hybridization using antisense probe against Ubc9. Gene expression is found ubiquitously distributed along the AP axis (left) and is dramatically reduced 24 hours after lethal exposure to gamma irradiation (6K rads) and remained at that level for 7 days post-irradiation (red arrows). Experiments involved three biological replicates with 10 animals per experiment. (B) Ubc9 gene expression levels in different cell populations (X1: proliferative cells, X2: early post-mitotic progeny, and Xins: late post-mitotic progeny). (C) t-SNE plot of single cells displaying clusters of neoblasts and differentiated cells (left), along with the overlaid Ubc9 expression (right). The respective reference for the level of expression based on the colored gradient scale blue to red (low-high, respectively). The gene expression result for “B and C” were obtained from the planaria single-cell database hosted by the Reddien Lab at the Whitehead Institute for Biomedical Research (https://radiant.wi.mit.edu/app/)[1]. (D) Time course of mitotic activity along the AP axis, expressed as fold change in reference to the control at each time point. (E) Spatial distribution of mitotic activity in whole mount immunostaining against phospho-histone H3 (Ser10) (H3P) at 25 dpf Ubc9(RNAi). Dashed line divides anterior and posterior regions of the animal. All scale bars = 200 μm. (F) Fold change mitotic counts obtained independently from the anterior or posterior regions. Mitotic levels involved three biological replicates and more than 40 animals. (G) FACS analysis using DRAQ5, a nuclear dye and Calcein, a live cell marker, in either anterior or posterior regions of control and Ubc9(RNAi) animals 25 dpf. Blue, green and red squares represent X1, X2 and Xins populations, respectively. (H) Cell cycle analysis using DRAQ5 DNA dye in AP regions of control and Ubc9(RNAi) animals 25 dpf. Red bars represent percentage of cells at different phases of cell cycle. All FACS analysis performed with more than 10,000 cells and results are representative of two experiments with about 40 animals total. (I) Gene expression levels of various cell cycle markers necessary for proper G1/S and G2/M transition in AP regions of control and Ubc9(RNAi) animals 25 dpf. Gene expression portrayed as fold change normalized to control. ** p<.01; *** p<.001; **** P<0.0001; two-way-ANOVA.
Fig 4.
Fig 4.. Ubc9-loss of function leads to regional cell death
(A) Whole mount immunostaining against caspase-3, a marker for cell death, in control and Ubc9(RNAi) animals. About 65% of experimental animals showed similar caspase-3 signal distribution at 25 dpf. Immunostainings involved three biological replicates and more than 40 animals. Scale bars = 200 μm. (B) Intensity of capase-3 signal from anterior to posterior (white line) of control and Ubc9(RNAi) animals. Intensity signal quantification involved three biological replicates and more than 20 animals. (C) FACS analysis staining against Annexin V, a marker for apoptosis, and 7AAD, a cell viability marker, in AP regions of control and Ubc9(RNAi) animals 25 dpf. Annexin V-/7 AAD- quadrant includes viable cells (outlined green). Annexin V+/7 AAD– and Annexin V+/7 AAD+ indicate cells that are in early and late (necrotic) stages of cell death, respectively (outlined red). Blue numbers in each quadrant indicate the percentage of cells with that staining profile. Data is representative of two experiments with n>40 each. (D) Fold change of BCL2 gene expression in anterior and posterior regions normalized to control group.
Fig 5.
Fig 5.. Ubc9 is required to maintain genomic integrity along the anteroposterior axis
(A) COMET assay, single gel electrophoresis under alkaline conditions, was performed in AP regions of control and Ubc9(RNAi) animals at10, 15, and 25 dpf. Visual scoring was used to quantify DNA damage. Color-coded key at bottom represents undamaged (green), moderate damage (orange) and extremely damaged DNA (red) shown in either anterior or posterior regions. DNA damage scale reference is similar to the one used in Peiris et al. 2016b. Downregulation of Ubc9 leads to systemic accumulation of DNA damage as early as day 15 when compared to controls. Approximately 40 cells were counted for each DNA condition in one biological replicate. (B) Chromosome quality (not damage and damaged) normalized to control in Ubc9(RNAi) 25 dpf animals. Experiment was repeated twice. (C) Whole mount immunostaining against gamma H2AX antibody in control and Ubc9(RNAi) animals 25 dpf. Total number of animals was 20 and n=2 biological replicates. (D) Whole mount immunostaining against human RAD51 antibody in control and Ubc9(RNAi) animals 25 dpf. Total number of animals was 15 and experiment was repeated twice. Scale bars = 200 μm and images are representative of approximately 70% of the animals in each condition. (E-F) Western-blot and subsequent quantification for RAD51 in control and Ubc9(RNAi) animals. Alpha tubulin was used as an internal control. Protein was extracted from n>30 animals. (G) Spatial distribution of RAD51 immunostaining (green) in reference to the cell nucleus (stained with DAPI, blue) in control and Ubc9(RNAi) 18 dpf and 5 days after sub-lethal irradiation (1.25k rads). RAD51 subcellular localization to the nucleus is at a maximum at this point in time. Scale bar = 10 μm. (H) Quantification of cells with RAD51+ foci in the nucleus and cytoplasm in control and Ubc9(RNAi) 18 dpf and 5 days after sub-lethal irradiation (1.25k rads). Approximately 50 cells were counted for each condition in two biological replicates and all RAD51 stainings were performed with human RAD51 antibody.
