step-wise evolution of neural patterning by hedgehog signalling in …10.1038... · 2020-07-06 ·...
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Articleshttps://doi.org/10.1038/s41559-020-1248-9
Step-wise evolution of neural patterning by Hedgehog signalling in chordatesQiongqiong Ren1,3, Yanhong Zhong1,3, Xin Huang1, Brigid Leung2, Chaofan Xing1, Hui Wang1, Guangwei Hu1, Yiquan Wang 1 ✉, Sebastian M. Shimeld 2 ✉ and Guang Li 1 ✉
1State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China. 2Department of Zoology, University of Oxford, Oxford, UK. 3These authors contributed equally: Qiongqiong Ren, Yanhong Zhong. ✉e-mail: [email protected]; [email protected]; [email protected]
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SUPPLEMENTARY INFORMATION 1
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Supplementary Figure 1. qPCR analysis of Ptch and Netrin expression in Hh-/- 5 and Ptch-/- larvae. (A). Phenotypes of Hh and Ptch larvae at the stages when gene 6 expression was analyzed. Scale bar is 100 µm. (B). Violin plot showing relative 7 expression levels of Ptch and Netrin to Gapdh in Hh and Ptch larvae. Statistical 8 analysis were performed with t-test incorporated in excel software (* p<0.05; ** 9 p<0.01, *** p<0.001). 10
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Supplementary Figure 2. Pharmacological manipulation of Hh signalling affects 13 but does not completely inhibit Ptch expression in amphioxus embryos. Whole 14 mounts with anterior to the left and the scale bar are 50 µm. All the embryos are in 15 lateral view. The control embryos were treated with DMSO (A, B) and experimental 16 embryos treated with LDE225 (C-H) or SAG (I-N) from the early gastrula stage, fixed 17 for in situ hybridization of Ptch at the stages indicated. 5S: embryos with 5 somites; 18 14S: embryos with 14 somites. Numbers in the bottom right corner of a panel show 19 the number of times the phenotype depicted shown, out of the total number of 20 embryos scored after in situ hybridisation. 21 22
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Supplementary Figure 3. The expression of Netrin gene in Hh-/- and Ptch-/- 24 embryos at 14-somite neurula stage. Anterior is to the left and the scale bar is 50 25 µm and the images are in lateral views. Top panels show digestion products from 26 PCR across the mutated region: mutation abolishes the restriction site, so the higher 27 band (uncut) indicates presence of the mutated allele, the lower band (cut) the wild 28 type allele. Lower panels show Netrin expression in embryos analyzed. Embryo 29 numbers and genotypes are shown on the top left corner of each image, with -/- 30 highlighted in red. 31 32
