We identified Olfml2b as a candidate downstream target of the tendon transcription factor Scleraxis (Scx). Olfml2b is expressed in mouse embryonic tenocytes, colocalizes with a tendon marker Mkx. Luciferase assay identified a Scx-responsive element approximately 5 kb upstream of Olfml2b transcription start site. To investigate its function, we generated an Olfml2b knockout mouse. Despite its tendon-specific expression, loss of Olfml2b did not affect embryonic tendon formation or the gross morphology of adult tendons. These findings indicate that Olfml2b is a possible Scx-regulated tendon gene but is dispensable for tendon development. Further study is required on its physiological role.
","acknowledgements":"We thank Dr. Hiroshi Asahara for kindly providing us Mkx-Ven reporter mouse. We thank Nobuhiro Yoshizawa for his technical contributions.
","authors":[{"affiliations":["Meiji University, Japan"],"departments":["School of Agriculgure"],"credit":["investigation"],"email":"ef231628@meiji.ac.jp","firstName":"Honoka","lastName":"Inoue","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Meiji University, Japan"],"departments":["School of Agriculture"],"credit":["investigation"],"email":"nanami.harry@icloud.com","firstName":"Nanami","lastName":"Ogawa","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Meiji University, Japan"],"departments":["School of Agriculture"],"credit":["investigation"],"email":"cf260415@meiji.ac.jp","firstName":"Rio","lastName":"Nakajima","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Meiji University, Japan"],"departments":["School of Agriculture"],"credit":["investigation"],"email":"arakawa_k@meiji.ac.jp","firstName":"Kohei","lastName":"Arakawa","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3454-2970"},{"affiliations":["Meiji University, Japan"],"departments":["School of Agriculture",""],"credit":["conceptualization","fundingAcquisition","supervision","writing_originalDraft","writing_reviewEditing"],"email":"inui_m@meiji.ac.jp","firstName":"Masafumi","lastName":"Inui","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-4720-007X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This Research is supported by MEXT KAKENHI (19K06697, 22H02636, 24K22257, 26K02842), for M.I., and TERUMO LIFE SCIENCE Foundation for M.I.
","image":{"url":"https://portal.micropublication.org/uploads/53aeee2e4b8de48f3ead5953eeb51ec0.png"},"imageCaption":"(A) Whole mount in situ hybridization of Olfml2b mRNA (top row) and Tnmd mRNA (bottom row) in E14.5 embryo (n=3). Scale bar: 1 mm. (B) Immunofluorescence of OLFML2B (top row) and Venus (Mkx-Ven) (bottom row) in forelimb section of E15.5 Mkx-Ven embryo (n=3). Scalebar: 250 um (C) Schematic drawing of Olfml2b gene and regulatory region candidates. Black boxes indicate Olfml2b exons. Blue boxes with numbers show the position of regulatory region candidates. Red arrowhead indicates the transcription start site of Olfml2b gene. (D) Left panel, a luciferase assay with pNL1.1-TK (Empty Vec) and pNL1.1-TK-candidate regions. Data is shown with mean + SD and a one-way ANOVA followed by Dunnett post hoc test was used for the statistical analysis. *p < 0.05. Right panel, a luciferase assay with pNL1.1-TK (Empty Vec), pNL1.1-TK-region2 vector with or without the overexpression of Scx and E47. Data is shown with mean + SD and a one-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. ***p < 0.001 (E) The deduced amino acid sequence (upper letters) from wildtype (WT) and mutant (MUT) DNA sequence (lower letters) around 1 base insertion (red letter) in Olfml2b mutant allele. Wildtype (WT) sequences are shown at the top and mutant (MUT) sequences are shown at the bottom. Red letters in DNA sequence and amino acid sequence indicate the inserted base and altered amino acids, respectively. (F) Western blotting of OLFML2B and GAPDH proteins in wildtype (WT) and Olfml2b KO (KO) embryonic limb (n=2). Red arrowhead and asterisk indicate Olfml2b protein and non-specific band, respectively. (G) Whole mount in situ hybridization of Tnmd mRNA in Wildtype (top row) and Olfml2b KO (bottom row) in E14.5 embryo (n=3). Scale bar: 1 mm. (H-I) H&E staining of posterior cruciate ligament (H) and Achilles’ tendon (I) of 11-weeks-old wildtype mice (top panels) and Olfml2b KO mice (bottom panels) cell density (cells/µm2) within tendon tissue are shown at right (n=2). Scale bar 50 µm.","imageTitle":"Olfml2b is expressed in embryonic tendon as possible direct target of Scx transcription factor, but is not essential for tendon formation","methods":"Methods
Animals
C57BL/6 mice, ICR mice, and Wister rats were purchased from Sankyo lab-service (Tokyo, Japan). MkxVen mouse (Ito et al., 2010) is kindly provided by Dr. Asahara. All animal experiments were approved by the animal experiment committees of Meiji University (approval No. MUIACUC2022-04).
