Chera Et Al. MAPK-Dependent Injury-Induced Apoptosis in Hydra

Chera et al. MAPK-dependent injury-induced apoptosis in Hydra

The MAPK/CREB pathway triggers apoptosis-induced compensatory proliferation in regenerating Hydra

Simona CHERA, Luiza GHILA, Yvan WENGER and Brigitte GALLIOT * Department of Genetics and Evolution, University of Geneva, Sciences III, 30 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland

 Correspoonding author: [email protected]

Running Title: MAPK-dependent injury-induced apoptosis in Hydra

SUPPLEMENTAL FIGURES

Figure S1: The Hydra RSK gene.

Figure S2: The Hydra CBP gene

Figure S3: Characterization of the cellular alterations in head-regenerating halves at 1 hpa.

Figure S4: Western blot analysis showing the RSK, CREB, CBP and actin protein levels in head-regenerating halves of hydra exposed 1x (A) or 3x (B) to dsRNAs.

SUPPLEMENTAL TABLES

Table S1: Modulations of CREB subcellular localisation in interstitial cells during early head regeneration.

Table S2: Inhibition of injury-induced apoptosis in RSK(RNAi), CREB(RNAi) or CBP(RNAi) Hydra (A) or in U0126-treated Hydra (B).

Table S3: Modulations in cell density and BrdU-labeling index in head-regenerating stumps after genetic or pharmacological inhibition of the MAPK/CREB pathway.

Table S4: Cell-type distribution among the BrdU positive cells found in the 3 regions of head- regenerating halves.

Table S5: Kinetics of head-regeneration in Hydra silenced for RSK, CREB or CBP.

1 Chera et al. MAPK-dependent injury-induced apoptosis in Hydra

FIGURE S1

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Figure S1: The Hydra RSK gene. A) Nucleotide and deduced protein RSK sequences from Hydra vulgaris obtained from RNA sequencing (454 and Illumina transcriptome, Yvan Wenger unpublished) B) Scheme depicting the general structure of the RSK proteins. The Hydra RSK cDNAs are represented as black bars. The Hv_RSK-609 cDNA was isolated with the est-RSK-for1 (gattgaaatcatgccacttgc), est-RSK-rev1 (ctacatctaacagaatattctctg) primers designed from the Hm_RSK EST-clone tac30b10.y1 (Genbank, Hydra EST sequencing project, Bode et al.). The N-moiety of the rat RSK2 protein was expressed to raise the anti-RSK antibody (Clontech).. C) Alignment of the Hydra RSK sequence with related sequences from metazoan and non metazoan species: Astpe: Asterina petcinifera (starfish), Brafl: Branchiostoma floridae (amphioxus), Caeel: Caenorhabditis elegans, Chick: Gallus gallus, Danre: Danio rerio (zebrafish), Hydvu: Hydra vulgaris, Monbe: Monosiga brevita (choanoflagellate); Schma: Schistosoma mansoni (Platyhelminthe, trematode), Xenla: Xenopus laevis (African clawed frog). See alignment on next page.

FIGURE S2

KIX domain (CREB interacting domain) CBP Hm QTNPSSTNEDREAWHKSVQEQRLIHLVERLVSAIIPKARNDPMTMKNKQMENLVLYAQKVEDDMYSTAASKEDYFHLLAEKIYKIQKDLEEKRMERLRKN CBP Gg TAAPPSSTGVRKAWHEHVTQDLRNHLVHKLVQAIFP--TPDPAALKDRRMENLVAYARKVEGDMYESANSRDEYYHLLAEKIYKIQKELEEKRRSRLHKQ CBP Hs TAAPPSSTGVRKGWHEHVTQDLRSHLVHKLVQAIFP--TPDPAALKDRRMENLVAYAKKVEGDMYESANSRDEYYHLLAEKIYKIQKELEEKRRSRLHKQ p300 Hs TAAQPSTTGIRKQWHEDITQDLRNHLVHKLVQAIFP--TPDPAALKDRRMENLVAYARKVEGDMYESANNRAEYYHLLAEKIYKIQKELEEKRRTRLQKQ CBP Br MANPAASGSMRKSWHEDITQDLRNHLVHKLVQAIFP--TPDPAALKDRRMENLVAYARKVEGDMYESANSRAEYYHFLAEKIYKIQKELEEKRRTRLQKQ CBP Dm GGNTGPPGDNEKDWRESVTADLRNHLVHKLVQAIFP--TSDPTTMQDKRMHNLVSYAEKVEKDMYEMAKSRSEYYHLLAEKIYKIQKELEEKRLKRKEQH CBP Ag PSMVAVPVQGTKDWHHSVTPDLRNHLVHKLVQAIFP--SPDPSAMFDKRMYNLVAYAKKVEGDMYEMANSRSEYYHLLAEKIYKIQKEL------CBP Ci GVGPMAQPRTVHQWHSQVKEELRNYLVQKLVQNIFP--TPDKISMTDVRMIKLLSFARKVEGSMYEQASSREEYYHLLAVFMHKYQCIIN------CBP Ac DIVKSIPVSSRKDWHAQVTQDLRNHLVHKLVQAIFP--TPDQATLRDSRMKNLVAYARKVEGDMYESANNRGQYYHLLAEKIYKIQKELEEKRIQRMKVQ CBP Ce NLPPPDMPDCTKEWHHQVTKDLRNHLVGKLVKAIFP--EPNQEAMNDNRLKDLIAYARKVEKEMFESANDREEYYHLLAEKIYKIQKELQEKKNSRLNQG

