Acute transcriptional up-regulation specific to osteoblasts/osteoclasts in medaka fish

Transcription

1 Aute transriptional up-regulation speifi to osteoblasts/osteolasts in medaka fish immediately after exposure to mirogravity Masahiro Chatani, 1, Hiroya Morimoto, 1 Kazuhiro Takeyama, 1 Akiko Mantoku, 1 Naoki Tanigawa, 2 Koji Kubota, 2 Hiromi Suzuki, 3 Satoko Uhida, 3 Fumiaki Tanigaki, 4 Masaki Shirakawa, 4 Oleg Gusev, 5, Vladimir Syhev, 6 Yoshiro Takano, 7 Takehiko Itoh, 1 and Akira Kudo 1 1 Graduate Shool of Biosiene and Biotehnology, Tokyo Institute of Tehnology, Yokohama , Japan 2 Chiyoda Corporation, Yokohama , Japan 3 Department of Siene and Appliations, Japan Spae Forum, Tokyo , Japan 4 Japan Aerospae Exploration Ageny, Tsukuba , Japan 5 Institute of Fundamental Mediine and Biology, Kazan Federal University, Kazan , Russia 6 SSC RF-Institute of Biomedial Problems RAS, Mosow, Russia 7 Setion of Biostrutural Siene, Graduate Shool of Medial and Dental Sienes, Tokyo Medial and Dental University, Tokyo , Japan Current address: Department of Pharmaology, Shool of Dentistry, Showa University, Tokyo , Japan Current address: RIKEN Innovation Center, RIKEN, Yokohama , Japan Corresponding author: Akira Kudo, PhD Department of Biologial Information, Tokyo Institute of Tehnology 1

2 B-33 Nagatsuta, Midori-ku, Yokohama , Japan Tel: , Fax: , Running title: Initial response of osteoblasts and osteolasts to mirogravity Supplementary Figure S1. Alizarin Red staining. (a-e) The larva, 5.0 mm in total length, was taken 3 days post hathing. Ventral view of pharyngeal bones in the head region is seen (a). Sale bar = 100 µm. Enlarged views are foused on the lower pharyngeal bone (b), upper pharyngeal bone () and leithrum (d). Lateral view of the head region is shown (e). Yellow arrows indiate the pharyngeal bones. Up, upper pharyngeal bone; lp, lower pharyngeal bone;, leithrum Supplementary Figure S2. No effet of hypergravity on osterix-dsred signals. (a) The fluoresent signals for osterix-dsred were observed 1 day before (day -1), immediately after (day 0), 1 day after (day 1), and 2 days after (day 2) hypergravity for mimiking the ondition of loaded gravity during the launh of Soyuz. Left olumn shows the 1G ontrol; and the right one, the hypergravity group. Sale bars = 100 µm. (b) Quantitative analysis of the intensity of osterix-dsred, whih revealed no signifiant differene between the 1G ontrol and hypergravity group. n.s., no signifiant differene Supplementary Figure S3. MMP9-positive ells overlap with TRAP-positive ells. (a) Fluoresent images from ventral view in the head region of larva 5.3 mm in total length at 3 days after hathing. White arrows show the pharyngeal bone region. Merge (left), TRAP-GFP (middle), MMP9-DsRed (right). p, pigment ell. Sale bar = 200 µm. 2

3 (b) TRAP staining and Von Kossa staining for MMP9-DsRed/TRAP-GFP transgeni line 9 days after hathing (7 mm total length). Blak arrows indiate TRAP-positive osteolasts. Sale bar = 200 µm. () Fluoresent image of the same setion seen in b. The green and red olors show the TRAP-GFP and MMP9-DsRed signals, respetively. The white arrows point to the TRAP- and MMP9-positive ells. (d-f) Enlarged views of the yellow-dotted square area in. The merged image (d) shows the o-loalization of MMP9-DsRed (e) and TRAP-GFP (f) in an osteolast (white arrows). Sale bar = 20 µm Supplementary Figure S4. No signifiant differene in fluoresent signals in RANKL-GFP line between ground and flight medaka. (a) Ventral view of RANKL-GFP transgeni line at day 1 after fertilization. RANKL was expressed at the pharyngeal bone region (yellow arrows in the dotted retangle), and the fluoresent signal of RANKL was enhaned. Sale bar = 200 µm. (b) Fluoresent images of RANKL-GFP in the pharyngeal bone region of the ground (left) and flight (right) medaka in a gel, whih images were aptured by the automati handling tehnique shown in Figure 1. The images were aptured under the same onditions between flight and ground samples shown in Supplementary Table S2. Yellow arrows point to the lower pharyngeal bone region. Sale bars = 50 µm. () Comparison of the intensity of RANKL-GFP signals. Values are shown as mean ± s.e.m. Ground, n=6 (days 3, 4, 5); flight, n=3 (days 3, 4, 5), as given in Supplementary Table 1. n.s.; no signifiane by Student s t-test. 3