Generation of Olfml2b knockout mouse
Olfml2b KO mouse was generated by genome editing (Hashimoto and Takemoto, 2015). Briefly, a crRNA targeting exon3 of the Olfml2b gene (5’- TCGGTTACCACTAAACTCGT -3’), tracrRNA, and S.p.Cas9 protein were purchased from IDT (Coralville, Iowa, USA). The fertilized eggs of C57BL/6 mice purchased from Ark-Resource (Kumamoto, Japan) were mixed with crRNA/tracrRNA/Cas9 protein and electroporation was performed using CUY21EDITⅡ electroporator (BEX, Tokyo, Japan). The electroporation conditions were 30 V (3 msec ON + 97 msec OFF) x 7 times. The surviving 2-cell-stage embryos were transferred to the oviducts of pseudo-pregnant ICR female mice. Genome sequencing of one F1 offspring revealed a 1-bp insertion in exon 3, predicted to cause a frameshift mutation.
Primary tenocyte culture
Primary tenocytes were isolated from 3-weeks-old male Wister rat and cultured as described previously (Tsuchiya et al., 2025).
Luciferase assay
Genomic sequences from 6 regions neighboring Olfml2b gene were PCR amplified and cloned into pNL1.1 vector (Promega, Wisconsin, USA) together with thymidine kinase (TK) promoter. Primer sequences used to the cloning are listed as below.
Region 1 fw: 5'-CAAATAGGACCCGTGAGAGC-3', rev: 5'-TGGAGCTCGGATTACAGAGG-3'
Region 2 fw: 5'-TTTTCCCGGTCCCTAGTCTC-3', rev: 5'-AGCCTGTTGATTCCATGTCC-3',
Region 3 fw: 5'-GGATGTGAACCCTCAGGAAA-3', rev: 5'-GTGGTGGGTGCTCAGAAGTT-3'
Region 4 fw: 5'-CCGAGGGTCAGTGATGTTTT-3', rev: 5'-CAAATGTTGCTCCCAAGTCA-3'
Region 5 fw: 5'-GCAACCTCCTCCTGGTTGTA-3', rev: 5'-TGGTCTAGCAGTGCAGGATG-3'
Region 6 fw: 5'-CCGATTCCACAGATGATAAGG-3', rev: 5'-TCCAAGATTCCTTTGCGTTC-3'
For overexpression plasmid, mouse Scleraxis coding sequence was PCR amplified and cloned into pcDNA3.1. Expression vector for mE47 was purchased from Addgene (ID34585) (Lee et al., 2011). Tenocytes were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) and extracted with lysis buffer (50 mM HEPES pH 7.8, 200 mM NaCl, 5 mM EDTA, 1%NP40, 5% Glycerol, 1 mM DTT) after 48 hours. Luciferase activities were detected using Nano-Glo Dual-Luciferase Reporter Assay (Promega) as described previously (Saotome et al., 2020). pGL4.53 (Promega) was used as internal control.
Immunofluorescence
Immunofluorescence were performed essentially as described before (Ono et al., 2023; Yamasaki et al., 2026). Briefly, mouse embryos were fixed with 4% paraformaldehyde (PFA) overnight at 4 °C, embedded in optimal cutting temperature (OCT) compound (Sakurafinetek, Tokyo, Japan) and sectioned at 10 µm. Sections were blocked with 10% fetal bovine serum (FBS)/Phosphate buffer saline (PBS) for 1 hour at room temperature and incubated with primary antibodies overnight at 4 °C. The sections were washed with PBST (PBS supplemented with 0.1 % Tween 20), incubated with secondary antibodies for 1 hour at room temperature, washed with PBST and mounted with fluoroshield mounting medium (Immunochemistry Technologies, Davis, CA, USA). α-GFP (Abcam, Cambridge, UK, ab13970, 1:1000), α-olfml2b (Merck HPA054136) were used as primary antibodies.
Whole-mount in situ hybridization (WISH)
WISH analysis was performed as described before (Sagasaki et al., 2026; Tanji et al., 2023). Olfml2b probe sequence were amplified from embryonic limb cDNA using primers below (fw 5’-cagtggagtggacttgctga-3’, rev 5’-cataacgctgcttcaggtca-3’) and cloned into pGEM T-easy vector (Promega). Tnmd probe was described previously (Shukunami et al., 2001).
Histology
Hindlimbs of 11-week-old mice were fixed with 4% PFA overnight at 4 °C, decalcified with 0.5 M EDTA solution, embedded with paraffin wax. Samples were sectioned at 6 µm and hematoxylin and Eosin (H&E) staining was performed as described before (Fukunaga et al., 2021). The cell density of the tendon/ligament tissue were calculated by counting hematoxylin positive nuclei within H&E image and normalized to the tendon area using Image J. Two individuals for each genotype were examined and both data were shown in the panel.