Figure S2: The Hydra CBP gene. A) Scheme depicting the general structure of the CBP proteins. The hydrozoan CBP cDNAs are represented as bars: Pc: Podocoryne carnea, Hm: Hydra magnipapillata; Hv: Hydra vulgaris. The TAZ-KIX-602 Hm cDNA that encodes a full KIX domain was obtained with the CBP-TAZ-for2 (YtWYtWcatgcWcataaRtgY) and CBP-KIX-rev3 (tgggggttaatcatatttgaactg) consensus primers, the CBP-3333 Hm cDNA with the consensus KIX-for3 (ccaggaacacagactaatccatct) and CBP-ZZ-rev1 (ctcactttcaatatctaatccaag) primers, the CBP-620 Hv cDNA with the CBP-est-for1 (ctaccgcattcaacacaagttaca) and CBP-est-rev1 (cataaagtttggactgttgaagag) primers designed from the CBP EST-clone tab48d04.x1 (Hm, Genbank, Hydra EST sequencing project, Bode et al.), the CBP- 453 Hv with the CBP-for1-Basel (cNaaRgcNBtNttYgcNttYga) and CBP-rev1-Basel (ccaRaaRtcNccYtcRaaRtaNgg) primers. The immunogen region used to raise the anti-CBP antibody (Santa Cruz) used in this study is noted. Accession numbers: FR796474, FR796475. B) Alignment of the KIX domain sequences from Hydra and various bilaterian species. Species code: Ac: Aplysia californica; Ag: Anopheles gambiae; Br: Brachydanio rerio (zebrafish); Ce: Caenorhabditis elegans; Ci: Ciona intestinalis; Dm: Drosophila melanogaster (fly); Gg: Gallus gallus (chicken); Hm: Hydra magnipapillata; Hs: Homo sapiens (human).

FIGURE S3

Figure S3: Characterization of the cellular alterations in head-regenerating halves at 1 hpa Cytological modifications detected in cells isolated from regenerating tissues. Cells from intact hydra (a-d), foot-regenerating tips (region4, e-h), head-regenerating tips (region1, i-l) and underlying body column (region2, m-p) were stained with Hoechst (blue), anti-RSK (green) and anti-CREB (red) antibodies. Note in region1 the modified shape of the endodermal epithelial cell (panel j, arrowhead) and the reduced size of the neuron (panel k, arrow). In contrast, the epithelial cells from the ectodermal layer (ecto. epith.) did not exhibit significant morphological modifications. Bar scale: 8 µm.

FIGURE S4

Figure S4: Western blot analysis showing the RSK, CREB, CBP and actin protein levels in head-regenerating halves of Hydra exposed 1x (A) or 3x (B) to dsRNAs.