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Supplementary Figure 4. TALEN target sites of amphioxus OligA, Mnxa, Err 34 and Smo gene and genotyping analysis of embryos of normal and abnormal 35 phenotypes. (A) Schematic of the coding sequence of amphioxus OligA gene. The 36 binding sits in E1 (1st exon) for the TALEN pairs used in this study are highlight in 37 grey. The SacI site in the spacer is underlined. (B) Schematic of the coding sequence 38 of amphioxus Mnxa gene. The binding sits in E1 for the TALEN pairs used in this 39 study are highlight in grey. The ClaI site in the spacer is underlined. (C) Schematic of 40 the coding sequence of amphioxus Err gene. The binding sits in E4 for the TALEN 41 pairs used in this study are highlight in grey. The NheI site in the spacer is 42 underlined. (D) Schematic of the coding sequence of amphioxus Smo gene. The 43 binding sits in E3 for the TALEN pairs used in this study are highlight in grey. The 44 BamHI site in the spacer is underlined. Sequencing results of cleaved PCR products 45 (WT) and uncleaved PCR products (-13bp, -8bp, -4bp and -10bp in A, B, C and D). 46 47
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Supplementary Figure 5. The expression of OligA, OligB and OligC in Hh-/- 50 amphioxus embryos at 11 somites stage. The probes used are designed to bind 51 the divergent 3’-untranslated regions (3’-UTR) of each gene. Whole mounts with 52 anterior to the left and in lateral view. Scale bar is 50 µm. The expression of OligA, 53 OligB and OligC in the neural tube of (A-C) control (Hh+/+ and Hh+/-) and (A’-C’) Hh-/- 54 embryos. Numbers in the bottom right corner of a panel show the number of times 55 the phenotype was seen, out of the number of embryos of that genotype analyzed. 56 57 58
59 Supplementary Figure 6. The expression of Vglut and Vgat in Ptch-/- amphioxus 60 embryos at 14 somites stage. Whole mounts with anterior to the left. Scale bar is 61 50 µm. The embryos are all in ventral view, focused on the dorsal side. Top panels 62 show digestion products from PCR across the mutated region: mutation abolishes 63 the restriction site, so the higher band (uncut) indicates presence of the mutated 64 allele, the lower band (cut) the wild type allele. Lower panels show Vglut and Vgat 65 expression in embryos analyzed. Embryo numbers and genotypes are shown on the 66 top left corner of each image, with -/- highlighted in red. 67 68
Supplementary Table 1. Primers used for amplifying gene fragments including TALEN target sites and the amplicon sequences 69 Genes Primer sequences (5’→3’) Amplicon sizes (bp) Amplicon sequences (5’→3’)