Western blotting
Western blotting was performed as described before (Inui et al., 2018; Saotome et al., 2020). α-Olfml2b (Merck, HPA054136, 1:500) and α-GAPDH (Millipore, MAB374, 1:2000) were used as primary antibodies and α-rabbit IgG-Peroxidase (SIGMA-Aldrich, A0545, 1:2000) and α-mouse IgG-Peroxidase (SIGMA-Aldrich, A2304, 1:2000) were used as secondary antibodies.
","reagents":"","patternDescription":"Tendon is a dense connective tissue that transmits the muscle contractile force to the bones. Scleraxis (Scx) is one of the essential transcription factors for tendon formation and maintenance (Brent et al., 2003; Huang et al., 2015; Liu et al., 2021; Schweitzer et al., 2001; Shukunami et al., 2018; Yoshimoto et al., 2017), however its direct target gene as well as their roles in tendon biology is insufficiently understood (Liu et al., 2021). Previously, we have identified genes that are downregulated in the embryo where Scx-lineage cells were ablated, and thus are possible Scx downstream targets (Ono et al., 2023). Among them, we found that Olfactomedin Like 2B (Olfml2b) gene is expressed in embryonic limb and tail at E14.5, in domains similar to those of Tnmd, a well-known Scx direct target gene (Fig. 1A). Immunofluorescence analysis in embryonic fore limb sections confirmed that OLFML2B protein colocalized with Mkx-Venus (MkxVen) tendon reporter (Ito 2010) (Fig. 1B). Specifically, OLFML2B signals were detected in the flexor and extensor tendons that are positive for MkxVen (Fig. 1B arrowheads). These results indicate that Olfml2b is expressed in embryonic tenocytes. Recent ChIP-seq study identified several Scx binding peaks near Olfml2b gene (Liu et al., 2021). We have examined the transcriptional regulatory activity of sequences around these Scx binding peaks by luciferase assay using primary rat tenocyte (Fig. 1C), and found that 800 bp sequence at 5 kbp upstream of Olfml2b transcription start site (region 2) has positive transcriptional activity (Fig. 1D). Moreover, overexpression of Scx together with E47 increased the luciferase activity, indicating that this sequence could respond to Scx (Fig. 1D). As Olfml2b is expressed in tenocyte as possible target of Scx transcription factor, we hypothesize that this gene could play some role in tendon formation and homeostasis. In line with this idea, OLFML2B mutation was identified in a family with twin sibling males diagnosed as an anterior cruciate ligament non-contact rupture (Caso et al., 2016). To elucidate the role of Olfml2b in tendon tissue, we have generated Olfml2b knockout (KO) mouse with genome editing technique. Established Olfml2b knockout mouse line has 1 bp insertion mutation in 3rd exon of Olfml2b gene that caused frameshift mutation and premature termination at 178th amino acid to OLFML2B protein (Fig. 1E). Western blotting analysis confirmed that OLFML2B protein in embryonic limb extract of Olfml2b KO mouse (Fig. 1F). Tnmd expression of Olfml2b KO embryo at E14.5 was similar to that of wild-type embryo, indicating that Olfml2b is not essential for the embryonic tendon formation (Fig. 1G). Histological examination of the Achilles’ tendon and cruciate ligaments in 11-weeks-old Olfml2b KO mice revealed that the gross morphology as well as cell density of these tissue are undistinguishable from those of wild-type mice (Fig. 1H). Altogether, our results indicated that Olfml2b is expressed in embryonic tendon cell but is not essential for the tendon/ligament tissue formation. We have identified possible regulatory sequence at ~5 kb upstream of Olfml2b gene. Further evaluation such as the narrowing down the crucial sequence and electrophoresis mobility shift assay (EMSA), would elucidate whether it contains direct regulatory element by Scx transcription factor (Shukunami et al., 2018). The role of Olfml2b in tendon tissue remains elusive. Other members of Olfml gene family could have redundant role that needs to be evaluated in future studies.
","references":[{"reference":"Brent AE, Schweitzer R, Tabin CJ. 2003. A somitic compartment of tendon progenitors. Cell 113(2): 235-48.
","pubmedId":"12705871","doi":""},{"reference":"Caso E, Maestro A, Sabiers CC, Godino M, Caracuel Z, Pons J, et al., Guerado E. 2016. Whole-exome sequencing analysis in twin sibling males with an anterior cruciate ligament rupture. Injury 47 Suppl 3: S41-S50.
","pubmedId":"27692106","doi":""},{"reference":"Fukunaga K, Tanji M, Hanzawa N, Kuroda H, Inui M. 2021. Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation. Biochem Biophys Rep 27: 101047.
","pubmedId":"34189280","doi":""},{"reference":"Hashimoto M, Takemoto T. 2015. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci Rep 5: 11315.
","pubmedId":"26066060","doi":""},{"reference":"Huang AH, Lu HH, Schweitzer R. 2015. Molecular regulation of tendon cell fate during development. J Orthop Res 33(6): 800-12.
