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Specific Functions of the Wnt Signaling System in Gene Regulatory Networks Throughout the Early Sea Urchin Embryo

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Specific Functions of the Wnt Signaling System in Gene Regulatory Networks Throughout the Early Sea Urchin Embryo Supplemental Appendix Specific functions of the Wnt signaling system in gene regulatory networks throughout the early sea urchin embryo Miao Cui, Natnaree Siriwon, Enhu Li, Eric H. Davidson and Isabelle S. Peter* * To whom correspondence may be addressed. Tel: +1 626 395 5887. E-mail: [email protected] Content Page Supplemental Materials and Methods 2-3 Supplemental Figures 4-13 Supplemental Figure Legends 14-16 Supplemental Tables 17-21 Supplemental References 22 Supplemental Materials and Methods Gene cloning and Whole mount in situ hybridization (WMISH). cDNAs of wnt, fzd, dkk, and sfrp genes were amplified by PCR using primers listed in Table S2, and cloned into pGEM-T constructs. cDNA prepared from various developmental stages were used as templates for PCR reactions. Sequences used to prepare WMISH probes of regulatory genes, i.e. alx, delta, foxa, hox11/13b, eve, lim1, emx, and foxq2, were previously described (1-5). Cloned constructs were linearized for transcription of antisense RNA probes labeled with digoxigenin (DIG) or fluorescein (FL). WMISHs were performed according to the standard methods (6). In brief, embryos were fixed in 2.5% glutaraldehyde, 32.5% sea water, 32.5 mM MOPS (pH 7) and 162.5 mM NaCl at 4°C overnight. 25 ng/µl of proteinase K in TBST was used to treat embryos at room temperature for 5-10 minutes, followed by 30-minute fixation in 4% paraformaldehyde at room temperature. Hybridizations were performed overnight at 58-60°C using a probe concentration of 0.5-1ng/µl. Probes were detected using anti-DIG or anti-FL Fab fragments conjugated to alkaline phosphatase (Roche). Color was developed using NBT/BCIP or INT/BCIP reagents (Roche). C59 treatment. The Porcupine inhibitor C59 was obtained from Cellagen Technology (C7641-2s). C59 was dissolved in DMSO at 10mM, and experiments were performed in the presence of C59 diluted in sea water to 0.5µM (see text), unless indicated otherwise. Except for the timing experiment in Fig. S6, embryos were treated with C59 at 1h post fertilization and were exposed until embryos were collected, at developmental times indicated in figure captions. MASO injection and RNA isolation. MASOs were provided by GeneTools. Sequences of gene-specific MASOs are shown in Table S4. MASOs were diluted in injection solution including 0.12M KCl at 300µM and injected into fertilized eggs in a volume of 2-4 pl. Randomized control MASOs (N25) were injected at same concentration. Experiments were performed on 2-5 independent embryonic batches. Embryos were cultured at 15°C, and approximately 300 MASO-injected embryos were collected at different time points. Total RNA was extracted using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. QPCR analysis. cDNA was synthesized using iScript cDNA synthesis kit (BioRad). QPCR was performed using Power SYBR Green Master Mix (Life Technology) with the primers listed in Table S3. Gene expression levels were normalized to those of poly- ubiquitin. Fold change was calculated by comparing normalized expression levels in the embryos injected with gene specific MASOs to expression levels in embryos injected with control MASOs. NanoString nCounter. Approximately 300 embryos were collected at different time points and lysed in 15µl of RLT buffer (Qiagen, Hilden, Germany). Hybridization reactions were performed according to manufacturer’s instructions in total 30µl solution with 10µl of detection probes, 10µl of hybridization buffer, 5µl of embryo lysate, and 5µl of capture probes. All hybridization reactions were incubated at 65 °C for a minimum of 18 h. Hybridized probes were recovered with the NanoString Prep Station and immediately evaluated with the NanoString nCounter. The resulting count for each gene was subtracted by counts of negative spikes for background correction and then normalized using the sum of all corrected counts for all genes in the codeset. Fold changes of normalized counts in C59 treated versus untreated control embryos is shown for genes with counts >200, corresponding to approximately 100 transcripts. Experiments were performed on 3 independent embryonic batches. Probe sequences and accession numbers for the genes included in the codeset are as same as previously reported (1). Supplemental Figures Supplemental Figure 1 A B 700 1800 700 wnt1 wnt3 wnt4 1600 wnt6 600 wnt5 600 wnt7 wnt16 1400 wnt9 500 500 wnt8 1200 wnt10 wntA 400 1000 400 300 800 300 600 ranscripts/Embryo 200 T ranscripts/Embryo 200 ranscripts/Embryo T 400 T 100 200 wnt8 100 0 0 0 0 20 40 60 80 0 20 40 60 80 C Time Post-fertilization (hrs) D Time Post-fertilization (hrs) 7000 7000 350 fzd1/2/7 sfrp1/5 fzdl4 srfp3/4 6000 6000 300 fzd5/8 dkk1 fzd9/10 dkk3 5000 5000 250 4000 4000 200 3000 3000 150 ranscripts/Embryo T 2000 2000 100 ranscripts/Embryo ranscripts/Embryo T T 1000 1000 50 0 0 0 0 20 40 60 80 0 10 20 30 40 50 60 70 80 dkk1 and dkk3 Time Post-fertilization (hrs) Time Post-fertilization (hrs) Supplemental Figure 2 wnt1 wnt4 wnt5 wnt8 wnt16 foxA lv lv lv lv lv lv vv vv vv vv vv vv 12hr lv lv lv lv lv lv vv vv vv vv vv vv 15hr lv lv lv lv lv lv vv vv vv vv vv vv 18hr lv lv lv lv lv vv vv vv vv vv vv 21hr lv lv lv lv lv lv vv vv vv vv vv vv 24hr 24hr Supplemental Figure 3 A 12hr 15hr 18hr 21hr 24hr lv lv lv lv lv hox7 vv vv av av av frizzled 1/2/7 frizzled hox7 lv lv lv lv lv frizzled 5/8 frizzled foxA lv lv lv lv lv vv vv vv vv vv frizzled 9/10 frizzled B lv 12h lv 15h lv 18h lv 21h lv 24h sfrp1/5 lv 12h lv 15h lv 18h lv 21h lv 24h sfrp3/4 lv 15h lv 24h lv 15h lv 24h dkk3 dkk1 Supplemental Figure 4 A Control * * 22h 48h 72h 72h B * * C59 0.1uM C59 22h 48h 72h * C59 0.5uM C59 * B 22h 48h 72h 72h 528 foxa (22hr) hox11/13b (22hr) 350 300 300 250 250 200 200 150 150 100 100 anscripts/Embryo ranscripts/Embryo T T 50 50 0 0 eve (22hr) gcm (22hr) 800 350 700 300 600 250 500 200 400 150 300 100 200 anscripts/Embryo ranscripts/Embryo T 100 T 50 0 0 Supplemental