Hh Forward: GTTATGGCGGGGGTACTAGC 313 GTTATGGCGGGGGTACTAGCGCGGTGGATGGTCACACTGGTCGCGATATCGGCGTTGGGAACGCACTGGGGACCGAGCGAGGCCTGCGGGCCCGGGGGAAGGTTCGGCCGGCGACGGCACCCTCGCAAGCTCACCCCCTTCGTGTACAAGCAACAGATGCCGGCGGTCTCCGAGAACACCTTTGGGGCCTCTGGCCTGTTCAACGGACGGATTACTCGCGACTCCGAGCGTTTCCATACTCTGAAACAAAATTTCAACACAGACATAATCTTCAAGGATGAGGAAAAAACGGGGGCTGACCGATTCATGACAC
Reverse: GTGTCATGAATCGGTCAGCC
Ptch Forward: GTTATTGGTGTGGCAACTTGG 441 gttattggtgtggcaacttgggggagggccaactgcagttgtcgttgtgataccacgtgttgacgcgagacaaaccagggttcctagggcaataaaatcgtacttacaagcgtcagatgatgccctagtacatgtactactacgttgggatattgttgatgccctaatgttcatgtatgtggggttggggagggcgaaaggttactaggtgtcaaatgaacacacaatcgccatgttgattacactgtagtgtttgtgaaaacgacagcttcgagctatagctctaagatgttatgtatcttacgatttcagGGCAGGGCGACTGGGAACAAGCAAGCGCTCTACCTCAGGGAGAAATGTGAGAGATGTCTGTTCAACACGGGAAAGATCATACAACTACACTGTGGAAAAGTTCTGTTTGTTGGAGTGCTTGTCATGGTT
Reverse: AACCATGACAAGCACTCCAAC
Err Forward: CTCAGACATGCAAGCATTGA 343 ctcagacatgcaagcattgatttcaggtgtttgaatcaagaaatcaatggtgtattaacgttagtaccgtcagattaactggtgtatgccaggaagcagtgctgcatgcattccatcatgtgactcggtgctgtaatgtacgtttccagGCCGCACGCGACATGTCCACAATTCTGTCCCCAAGAAGTTTGTGCTAGCGGGCGGTGGTACCAAAACAAAACGACAGGTCGTCACTTGTCCAACCAACTCACACAAGCAGCATGGCAGAAAAGgcaagccacttcctgtgtcaaccactgttctatacacttctacaactgtcctcttccaagcaactaaccgt
Reverse: ACGGTTAGTTGCTTGGAAGA
Mnxa Forward: ACCTCGAAGAGAAGGGAAGC 487 ACCTCGAAGAGAAGGGAAGCgccTTTTTCgGtTGAAAAGAGtAAGAGGTCTCAACATGATGACGGAAAGTATCAAGTCGTTTCGTATCGATGACCTCCTgGCGTCGGACCGGCGCAAGCTaCAGCGCTCCTCGGGGCTaGGcGTGTGGAGCCCCCGCCAGCGAAGCCCTACCGCCTCGGAGCCAAGTCCACGGGGCCAGCCCGACACGCCGCCCGTCTCACCCCTAGCGGAgGAGAGTCCCAAGATGTCCCCGCCGTCTAGTTTtGTCCCcAGACCGGGACTACTtCACCTGCACCCGTCaGCCGGtCTcCCCCTGCACCCGCACCCGGCGTACGGTCAGCACCACTTCCCGTTGACTGCTAACGGGACGCACCACTTCGGGTTCTCGGGCTTCCAcCCACATCACCCCGACCATCTCAAGACGGCCATGCCCTTCGAaTGGGTCCGTGCAGCGGGAATGATCATGCCAAGAATGGATTACGGAGgt
Reverse: ACCTCCGTAATCCATTCTTGGC
OligA Forward: GACTGCCAAGTGGGTTCTGA 554 GACTGCCAAGTGGGTTCTGAAGATGGAACAGAGTCCGGACGCCGCCATGTTCAACAGAACCTCCCCGTCTGTGTCTCCCCGGCCGTCCTCACCGACATCGTCCTGGTCCAGCGGCGGCTCGCCCGCCCCCTCCTCACCCTCCTCCCGCTCCCGAACGGGGAAGATCAGGAGGAGCAAGAGTATGAAGAAAGCCGAGAAGCAGTTATCGGAGGAAGAACTCCAGGAGCTCCGTCTGAAAGTCAACCATCGGGAGAGGAAGAGAATGCACGACCTGAACTCGGCGCTGGACGGGTTGCGGGAAGTCATGCCGTACGCTCACGGTCCGTCCGTCAGGAAACTCTCCAAAATCGCCACGCTGCTGCTGGCCAAGAATTACATCCTGATGCTCAACAGTTCCTTGGAGGAGATGAAGCGGCTCGTGAGTGACATCCAACACACTCGCCGCGCTGGACTGCCTTCAACCACCCTCCCCACGCCATCAGGGGCCGCCGTACCGCCCTTCCACAGGCTGGCCATGCCTCCGACCCCGATCGTTCCTaCACACACTCCGGTGA
Reverse: TCACCGGAGTGTGTGTAGGA
Smo Forward: CTTTTTCTAGAACGAAGTGAAG 234 ctttttctagAACGAAGTGAAGAAgATCAAGTTCAATGTAACTGGTGGTTGTGAGCGCCCTCTGGTGGAAACAGACAATGAGAAAAGCTTCTACGAAGGTGTTGAAGGCTGTGGGATCCAGTGTCAAAATCCACTCTTCACAGACGCTGAACATGAGCGCATCCACAAGTTCATAGCTGGGTTTGGAGCCGTGACTGTTGTTTTTACATTCTTCACTTTGgtgagtggtcagaa Reverse: TCTGACCACTCACCAAAGTGA