","pubmedId":"25664867","doi":""},{"reference":"Inui M, Mokuda S, Sato T, Tamano M, Takada S, Asahara H. 2018. Dissecting the roles of miR-140 and its host gene. Nat Cell Biol 20(5): 516-518.
","pubmedId":"29695789","doi":""},{"reference":"Ito Y, Toriuchi N, Yoshitaka T, Ueno-Kudoh H, Sato T, Yokoyama S, et al., Asahara H. 2010. The Mohawk homeobox gene is a critical regulator of tendon differentiation. Proc Natl Acad Sci U S A 107(23): 10538-42.
","pubmedId":"20498044","doi":""},{"reference":"Lee K, Gjorevski N, Boghaert E, Radisky DC, Nelson CM. 2011. Snail1, Snail2, and E47 promote mammary epithelial branching morphogenesis. EMBO J 30(13): 2662-74.
","pubmedId":"21610693","doi":""},{"reference":"Liu H, Xu J, Lan Y, Lim HW, Jiang R. 2021. The Scleraxis Transcription Factor Directly Regulates Multiple Distinct Molecular and Cellular Processes During Early Tendon Cell Differentiation. Front Cell Dev Biol 9: 654397.
","pubmedId":"34150754","doi":""},{"reference":"Ono Y, Schlesinger S, Fukunaga K, Yambe S, Sato T, Sasaki T, et al., Inui M. 2023. Scleraxis-lineage cells are required for correct muscle patterning. Development 150(10): pii: dev201101. 10.1242/dev.201101.
","pubmedId":"37246520","doi":""},{"reference":"Sagasaki R, Taniguchi A, Oka S, Mishima M, Yamasaki Y, Kawa Y, et al., Inui M. 2026. Three-dimensional observation of the muscle-tendon integration process in mouse embryos. Dev Dyn: 10.1002/dvdy.70167.
","pubmedId":"42383507","doi":""},{"reference":"Saotome H, Ito A, Kubo A, Inui M. 2020. Generation of a Quantitative Luciferase Reporter for Sox9 SUMOylation. Int J Mol Sci 21(4): 10.3390/ijms21041274.
","pubmedId":"32070068","doi":""},{"reference":"Schweitzer R, Chyung JH, Murtaugh LC, Brent AE, Rosen V, Olson EN, Lassar A, Tabin CJ. 2001. Analysis of the tendon cell fate using Scleraxis, a specific marker for tendons and ligaments. Development 128(19): 3855-66.
","pubmedId":"11585810","doi":""},{"reference":"Shukunami C, Oshima Y, Hiraki Y. 2001. Molecular cloning of tenomodulin, a novel chondromodulin-I related gene. Biochem Biophys Res Commun 280(5): 1323-7.
","pubmedId":"11162673","doi":""},{"reference":"Shukunami C, Takimoto A, Nishizaki Y, Yoshimoto Y, Tanaka S, Miura S, et al., Hiraki Y. 2018. Scleraxis is a transcriptional activator that regulates the expression of Tenomodulin, a marker of mature tenocytes and ligamentocytes. Sci Rep 8(1): 3155.
","pubmedId":"29453333","doi":""},{"reference":"Tanji M, Wada K, Sakamoto K, Ono Y, Inui M. 2023. Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization. Dev Growth Differ 65(1): 48-55.
","pubmedId":"36517457","doi":""},{"reference":"Tsuchiya Y, Matsuo H, Asahara H, Inui M. 2025. Matrix stiffness regulates Mkx expression in rat tenocyte through TRPM7. Biochem Biophys Rep 43: 102178.
","pubmedId":"40740470","doi":""},{"reference":"Yamasaki Y, Sakamoto K, Yashiro S, Kawa Y, Kondow A, Kubo A, Hitachi K, Inui M. 2026. SMAD2 ubiquitination through PY motif regulates skeletal muscle mass and fibrotic degeneration. Sci Rep 16(1): 6666.
","pubmedId":"41617874","doi":""},{"reference":"Yoshimoto Y, Takimoto A, Watanabe H, Hiraki Y, Kondoh G, Shukunami C. 2017. Scleraxis is required for maturation of tissue domains for proper integration of the musculoskeletal system. Sci Rep 7: 45010.
","pubmedId":"28327634","doi":""}],"title":"Olfml2b is expressed in embryonic tenocyte, but not essential for tendon formation
","reviews":[{"reviewer":{"displayName":"Yayoi Izu"},"openAcknowledgement":false,"status":{"submitted":true}},{"reviewer":{"displayName":"Yayoi Izu"},"openAcknowledgement":null,"status":{"submitted":false}}],"curatorReviews":[]},{"id":"2bb32033-8754-4690-8d95-ea126d591852","decision":"revise","abstract":"We identified Olfml2b as a candidate downstream target of the tendon transcription factor Scleraxis (Scx). Olfml2b is expressed in mouse embryonic tenocytes, colocalizes with a tendon marker Mkx. Luciferase assay identified a Scx-responsive element approximately 5 kb upstream of Olfml2b transcription start site. To investigate its function, we generated an Olfml2b knockout mouse. Despite its tendon-specific expression, loss of Olfml2b did not affect embryonic tendon formation or the gross morphology of adult tendons. These findings indicate that Olfml2b is a possible Scx-regulated tendon gene but is dispensable for tendon development. Further study is required on its physiological role.