For explanations concerning the epistatic relationships between RSK, CREB and CBP, please refer to Figure 3C and to the result section of the paper (Silencing of the RSK, CREB and CBP genes).TABLE S1:

CREB subcellular localisation 1 hour pa 2 hours pa 4 hours pa

In pairs of interstitial cells nuclear 10.2% ± 1.2 15.1% ± 4.2 16.1% ± 8.2 nuclear + cytoplasmic 20.0% ± 2.2 41.3% ± 2.2 19.3% ± 6.3 cytoplasmic 69.7% ± 2.0 43.7% ± 2.0 64.6% ± 8.1

In single interstitial cells nuclear 14.4% ± 1.9 34.4% ± 6.7 46.5% ± 10.7 nuclear + cytoplasmic 4.1% ± 3.6 46.2% ± 7.3 30.8% ± 3.9 cytoplasmic 81.5% ± 4.6 19.4% ± 3.6 22.8% ± 11.2

Table S1: Modulations of CREB subcellular localisation in interstitial cells during early head regeneration. Head-regenerating tips (Hv Zürich strain) were macerated at various time points post- amputation (pa) and CREB was detected by immunostaining. Note that the high level of cytoplasmic CREB at the time the interstitial cells undergo apoptosis.

TABLE S2

A RSK(RNAi) CREB(RNAi) CBP(RNAi) L4440 (RNAi) Number of cells counted n = 708, 631, 642 n = 689, 621, 613 n = 647, 601, 605 n = 600, 691 ,611 in region1 of each animal Apoptotic (%) 2.4% ± 1.61 2.7% ± 1.52 0.43% ± 0.41 51.6% ± 4.14

Non-apoptotic (%) 97.6% ± 1.61 97.3% ± ±1.52 99.6% ± 0.41 48.4% ± 4.14 N Endo. epith. 27.9% ± 4.94 23.5% ± 3.54 26.7% ± 6.5 47.7% ± 6.86 on 23.4% ± 5.7 20.1% ± 3.82 20.7% ± 3.74 24.4% ± 5.59 - Ecto. epith. ap Interstitial cells 24.5% ± 2.96 30.6% ± 6.48 27.2% ± 0.8 27.6% ± 7.15 op to Neurons 8.6% ± 1.5 11.0% ± 0.98 10.6% ± 3.19 0.33% ± 0.02 tic Nematoblasts 7.2% ± 2.08 7.3% ± 2.98 7.0% ± 2 0.16% ± 0.17 Gland cells 8.4% ± 2.3 7.4% ± 2.47 7.7% ± 1.53 0.16% ± 0.17

B DMSO (0.01%) U0126 (20 µm) Number of cells counted in region1 of each animal 706 684 676 mean value 654 666 689 mean value Apoptotic (%) 43.6 51.1 49.6 48.1% ± 3.9 10.5 15.1 8 11.2% ± 3.6

Non-apoptotic (%) 56.4 48.9 50.4 51.9% ± 3.9 89.5 84.9 92 88.8% ± 3.6 Non Endo. epith. 45.9 55.1 49.2 50.1% ± 4.6 31.4 40.3 34.4 35.4% ± 4.5 - Ecto. epith. 26.2 21.4 26.0 24.6% ± 2.7 29.9 31.2 26.1 29.1% ± 2.6 apo ptot Interstitial cells 24.8 22.1 18.9 21.7% ± 3.3 20.8 15.7 23.5 20% ± 4.0 ic Neurons 0.9 0.7 1.3 0.93% ± 0.3 9.1 6.1 7.4 7.5% ± 1.5 Nematoblasts 0.9 0 0.9 0.60% ± 0.5 3.6 3.3 4.7 3.7% ± 0.5 Gland cells 1.4 0.7 4.4 2.16% ± 1.9 5.6 3.5 4.7 4.3% ± 0.9

Table S2: Inhibition of injury-induced apoptosis in RSK(RNAi), CREB(RNAi) or CBP(RNAi) Hydra (A) or in U0126-treated Hydra (B). Apoptotic and non-apoptotic cells were counted in head-regenerating tips (region-1) after nuclear staining (Hoechst) and anti-RSK/anti-CREB immunostaining performed one hour after amputation. Hydra (Hv Zürich strain) were either exposed once to dsRNAs produced from the empty(L4440), RSK(L4440), CREB(L4440), CBP(L4440) constructs (A) or treated with U0126 for 90 minutes before and one hour after amputation. The corresponding graphs are depicted in Figure 4B and 4C. Note that the cell type populations that undergo apoptosis in control animals (L4440(RNAi) or DMSO, i.e. neurons, nematoblasts, gland cells) remain unaffected in RSK(RNAi), CREB(RNAi), CBP(RNAi) and U0126-treated Hydra.