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Supplementary Table 2. Primers used for making pGEM-T Easy or pXT7 72 constructs 73
Genes Primer sequences (5’→3’) Enzyme site added
GenBank Position
Pax3/7
F: AGTCTTCATCAACGGCCGCC -
MF979123.1 99-982
R: CGTACATGCTGGCTGTGTCT -
Dlx
F: CGGAATTCATGGAGATCCCGCCGGTGC -
U47058.1 293-1090
R: GGACTAGTTCAAAGTCTCTGTAAGAAGTTGACAG SpeI
Pax4/6
F: GAATTCCGGGAGTTTTCACCAGACAA EcoRI
AJ223442.1 501-1971
R: ACTAGTCGGCCAGTAGGGTTGTG -
Nkx2.2
F: GAATTCCTGACGGGATTCTGTTTGG EcoRI
AF032999.1 91-1187
R: ACTAGTGACCGACTACCTCATCTTAT SpeI
Nkx2.1 F: CGGAATTCATGGAGTCCATAAGCCCTAAGC EcoRI
AF077840.1 164-1083 R: GGACTAGTTCACCACGCTCTGCCCTGCTGT SpeI
Netrin
F: TGTAAACAGTGACCCATTCCG -
AJ252166.1 117-1832
R: CACATGGCATGAAGGTTGA -
Err
F: CCAGACTTCAGTGGACGATGAC -
AY738654.1 1-1491
R: GGGTCCCTATGTCCCTATGC -
IrxA
F: GGGGTACCATGTCGTACGCACAGTTCGGT KpnI
EU754746.1 1-1394
R: GGACTAGTTTACCTCTTTTGTACCGGTTTG SpeI
IrxB
F: GGGGTACCATGTCCAGCATGTCCTATGCAC KpnI
EU754748.1 1-1491
R: GGACTAGTCTATCTGGACGATAGGCTGGTCAG SpeI
IrxC
F: GGGGTACCATGTCCTACCCACACTTTGGATACT KpnI
EU754750.1 1-1485 R: GGACTAGTCTATCTTGAAGACAGATTGGTGAG SpeI
Nkx6
F: CGGAATTCATGCTTGTATCGGAGACTGC EcoRI
XM_002609024.1 82-957
R: GGACTAGTCTACGTACCGATGTGTGAAG SpeI
OligA
F: GGTACCATGGTAATATACGCCATCGA KpnI
MT166306 1-857
R: ACTAGTTCACCTGGTGAAGGTTGGGA SpeI
Vacht
F: CGGAATTCATGACCGTGCTGGGATTCGA EcoRI
EF118170.1 200-1750 R: GGACTAGTTCAGTATTCGTCGTCCCACG SpeI
Vglut
F: CGGAATTCCCTCGCTCAACATGCTCATC EcoRI
XM_002597566.1 61-975
R: GGACTAGTTACAACTTACTTCTCTCCAT SpeI
Vgat
F: CGGAATTCACAACATGGCGTGGCGCTGG EcoRI
XM_002609689.1 468-1414
R: GGACTAGTCTGTACGAGATGGTCAGTCG SpeI
Lhx3
F: ATGATGACCACAGTTCTACC -
XM_002591792.1 88-1296
R: TCAGTAGTGGCTGTCTACGTC -
Mnxa
F: GGTACCGTCTCAACATGATGACGGAAAG KpnI
AF308821.1 63-968
R: ACTAGTTAATGGAATCATCTTGTTGT SpeI
Islet
F: GGTACCTGAACCACTCACCACCCA KpnI
AF226616.1 41-1341
R: CCTGTACCTAACCACCTGCC -
OligA-3’UTR
F: CCGGACTGCCCTCAACCACC -
MT166305 1-548
R: ACATTTGAAACAAAGGCACT -
OligB-3’UTR
F: ACCGTGGTCACCACGCGGG -
MT166304 1-621
R: TGTGAGCCGTCAGCTATCC -
OligC-3’UTR
F: AAGCTGCCCCCCTCACCC -
MT166303 1-571
R: CAATCTCACTACATACAGAG -
74 75 76
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Supplementary Table 3. Primers used RT-qPCR 79
Genes Primer sequences (5’→3’) Amplicon sizes
Netrin F: AGTCCACGCCATGAAGACAC
191 bp R: TTGAGGTCGAACCCTCCTGA
Gapdh F: GGTGGAAAGGTCCTGCTCTC
169 bp R: CTGGATGAAAGGGTCGTTAATGG
Ptch F: GTCATGGTTCTCTTGGCCGT
187 bp R: GCACATTGTCCCCTTCGTTC
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