","acknowledgements":"We thank Dr. Hiroshi Asahara for kindly providing us Mkx-Ven reporter mouse. We thank Nobuhiro Yoshizawa and Atsumi Sawada for their technical contributions.
","authors":[{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University"],"departments":[""],"credit":["investigation"],"email":"ef231628@meiji.ac.jp","firstName":"Honoka","lastName":"Inoue","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University"],"departments":[""],"credit":["investigation"],"email":"nanami.harry@icloud.com","firstName":"Nanami","lastName":"Ogawa","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University"],"departments":[""],"credit":["investigation"],"email":"cf260415@meiji.ac.jp","firstName":"Rio","lastName":"Nakajima","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University"],"departments":[""],"credit":["investigation"],"email":"arakawa_k@meiji.ac.jp","firstName":"Kohei","lastName":"Arakawa","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3454-2970"},{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University"],"departments":["",""],"credit":["conceptualization","fundingAcquisition","supervision","writing_originalDraft","writing_reviewEditing"],"email":"inui_m@meiji.ac.jp","firstName":"Masafumi","lastName":"Inui","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-4720-007X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This Research is supported by MEXT KAKENHI (19K06697, 22H02636, 24K22257, 26K02842), for M.I., and TERUMO LIFE SCIENCE Foundation for M.I.
","image":{"url":"https://portal.micropublication.org/uploads/660abcdab85de7d578de1b321f8ad765.png"},"imageCaption":"(A) Whole mount in situ hybridization of Olfml2b mRNA (top row) and Tnmd mRNA (bottom row) in E14.5 embryo (n=3). Scale bar: 1 mm. (B) Immunofluorescence of OLFML2B (top row) and Venus (Mkx-Ven) (bottom row) in forelimb section of E15.5 Mkx-Ven embryo (n=3). Scalebar: 250 um (C) Schematic drawing of Olfml2b gene and regulatory region candidates. Black boxes indicate Olfml2b exons. Blue boxes with numbers show the position of regulatory region candidates. Red arrowhead indicates the transcription start site of Olfml2b gene. (D) Left panel, a luciferase assay with pNL1.1-TK (Empty Vec) and pNL1.1-TK-candidate regions. Data is shown with mean + SD and a one-way ANOVA followed by Dunnett post hoc test was used for the statistical analysis. *p < 0.05. Right panel, a luciferase assay with pNL1.1-TK (Empty Vec), pNL1.1-TK-region2 vector with or without the overexpression of Scx and E47. Data is shown with mean + SD and a one-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. ***p < 0.001 (E) The deduced amino acid sequence (upper letters) from wildtype (WT) and mutant (MUT) DNA sequence (lower letters) around 1 base insertion (red letter) in Olfml2b mutant allele. Wildtype (WT) sequences are shown at the top and mutant (MUT) sequences are shown at the bottom. Red letters in DNA sequence and amino acid sequence indicate the inserted base and altered amino acids, respectively. (F) Western blotting of OLFML2B and GAPDH proteins in wildtype (WT) and Olfml2b KO (KO) embryonic limb (n=2). Red arrowhead and asterisk indicate Olfml2b protein and non-specific band, respectively. (G) Whole mount in situ hybridization of Tnmd mRNA in Wildtype (top row) and Olfml2b KO (bottom row) in E14.5 embryo (n=3). Scale bar: 1 mm. (H-I) H&E staining of posterior cruciate ligament (H) and Achilles tendon (I) of 11-weeks-old wildtype mice (top panels) and Olfml2b KO mice (bottom panels) cell density (cells/µm2) within tendon tissue are shown at right (n=2). Scale bar 50 µm.
","imageTitle":"Olfml2b is expressed in embryonic tendon as possible direct target of Scx transcription factor
","methods":"Methods
Animals
C57BL/6 mice, ICR mice, and Wistar rats were purchased from Sankyo lab-service (Tokyo, Japan). MkxVen mouse (Ito et al., 2010) is kindly provided by Dr. Asahara. All animal experiments were approved by the animal experiment committees of Meiji University (approval No. MUIACUC2022-04).
Generation of Olfml2b knockout mouse
Olfml2b KO mouse was generated by genome editing (Hashimoto and Takemoto, 2015). Briefly, a crRNA targeting exon3 of the Olfml2b gene (5’- TCGGTTACCACTAAACTCGT -3’), tracrRNA, and S.p.Cas9 protein were purchased from IDT (Coralville, Iowa, USA). The fertilized eggs of C57BL/6 mice purchased from Ark-Resource (Kumamoto, Japan) were mixed with crRNA/tracrRNA/Cas9 protein and electroporation was performed using CUY21EDITⅡ electroporator (BEX, Tokyo, Japan). The electroporation conditions were 30 V (3 msec ON + 97 msec OFF) x 7 times. The surviving 2-cell-stage embryos were transferred to the oviducts of pseudo-pregnant ICR female mice. Genome sequencing of one F1 offspring revealed a 1-bp insertion in exon 3, predicted to cause a frameshift mutation.