TABLE S3:

A untreated Hydra CREB(RNAi) 1X Regio Density BLI n Regio Density BLI n n n 1 nd nd - 1 1.58 ± 0.20 20.6 ± 1.3 3 BrdU: -2 -> 0 hrs 2 1.65 ± 0.21 43 ± 2.0 4 2 0.85 ± 0.44 39.9 ± 3.6 3 Maceration @ 4 hpa 3 1.70 ± 0.09 44 ± 1.0 4 3 0.74 ± 0.05 30.2 ± 3.3 3 1 1.02 ± 0.09 19 ± 5.0 3 1 2.03 ± 0.17 75.7 ± 2.2 4 BrdU: -2 -> 0 hrs Maceration @ 8 hpa 2 2.95 ± 0.10 34 ± 2.0 3 2 1.27 ± 0.13 37.0 ± 3.5 4 3 1.16 ± 0.08 24 ± 1.0 3 3 0.52 ± 0.07 9.4 ± 1.8 4 1 1.62 ± 0.12 20 ± 1.0 3 1 1.56 ± 0.08 83.0 ± 4.5 3 BrdU: 0 -> +2 hpa 2 1.62 ± 0.08 54 ± 3.6 3 2 0.85 ± 0.13 22.3 ± 2.1 3 Maceration @ 4 hpa 3 1.69 ± 0.03 18 ± 2.6 3 3 0.75 ± 0.11 18.4 ± 0.6 3 1 1.71 ± 0.07 10 ± 1.0 4 1 1.65 ± 0.20 91.3 ± 1.7 3 BrdU: 2 -> 4 hpa 2 1.67 ± 0.11 9 ± 2.0 4 2 0.88 ± 0.09 27.3 ± 4.1 3 Maceration @ 4 hpa 3 1.68 ± 0.03 6 ± 4.0 4 3 0.77 ± 0.04 29.3 ± 0.8 3

B U0126 (20 µM) ZVAD (20 µM) Regio Density BLI n Regio Density BLI n n1 1.13 ± 0.09 8.5 ± 1.5 3 n1 1.15 ± 0.14 26.2 ± 3.8 5 BrdU: -2 -> 0 hrs 2 1.17 ± 0.04 14.6 ± 2.2 3 2 1.17 ± 0.15 16.6 ± 4.4 5 Maceration @ 4 hpa 3 1.20 ± 0.03 6.6 ± 2.3 3 3 1.19 ± 0.19 12.1 ± 5.0 5 1 1.25 ± 0.06 14.3 ± 2.4 3 1 1.53 ± 0.05 27.1 ± 4.5 4 BrdU: -2 -> 0 hrs 2 1.261.2633 ± 0.06 ± 19.9 ± 3.3 3 2 0.98 ± 0.09 20.0 ± 4.7 4 Maceration @ 8 hpa 3 1.25 ± 0.09 10.4 ± 5.3 3 3 1.10 ± 0.10 18.1 ± 3.3 4 1 1.08 ± 0.03 11.1 ± 2.6 3 1 1.17 ± 0.09 53.2 ± 4.0 4 BrdU: 0 -> +2 hpa 2 1.21 ± 0.07 20.3 ± 0.8 3 2 1.20 ± 0.15 15.3 ± 1.9 4 Maceration @ 4 hpa 3 1.22 ± 0.13 8.4 ± 3.3 3 3 1.25 ± 0.18 8.1 ± 1.3 4 1 1.13 ± 0.10 19.6 ± 4.5 3 1 1.12 ± 0.24 26.0 ± 2.8 3 BrdU: 2 -> 4 hpa 2 1.26 ± 0.14 26.1 ± 4.7 3 2 1.17 ± 0.09 22.9 ± 2.4 3 Maceration @ 4 hpa 3 1.23 ± 0.12 1.4 ± 1.4 3 3 1.20 ± 0.09 12.0 ± 0.1 3

Table S3: Modulations in cell density and BrdU-labeling index in head-regenerating stumps after genetic or pharmacological inhibition of the MAPK/CREB pathway. A) Cell density and BrdU-labeling indexes (BLI) were measured in the 3 regions of the head- regenerating halves (see Fig. 1E) of animals either untreated or exposed once to CREB dsRNAs the day before amputation. BrdU labeling was performed for 2 hours either before bisection (-2 -> 0 hrs), or immediately after (0 -> 2 hpa) or between 2 and 4 hpa. nd: cells submitted to apoptosis, no BrdU incorporation. The corresponding graphs are shown in Figure 6G and 6H. B) Cell density and BrdU-labeling indexes (BLI) were measured in the 3 regions of head-regenerating halves of animals exposed to U0126 or ZVAD treatment. Conditions of BrdU labeling are as above. The corresponding graphs are shown in Figure 6I and 6J. n: number of animals counted in each condition; around 100 CREB+ cells were counted in each region.