4 a b lp lp up up up up d lp lp e up lp Supplementary Figure S1

5 a Control osterix Hypergravity osterix day -1 day 0 day 1 day 2 b Fluoresene intensity G Hyper G day -1 day 0 day 1 day 2 Observation day Supplementary Figure S2

6 a TRAP-GFP/MMP9-DsRed TRAP-GFP MMP9-DsRed p p p p b d e f Supplementary Figure S3

7 a b Ground Flight RANKL RANKL Day 5 Day 5 Fluoresene intensity Ground Flight 0 Day 3 Day 4 Day 5 Observation day Supplementary Figure S4

8 Ground Flight Day N Animal ID for Osterix-DsRed Day N Animal ID for Osterix-DsRed Day 1 9 A1, A2, A4, A6, A7, A8, A9, A10, A11 Day 1 6 a1, a2, a3, a4, a5, a6 Day 2 10 A1, A2, A4, A5, A6, A7, A8, A9, A10, A11 Day 2 6 a1, a2, a3, a4, a5, a6 Day 3 10 A1, A2, A4, A5, A6, A7, A8, A9, A10, A11 Day 3 6 a1, a2, a3, a4, a5, a6 Day 4 10 A1, A2, A4, A5, A6, A7, A8, A9, A10, A11 Day 4 7 a1, a2, a3, a4, a5, a6, a7 Day 5 11 A1, A2, A4, A5, A6, A7, A8, A9, A10, A11, A12 Day 5 7 a1, a2, a3, a4, a5, a6, a7 Day 6 10 A1, A4, A5, A6, A7, A8, A9, A11, A12, A13 Day 6 6 a1, a3, a4, a5, a7, a8 Day 7 11 A1, A4, A5, A7, A8, A9, A12, A14, A15, A16, A17 Day 7 5 a1, a3, a5, a7, a8 Day 8 11 A1, A4, A7, A9, A12, A14, A15, A16, A17, A18, A19 Day 8 4 a3, a5, a7, a8 Day N Animal ID for Osteoalin-DsRed Day N Animal ID for Osteoalin-DsRed Day 1 6 B1, B2, B3, B4, B5, B6 Day 1 3 b1, b2, b3 Day 2 7 B1, B2, B3, B4, B5, B6, B8 Day 2 3 b1, b2, b3 Day 3 7 B1, B2, B3, B4, B5, B6, B8 Day 3 5 b1, b2, b3, b4, b5 Day 4 6 B2, B4, B5, B6, B8, B10 Day 4 4 b2, b3, b4, b5 Day 5 3 B4, B8, B10 Day 5 4 b2, b3, b4, b5 Day N Animal ID for TRAP-GFP Day N Animal ID for TRAP-GFP Day 4 14 A1, A2, A3, A4, A6, A7, A8, A9, A10, A11, B4, B5, B8, B10 Day 4 9 a1, a2, a3, a4, a5, a6, a7, b2, b3 Day 6 16 A1, A2, A4, A6, A7, A8, A9, A11, A12, B4, B8, B10, B12, B13, Day 6 7 a1, a3, a4, a5, a7, a8, b3 B14, B15 Day N Animal ID for MMP9-DsRed Day N Animal ID for MMP9-DsRed Day 4 6 C1, C2, C3, C4, C5, C6 Day 4 4 1, 2, 3, 4 Day 6 6 C1, C2, C3, C4, C5, C6 Day 6 3 2, 3, 4 Day N Animal ID for RANKL-GFP Day N Animal ID for RANKL-GFP Day 3 6 C1, C2, C3, C4, C5, C6 Day 3 3 1, 2, 3 Day 4 6 C1, C2, C3, C4, C5, C6 Day 4 3 1, 3, 4 Day 5 6 C1, C2, C3, C4, C5, C6 Day 5 3 1, 3, 4 Day N Animal ID for Osterix-DsRed (Whole-body) Day N Animal ID for Osterix-DsRed (Whole-body) Day 1 5 A1, A2, A3, A4, A5 Day 1 3 a1, a4, a8 Day 2 5 A1, A2, A3, A4, A5 Day 2 3 a1, a4, a8 Day 4 5 A1, A2, A3, A4, A5 Day 4 4 a1, a2, a4, a8 Day 6 5 A1, A2, A3, A4, A5 Day 6 3 a1, a2, a8 Day 7 5 A2, A3, A4, A6, A7 Day 7 3 a1, a2, a8 Supplementary Table S1 The reord of observation for individual medaka samples (n 3) in the ground ontrol (left) and flight (right) group A-C and a- show the kind of transgeni lines for observation; Osterix-DsRed/TRAP-GFP transgeni line in the ground (A) and flight (a), Osteoalin-DsRed/TRAP-GFP transgeni line in the ground (B) and flight (b), and MMP9-DsRed/RANKL-GFP transgeni line in the ground (C) and flight (). Day means the observation day. N means the number of observed fish. The images of ground ontrol samples were aptured during 2014/06/ /0616, and those of flight samples were aptured during 2014/02/ /02/14.