Primary tenocyte culture
Primary tenocytes were isolated from 3-weeks-old male Wistar rat and cultured as described previously (Tsuchiya et al., 2025).
Luciferase assay
Genomic sequences from 6 regions neighboring Olfml2b gene were PCR amplified and cloned into pNL1.1 vector (Promega, Wisconsin, USA) together with thymidine kinase (TK) promoter. Primer sequences used to the cloning are listed as below.
Region 1 fw: 5'-CAAATAGGACCCGTGAGAGC-3', rev: 5'-TGGAGCTCGGATTACAGAGG-3'
Region 2 fw: 5'-TTTTCCCGGTCCCTAGTCTC-3', rev: 5'-AGCCTGTTGATTCCATGTCC-3',
Region 3 fw: 5'-GGATGTGAACCCTCAGGAAA-3', rev: 5'-GTGGTGGGTGCTCAGAAGTT-3'
Region 4 fw: 5'-CCGAGGGTCAGTGATGTTTT-3', rev: 5'-CAAATGTTGCTCCCAAGTCA-3'
Region 5 fw: 5'-GCAACCTCCTCCTGGTTGTA-3', rev: 5'-TGGTCTAGCAGTGCAGGATG-3'
Region 6 fw: 5'-CCGATTCCACAGATGATAAGG-3', rev: 5'-TCCAAGATTCCTTTGCGTTC-3'
For overexpression plasmid, mouse Scleraxis coding sequence was PCR amplified and cloned into pcDNA3.1. Expression vector for mE47 was purchased from Addgene (ID34585) (Lee et al., 2011). Tenocytes were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) and extracted with lysis buffer (50 mM HEPES pH 7.8, 200 mM NaCl, 5 mM EDTA, 1%NP40, 5% Glycerol, 1 mM DTT) after 48 hours. Luciferase activities were detected using Nano-Glo Dual-Luciferase Reporter Assay (Promega) as described previously (Saotome et al., 2020). pGL4.53 (Promega) was used as internal control.
Immunofluorescence
Immunofluorescence were performed essentially as described before (Ono et al., 2023; Yamasaki et al., 2026). Briefly, mouse embryos were fixed with 4% paraformaldehyde (PFA) overnight at 4 °C, embedded in optimal cutting temperature (OCT) compound (Sakurafinetek, Tokyo, Japan) and sectioned at 10 µm. Sections were blocked with 10% fetal bovine serum (FBS)/Phosphate buffer saline (PBS) for 1 hour at room temperature and incubated with primary antibodies overnight at 4 °C. The sections were washed with PBST (PBS supplemented with 0.1 % Tween 20), incubated with secondary antibodies for 1 hour at room temperature, washed with PBST and mounted with fluoroshield mounting medium (Immunochemistry Technologies, Davis, CA, USA). α-GFP (Abcam, Cambridge, UK, ab13970, 1:1000), α-olfml2b (Merck HPA054136) were used as primary antibodies.
Whole-mount in situ hybridization (WISH)
WISH analysis was performed as described before (Sagasaki et al., 2026; Tanji et al., 2023). Olfml2b probe sequence were amplified from embryonic limb cDNA using primers below (fw 5’-cagtggagtggacttgctga-3’, rev 5’-cataacgctgcttcaggtca-3’) and cloned into pGEM T-easy vector (Promega). Tnmd probe was described previously (Shukunami et al., 2001).
Histology
Hindlimbs of 11-week-old mice were fixed with 4% PFA overnight at 4 °C, decalcified with 0.5 M EDTA solution, embedded with paraffin wax. Samples were sectioned at 6 µm and hematoxylin and Eosin (H&E) staining was performed as described before (Fukunaga et al., 2021). The cell density of the tendon/ligament tissue were calculated by counting hematoxylin positive nuclei within H&E image and normalized to the tendon area using Image J. Two individuals for each genotype were examined and both data were shown in the panel.
Western blotting
Western blotting was performed as described before (Inui et al., 2018; Saotome et al., 2020). α-Olfml2b (Merck, HPA054136, 1:500) and α-GAPDH (Millipore, MAB374, 1:2000) were used as primary antibodies and α-rabbit IgG-Peroxidase (SIGMA-Aldrich, A0545, 1:2000) and α-mouse IgG-Peroxidase (SIGMA-Aldrich, A2304, 1:2000) were used as secondary antibodies.