Fig 6.
Fig 6.. Regional defects after Ubc9(RNAi) are mediated through repression of Hedgehog pathway
(A) Whole mount in situ hybridization expression of sfrp-1 (anterior polarity marker) and fz4 (posterior polarity marker) in control and 10, 15 and 25 dpf Ubc9(RNAi) animals. Red arrows indicate abnormal gene expression. Scale bars = 200 μm. Note that controls for each time point were executed but no apparent change in gene expression was observed over time –not shown. (B) Gene expression levels of Patched, Hedgehog and Smoothened after Ubc9(RNAi) animals at 10, 15 and 25 dpf. Gene expression is given in fold change normalized to control. (C) Whole mount in situ hybridization expression of Patched and Hedgehog in control and 10, 15 and 25 dpf Ubc9(RNAi) animals. Scale bars = 200 μm. (D). RNAi schedule based on feeding with bacterially expressed dsRNA to perform double knockdown of Hedgehog or Patched and Ubc9. All controls were fed either Unc22 or gfp. Black bars represent feeding days and red represents fixation. (E) Histogram depicts occurrence of tail abnormalities in Hedgehog + Ubc9 and Patched + Ubc9 animals. Data is representative of n=30 animals consisting of three biological replicates.
Fig 7.
Fig 7.. Ubc9 is required for proper regeneration.
(A) Schematic representation of regeneration experiments on control and Ubc9(RNAi) animals 18 dpf. (B) Live pictures of control and Ubc9(RNAi) 18 dpf animals 7 days post amputation (7dpa). (C) Blastema size in control and Ubc9(RNAi) animals expressed as fold change relative to control. In all experiments used more than 30 animals in three biological replicates. (D) Trunk fragments generated from control and Ubc9(RNAi) 18dpf animals were fixed at 0, 6, 10, 20, 30 and 48 hours post amputation. Mitotic cell numbers were determined via H3P staining and expressed as H3P+ foci divided by surface area in mm2. Three biological replicates with more than 15 animals per time point were used. (E) Whole mount immunostaining against SYNORF-1 (synapsin), a marker for the nervous system, in control and Ubc9(RNAi) 18 dpf 7DPA. Green signal denotes the planarian central nervous system and insets provide detailed amplification of anterior and posterior ends including brain and tip of the tail, respectively. White dotted lines underscore differences between control and experimental group regarding brain morphology (top) and ventral nerve cords (bottom). More than 20 animals were stained in three biological replicates. (F) Regenerated brain size obtained from determination of surface area in both control and experimental group. Differences in brain size were calculated with Two way-ANOVA. (G) RNAi schedule based on feeding with bacterially expressed dsRNA to perform double knockdown of Hedgehog or Patched and Ubc9. All controls were fed either Unc22 or gfp. Black bars represent feeding days and red represents amputation. (H) Histogram depicts occurrence of regenerative defects in Patched + Ubc9 and Hedgehog + Ubc9 animals 7 days after amputation. Representative images of defects is shown on the right side. (I) Schematic summary representing Smed-Ubc9 acts as an upstream regulator of different functions in the adult body. At the organismal level, downregulation of Ubc9 lead to a systemic increase in DNA damage and decrease in in both stem cell renewal and cell cycle progression. However, it remains unclear whether inhibition of Hedgehog (Hh) signaling is associated with any of the systemic effects. Regionally, dysfunctional Ubc9 triggers collective cell death and posterior specific abrogation of tissue regeneration, which are mediated through Hh signaling. At the organ level, Ubc9 also regulates proper brain regeneration likely through disruption of Hh signaling that is required for neurogenesis. It is possible that lack of specific neurons in Ubc9(RNAi) animals also affect neural regulation of cell cycle progression but additional experiments are required. **** P<0.0001 and all other statistical comparisons were made with Sidak’s multiple comparisons test NS (no significance) p>.05; ** p<.01; *** p<.001; **** P<0.0001. Scale bars = 200 μm.

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