9 Figure number Objet Gain value Ground Exposure time (mse) Gain value Flight Exposure time (mse) Objetive lens Figure 2, d Osterix-DsRed (Whole-body) x Figure 3a, b Osterix-DsRed x Figure 3e, f Osteoalin-DsRed x Figure 4a, b TRAP-GFP x Figure 4d, e MMP9-DsRed x Figure S4b RANKL-GFP x Supplementary Table S2 Fluoresene observation for the flight and ground ontrol samples Both samples were observed under the same onditions.

10 Up-regulated GO annotations q-value Down-regulated GO annotations q-value Moleular funtion Moleular funtion DNA binding extraelullar matrix strutural onstituent Heteroyli ompound binding phosphotransferase ativity, alohol group as aeptor Nulei aid binding protein kinase ativity Organi yli ompound binding reeptor ativity Biologial proess Biologial proess Cellular aromati ompound metaboli proess musle tissue development Cellular nitrogen ompound metaboli proess phosphorylation DNA metaboli proess signal transdution DNA repliation striated musle tissue development DNA repliation initiation Cellular omponent Endoderm formation ollagen trimer hematopoieti or lymphoid organ development ontratile fiber part heteroyle metaboli proess extraellular matrix nitrogen ompound metaboli proess integral omponent of membrane nulei aid metaboli proess integrin omplex nuleobase-ontaining ompound metaboli proess membrane organi yli ompound metaboli proess membrane part Cellular omponent plasma membrane MCM omplex protein omplex involved in ell adhesion nuleus proteinaeous extraellular matrix reeptor omplex Supplementary Table S3 Enrihed systems by GO analysis for whole-body in-flight medaka at days 2 and 6

11 Gene ID Gene title FC FC p-value p-value (Day 2, F/G) (Day 6, F/G) (Day 2) (Day 6) LOC Krueppel-like fator 9-like LOC TNFAIP3-interating protein 1-like LOC GATA-binding fator 5-A-like LOC G0/G1 swith protein 2-like LOC AMP-responsive element modulator-like LOC tubulin beta-6 hain-like LOC serine/threonine-protein kinase SIK2-like LOC early growth response protein 1-B-like LOC unonventional myosin-xv-like LOC transription fator Maf-like LOC PR domain zin finger protein 1-like foxa3 Me-FKH gata-1 hematopieti transription fator GATA LOC Krueppel-like fator 2-like LOC CCAAT/enhaner-binding protein beta-like LOC transription fator jun-b-like LOC jun dimerization protein 2-like LOC neuronal PAS domain-ontaining protein 4-like LOC transription fator AP-1-like LOC early growth response protein 1-like LOC nulear reeptor subfamily 0 group B member 2-like fos -fos LOC dnaj homolog subfamily B member 5-like LOC nulear reeptor subfamily 1 group D member 2-like LOC r3hdm1 R3H domain ontaining LOC CCAAT/enhaner-binding protein delta-like LOC C8orf LOC TSC22 domain family, member LOC NF-kappa-B inhibitor alpha-like LOC signal-transduing adaptor protein 1-like LOC vesile-assoiated membrane protein 2-like LOC GADD45 beta-like LOC zin finger protein 143-like Supplementary Table S4 Up-regulation of nuleus-related genes in whole-body in flight (F) versus ground (G) at days 2 and 6 *FC means fold hange

12 Gene ID Gene title FC p-value q-value (Day 6, F/G) LOC mm LOC mm LOC mm LOC mmbp LOC mm LOC mm LOC mm Supplementary Table S5 Up-regulation of MCM family in whole-body medaka at day 6 in flight (F) versus ground (G) *FC means fold hange

13 Gene ID Gene title FC FC p-value p-value Day 2 Day 6 Day 2 Day 6 Cell Adhesion LOC laudin-5-like LOC laudin-7-like LOC laudin-like protein ZF-A89-like LOC desmoglein-4-like LOC desmoplakin-like LOC neural ell adhesion moleule L1-like protein-like LOC arinoembryoni antigen-related ell adhesion moleule 1-like LOC integrin alpha-2-like Channel, Transporter LOC solute arrier family 13 member 5-like LOC solute arrier family 25 member 38-A-like LOC solute arrier family 12 member 3-like LOC aquaporin-3-like LOC ammonium transporter Rh type B-A-like Reeptor LOC sphingosine 1-phosphate reeptor 1-like LOC marophage mannose reeptor 1-like Membrane-related LOC transmembrane protease serine 4-like LOC lass I histoompatibility antigen, F10 alpha hain-like LOC hondroitin sulfate proteoglyan 4-like LOC regulator of G-protein signaling 1-like LOC ell yle ontrol protein 50B-like LOC gastri intrinsi fator-like LOC C-type letin domain family 4 member M-like LOC aba12 ATP-binding assette, sub-family A (ABC1), member LOC probable G-protein oupled reeptor LOC alpain-5-like LOC kunitz-type protease inhibitor 1-like LOC transmembrane protein 54-like Supplementary Table S6 Alteration of the expression of membrane-related genes in whole-body medaka at days 2 and 6 in flight (F) versus ground (G) *FC means fold hange