","reagents":"","patternDescription":"Tendon is a dense connective tissue that transmits the muscle contractile force to the bones. Scleraxis (Scx) is one of the essential transcription factors for tendon formation and maintenance (Brent et al., 2003; Huang et al., 2015; Liu et al., 2021; Schweitzer et al., 2001; Shukunami et al., 2018; Yoshimoto et al., 2017), however its direct target gene as well as their roles in tendon biology is insufficiently understood (Liu et al., 2021). Previously, we have identified genes that are downregulated in the embryo where Scx-lineage cells were ablated, and thus are possible Scx downstream targets (Ono et al., 2023). Among them, we found that Olfactomedin Like 2B (Olfml2b) gene is expressed in embryonic limb and tail at E14.5, in domains similar to those of Tnmd, a well-known Scx direct target gene (Fig. 1A). Immunofluorescence analysis in embryonic fore limb sections confirmed that OLFML2B protein colocalized with Mkx-Venus (MkxVen) tendon reporter (Ito et al., 2010) (Fig. 1B). Specifically, OLFML2B signals were detected in the flexor and extensor tendons that are positive for MkxVen (Fig. 1B arrowheads). These results indicate that Olfml2b is expressed in embryonic tenocytes. Because Olfml2b encodes a secreted extracellular matrix (ECM) associated protein (Furutani et al., 2005), we hypothesized that it could contribute to the development and function of tendon tissue which is rich in ECMs.
Recent ChIP-seq study identified several Scx binding peaks near Olfml2b gene (Liu et al., 2021). We have examined the transcriptional regulatory activity of sequences around these Scx binding peaks by luciferase assay using primary rat tenocyte (Fig. 1C), and found that 800 bp sequence at 5 kbp upstream of Olfml2b transcription start site (region 2) has positive transcriptional activity (Fig. 1D). Moreover, overexpression of Scx together with E47 increased the luciferase activity, indicating that this sequence could respond to Scx (Fig. 1D).
As Olfml2b is expressed in tenocyte as possible target of Scx transcription factor, we hypothesize that this gene could play some role in tendon formation and homeostasis. In line with this idea, OLFML2B mutation was identified in a family with twin sibling males diagnosed as an anterior cruciate ligament non-contact rupture (Caso et al., 2016). To elucidate the role of Olfml2b in tendon tissue, we have generated Olfml2b knockout (KO) mouse with genome editing technique. Established Olfml2b knockout mouse line has 1 bp insertion mutation in 3rd exon of Olfml2b gene that caused frameshift mutation and premature termination at 178th amino acid to OLFML2B protein (Fig. 1E). Western blotting analysis confirmed that OLFML2B protein is absent in embryonic limb extract of Olfml2b KO mouse (Fig. 1F). Tnmd expression of Olfml2b KO embryo at E14.5 was similar to that of wildtype embryo, indicating that Olfml2b is not essential for the embryonic tendon formation (Fig. 1G). Histological examination of the Achilles tendon and cruciate ligaments in 11-weeks-old Olfml2b KO mice revealed that the gross morphology undistinguishable from those of wildtype mice (Fig. 1H, I). Cell density of cruciate ligaments, but not of Achilles tendon, was slightly lower in Olfml2b KO mice (Fig. 1H, I). Further analysis with larger numbers of mice could Altogether, our results indicated that Olfml2b is expressed in embryonic tendon cell but is not essential for the tendon/ligament tissue formation. We have identified possible regulatory sequence at ~5 kb upstream of Olfml2b gene. Further evaluation such as the narrowing down the crucial sequence and electrophoresis mobility shift assay (EMSA), would elucidate whether it contains direct regulatory element by Scx transcription factor (Shukunami et al., 2018). The role of Olfml2b in tendon tissue remains elusive. Considering the ECM associating nature of Olfml2b, it could be involved in collagen fiber organization in tendon tissue. Given the trend toward reduced cell density in Olfml2b KO cruciate ligament, further analyses with increased sample number and extending to other tendon/ligament tissues may help elucidate the function of Olfml2b. Other members of Olfml gene family could have redundant role that needs to be evaluated in future studies.
","references":[{"reference":"Brent AE, Schweitzer R, Tabin CJ. 2003. A somitic compartment of tendon progenitors. Cell 113(2): 235-48.
","pubmedId":"12705871","doi":""},{"reference":"Caso E, Maestro A, Sabiers CC, Godino M, Caracuel Z, Pons J, et al., Guerado E. 2016. Whole-exome sequencing analysis in twin sibling males with an anterior cruciate ligament rupture. Injury 47 Suppl 3: S41-S50.
","pubmedId":"27692106","doi":""},{"reference":"Fukunaga K, Tanji M, Hanzawa N, Kuroda H, Inui M. 2021. Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation. Biochem Biophys Rep 27: 101047.
","pubmedId":"34189280","doi":""},{"reference":"Furutani Y, Manabe R, Tsutsui K, Yamada T, Sugimoto N, Fukuda S, et al., Sekiguchi K. 2005. Identification and characterization of photomedins: novel olfactomedin-domain-containing proteins with chondroitin sulphate-E-binding activity. Biochem J 389(Pt 3): 675-84.
