Genome Data Viewer (GDV) (https://www.ncbi.nlm.nih.gov/genome/gdv/ ) [18 (link)] and Ensembl Genome Browser (EGB) 105 (https://www.ensembl.org/index.html ) were used to search gene sequence databases to identify gene orthologues and examine the expression of scpp genes in medaka (ASM223467v1). Genomic synteny analysis was also conducted using GDV and EGB105. Each scpp gene assembly number in medaka was referenced to GDV and EGB105, as listed in availability of data. For scpp4, scpp7, scpp9, and enam, similar sequences were identified in the medaka genome by performing genomic alignment with zebrafish (GRCz11) and fugu (fTakRub1.2) in EGB105. Those sequences were then obtained by searches using BLAST (blastin) (https://blast.ncbi.nlm.nih.gov/Blast.cgi ). Genomic locations of the scpp genes were extracted from publicly available annotations in EGB105. Specific primers were designed using Primer3Plus (https://primer3plus.com ), and the primer sequences are listed in Table 1 . Brain, liver, fin ray bone, and pharyngeal tissues (teeth and bone) were dissected from the medaka, and total RNA was extracted from each tissue using TRI REAGENT (Molecular Research Center Inc., Cincinnati, OH, USA). Total RNA from the fin ray bone was extracted after dissecting the fin ray from the body without removing the epithelium. cDNAs were generated using a 1st Strand cDNA Synthesis kit for RT-PCR® (Roche, Basel, Switzerland).
Specific primers for scpp genes and mineralized tissue-related genes used in qPCR and ISH analyses
| Gene | Primer sequences (5' → 3') | Size | Primer sequences (5' → 3') | Size |
|---|---|---|---|---|
| qPCR | ISH | |||
| scpp1 | F: GCCAGCAAGAGTTCAAAGGA | 139 bp | F: CAGACAACATCGTGAGACTTCTTC | 396 bp |
| R: CATCTCCTACGCTCTGGACAA | R: GAAAACACCCTTACCCAACTCTG | |||
| scpp2 | F: TGCCAGCAATAGCAATGAGA | 148 bp | F: CCTTCTGTTGATCTGCCTTTTC | 340 bp |
| R: CTGGGGGTTTAGATTCAGCA | R: CTGAGGGATGTTGGGATTATTG | |||
| scpp5 | F: CATGCCTCAGCAAACCATAAA | 149 bp | F: GTGTTTTGTTTCTGAGGGGACA | 443 bp |
| R: GGCAGATAATGACGGAGGAG | R: GTTTGCTGAGGCATGTTTTGAG | |||
| sparc | F: TGAGGGACTGGCTGAAGAAC | 150 bp | F: AGCAGAAGCTCAGGGTAAAGAAG | 352 bp |
| R: CCAACAGGTCCAAAGAGTGG | R: GTCAGAGGAGTTTAGATGACGAGA | |||
| spp1 | F: CACTGACTTTCTGGAGGAGGA | 84 bp | F: AAGAAGACGAAGACGAAACCAC | 252 bp |
| R: TGGTCTTGAGATACGCTGGA | R: AGGGAGAGGGAACTTTGTGATAG | |||
| col1a1 | F: CAAGAACAGCGTTGCCTACA | 95 bp | F: TGAAGTGGTGTGTGAAGAAGTG | 284 bp |
| R: CCTCGGCTCTGATCTCAATC | R: GCATAGCTGGAGATTTGTCATC | |||
| bglap | F: TGAAATGGCTGACACTGAGG | 52 bp | F: CTACTGCTTCTGCCTCATCATC | 215 bp |
| R: TCCGTAGTAGGCGGTGTATG | R: AGTGTCAGCCATTTCATCACAG | |||
| runx2 | F: GGAAAGGATGCGAGTGAGAG | 105 bp | F: GCACACCCTATCTCTACTACGG | 265 bp |
| R: TCTGTGATCTGCGTCTGACC | R: CTCCACCCACTTTTCCTCTAA | |||
| sp7 | F: GCTCACGGTTTAAGGAGGTG | 73 bp | F: CGCATCTATTCTGGAGGTAGGAAG | 351 bp |
| R: AATCAGGGATGGAGGGAAAC | R: GAGTGGGAGAAGGGGTTATATTCTG | |||
| β-actin | F: GCCAACAGGGAGAAGATGAC | 133 bp | ||
| R: CATCACCAGAGTCCATGACG |
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