Figure 5 A B Porcupine MASO 24hfp 2 Porcupine MASO erence (log2) 72hpf f 0 old dif F 2 e a r Control MASO xa v b o e xQ2 f gcm o 72hpf f limp1b x11/13b b o C 10 20 30 40h Wnt1/199-245 NL HN NE AG RK TV SSE MR RE CK CH GM SG SC TI ET CWMR LP TF RT VG EL Wnt16/265-311 NL HN NE AG RQ LI KS GM KT LC RC HG VS AS CS LK TC WK AM PS FK EI GD L Wnt4/15-61 NL HN NE AG RRT IE D NM RM EC KC HG VS GS CE MK TC WK SM PT FG D IG QV Wnt5/254-300 NL HN NE AG RK AV YD NA GT EC KC HG VS GS CS LK TC WL QL SP FN RV GT I Wnt8/163-209 TL HN NE AG RK AV KQ TL QR TC KC HG VS GS CS LQ TC WN HV AN FR VI GD E Conservation + ******** 7 5 9 3 6 4 7 4 5 4 * 9 *** 8 * + ** 6 9 6 *** 4 3 8 6 7 * 4 3 9 * 7 4 Quality Consensus NL HN NE AG RK + V K + NM R + EC KC HG VS GS CS LK TC WK - + PT FR -I GD L Supplemental Figure 6 A hox1113b foxa 1 0 1 2 3 4 blimp1b brachyury erence [Log2] f 1 old dif 0 F 1 2 3 4 1 24 15 24 18 24 21 24 1 24 15 24 18 24 21 24 Time duration of C59 treatment [hfp] B foxQ2 nk2.1 2 1 0 1 erence[Log2] nkx3-2 fzd5/8 f 2 old dif F 1 0 1 1 24 12 2415 24 18 24 1 24 12 2415 24 18 24 Time duration of C59 treatment [hfp] Supplemental Figure 7 A Control Dn-cadherin mRNA 72h 72h B Veg2 endoderm genes Veg1 endoderm genes Apical genes 2.5 2.5 2.5 0 0 0 -2.5 -2.5 -2.5 -5 -5 -5 blimp1b bra dkk3 2.5 2.5 2.5 0 0 0 -2.5 -2.5 -2.5 -5 -5 -5 foxa hnf1 foxQ2 2.5 Fold change (log2) 2.5 Fold change (log2) 2.5 Fold change (log2) 0 0 0 -2.5 -2.5 -2.5 -5 -5 -5 tgif hox11/13b fzd5/8 09 12 15 18 21 24 09 12 15 18 21 24 09 12 15 18 21 24 Supplemental Figure 8 hox7 20h irxa 19h msx 18h hox7 24h irxa 24h msx 24h nk1 24h sp5 24h unc4.1 24h Supplemental Figure 9 A DMSO C59 DMSO C59 DMSO a a’ e e’ alx alx eve eve b b’ f f’ C59 delta delta lim1 lim1 c c’ g g’ foxA foxA emx emx apical plate d d’ h h’ non-apical ectoderm Veg1 ectoderm Veg1 endoderm Veg2 endoderm Veg2 Mesoderm hox11/13b hox11/13b foxQ2 foxQ2 B Control Wnt1 MO Wnt4 MO Wnt5 MO Wnt8 MO Wnt16 MO * foxA * foxA * foxA * foxA * foxA * foxA hox11/13b hox11/13b hox11/13b hox11/13b hox11/13b hox11/13b eve eve eve eve eve eve lim1 lim1 lim1 lim1 lim1 lim1 emx emx emx emx emx emx foxQ2 foxQ2 foxQ2 foxQ2 foxQ2 foxQ2 Supplemental Figure 10 control MASO injected embryo embryo wnt1 wnt16 MASO foxa wnt1 wnt16 MASO hox11/13b wnt1 wnt16 MASO eve wnt1 wnt16 MASO lim1 wnt1 wnt16 MASO emx wnt8 MASO wnt8 MASO foxq2 Supplemental Figure 11 SM Veg2 Meso Veg2 Endo Veg1 Endo Veg1 Ecto wnt1 wnt4 wnt5 wnt8 wnt16 blimp1b bra eve gataE hox11/13b 2 foxA 12h 15h 18h 21h 24h 12h 15h 18h 21h 24h 12h 15h 18h 21h 24h 12h 15h 18h 21h 24h 12h 15h 18h 21h 24h Supplemental Figure 12 2 2 Hox11/13b MASO 24hr 0 erence [log2] f 2 old dif 4 F 2 Eve MASO 24hr 0 erence [log2] f 2 old dif 4 F wnt1 wnt4 wnt5 wnt8 wnt16 hox11/13b Supplemental Figure 13 2 2 FoxA MASO 24h hox11/13b Bra MASO 24h 1 1 0 0 erence (log2) erence (log2) f f 1 1 old dif old dif F F 2 2 2 2 Blimp1b MASO 24h GataE MASO 24h 1 1 0 0 erence (log2) f erence (log2) f old dif 1 1 F old dif F 2 2 Wnt1 Wnt4 Wnt5 Wnt8 Wnt1 Wnt4 Wnt5 Wnt8 Wnt16 Wnt16 Supplemental Figure Legends Supplemental Figure 1.
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