TABLE S4 number of % large i-cells % small i-cells % epith. cells BrdU+ cells untreated Hydra region 1 34, 36, 29 95.0 ± 1.3 0 ± 0 5.0 ± 1.3 region 2 88, 85, 89 5.4 ± 1.5 91.6 ± 1.5 3.1 ± 1.3 region 3 26, 35, 34 91.7 ± 2.8 0 ± 0 8.3 ± 2.8 CREB(RNAi) 1x region 1 69, 64, 54 19.8 ± 9.7 77.8 ± 7.2 2.4 ± 4.2 region 2 20, 19, 23 71.8 ± 12.6 23.4 ± 12.9 4.9 ± 0.4 region 3 14, 17, 12 31.6 ± 6.9 63.7 ± 8.1 4.8 ± 6.7 ZVAD region 1 79, 51, 42 26.7 ± 16.4 65.4 ± 16.9 8.4 ± 2.9 region 2 19, 17, 25 42.6 ± 5.2 45.1 ± 6.0 12.3 ± 8.5 region 3 24, 9, 13 58.8 ± 25.6 28.0 ± 24.7 13.2 ± 6.8 U0126 region 1 9, 16, 17 50.6 ± 11.5 37.2 ± 11.9 12.1 ± 10.5 region 2 25, 23, 21 31.8 ± 5.1 65.0 ± 8.4 3.2 ± 5.5 region 3 5, 14, 17 44.4 ± 18.5 43.0 ± 27.0 12.6 ± 10.9

Table S4: Cell-type distribution among the BrdU positive cells found in the 3 regions of head- regenerating halves. Animals were BrdU-labeled for 2 hours immediately after mid-gastric bisection, washed, left to regenerate for 2 hours more and fixed for CREB and BrdU immunostaining. Three regenerating halves were counted for each condition. See the corresponding graph in Figure 7C.

TABLE S5

RNAi Feedings Phase 1 Phase 2 Phase 3 Phase 4 TOTAL target number (hours) (hours) (hours) (hours) duration empty RNAi 1x 24.7 ± 5.2 14.7 ± 2.1 8.0 ± 1.3 10.7 ± 2.7 58.1 hrs L4440 RNAi 2x 25.7 ± 6.4 13.7 ± 1.5 3.7 ± 1.5 7.0 ± 1.1 50.1 hrs

RSK RNAi 1x 67.3 ± 8.8 12.3 ± 2.7 13.0 ± 3.3 6.7 ± 1.6 99.3 hrs RNAi 2x 115.3 ± 1.2 31 ± 2.8 35.7 ± 3.9 6.0 ± 0.0 188.0 hrs CREB RNAi 1x 89.3 ± 6.7 8.3 ± 1.5 3.3 ± 1.0 7 ± 1.1 107.9 hrs RNAi 2x 118.0 ± 2.0 29.7 ± 3.5 45.0 ± 2.1 6.7 ± 1.0 199.4 hrs CBP RNAi 1x 100.3 ± 7.1 7 ± 2.1 7.3 ± 1.6 7.3 ± 1.0 121.9 hrs RNAi 2x 136.0 ± 2.0 15.5 ± 3.0 44.3 ± 2.7 8.3 ± 2.0 204.1 hrs

Table S5: Kinetics of head-regeneration in Hydra silenced for RSK, CREB or CBP. Each phase of head regeneration was measured in Hydra (Hv Basel strain) exposed once or twice to dsRNAS produced from the empty(L4440), RSK(L4440), CREB(L4440), CBP(L4440) constructs. For each condition the mean value was calculated from 10 head-regenerating hydra. During phase 1, the apical tip remains smoothly obtuse; during phase 2, the apical tip becomes inflated and square, during phase 3, the first tentacle rudiments emerge, and from phase 4, new tentacles appear while the first ones elongate, becoming longer than the diameter of the head region. The corresponding graphs are depicted in Figure 8A.