","pubmedId":"15836428","doi":""},{"reference":"Hashimoto M, Takemoto T. 2015. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci Rep 5: 11315.
","pubmedId":"26066060","doi":""},{"reference":"Huang AH, Lu HH, Schweitzer R. 2015. Molecular regulation of tendon cell fate during development. J Orthop Res 33(6): 800-12.
","pubmedId":"25664867","doi":""},{"reference":"Inui M, Mokuda S, Sato T, Tamano M, Takada S, Asahara H. 2018. Dissecting the roles of miR-140 and its host gene. Nat Cell Biol 20(5): 516-518.
","pubmedId":"29695789","doi":""},{"reference":"Ito Y, Toriuchi N, Yoshitaka T, Ueno-Kudoh H, Sato T, Yokoyama S, et al., Asahara H. 2010. The Mohawk homeobox gene is a critical regulator of tendon differentiation. Proc Natl Acad Sci U S A 107(23): 10538-42.
","pubmedId":"20498044","doi":""},{"reference":"Lee K, Gjorevski N, Boghaert E, Radisky DC, Nelson CM. 2011. Snail1, Snail2, and E47 promote mammary epithelial branching morphogenesis. EMBO J 30(13): 2662-74.
","pubmedId":"21610693","doi":""},{"reference":"Liu H, Xu J, Lan Y, Lim HW, Jiang R. 2021. The Scleraxis Transcription Factor Directly Regulates Multiple Distinct Molecular and Cellular Processes During Early Tendon Cell Differentiation. Front Cell Dev Biol 9: 654397.
","pubmedId":"34150754","doi":""},{"reference":"Ono Y, Schlesinger S, Fukunaga K, Yambe S, Sato T, Sasaki T, et al., Inui M. 2023. Scleraxis-lineage cells are required for correct muscle patterning. Development 150(10): pii: dev201101. 10.1242/dev.201101.
","pubmedId":"37246520","doi":""},{"reference":"Sagasaki R, Taniguchi A, Oka S, Mishima M, Yamasaki Y, Kawa Y, et al., Inui M. 2026. Three-dimensional observation of the muscle-tendon integration process in mouse embryos. Dev Dyn: 10.1002/dvdy.70167.
","pubmedId":"42383507","doi":""},{"reference":"Saotome H, Ito A, Kubo A, Inui M. 2020. Generation of a Quantitative Luciferase Reporter for Sox9 SUMOylation. Int J Mol Sci 21(4): 10.3390/ijms21041274.
","pubmedId":"32070068","doi":""},{"reference":"Schweitzer R, Chyung JH, Murtaugh LC, Brent AE, Rosen V, Olson EN, Lassar A, Tabin CJ. 2001. Analysis of the tendon cell fate using Scleraxis, a specific marker for tendons and ligaments. Development 128(19): 3855-66.
","pubmedId":"11585810","doi":""},{"reference":"Shukunami C, Oshima Y, Hiraki Y. 2001. Molecular cloning of tenomodulin, a novel chondromodulin-I related gene. Biochem Biophys Res Commun 280(5): 1323-7.
","pubmedId":"11162673","doi":""},{"reference":"Shukunami C, Takimoto A, Nishizaki Y, Yoshimoto Y, Tanaka S, Miura S, et al., Hiraki Y. 2018. Scleraxis is a transcriptional activator that regulates the expression of Tenomodulin, a marker of mature tenocytes and ligamentocytes. Sci Rep 8(1): 3155.
","pubmedId":"29453333","doi":""},{"reference":"Tanji M, Wada K, Sakamoto K, Ono Y, Inui M. 2023. Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization. Dev Growth Differ 65(1): 48-55.
","pubmedId":"36517457","doi":""},{"reference":"Tsuchiya Y, Matsuo H, Asahara H, Inui M. 2025. Matrix stiffness regulates Mkx expression in rat tenocyte through TRPM7. Biochem Biophys Rep 43: 102178.
","pubmedId":"40740470","doi":""},{"reference":"Yamasaki Y, Sakamoto K, Yashiro S, Kawa Y, Kondow A, Kubo A, Hitachi K, Inui M. 2026. SMAD2 ubiquitination through PY motif regulates skeletal muscle mass and fibrotic degeneration. Sci Rep 16(1): 6666.
","pubmedId":"41617874","doi":""},{"reference":"Yoshimoto Y, Takimoto A, Watanabe H, Hiraki Y, Kondoh G, Shukunami C. 2017. Scleraxis is required for maturation of tissue domains for proper integration of the musculoskeletal system. Sci Rep 7: 45010.
","pubmedId":"28327634","doi":""}],"title":"Embryonic tenocytes express Olfml2b, a potential direct target of Scleraxis
","reviews":[{"reviewer":{"displayName":"Yayoi Izu"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"9ffdc9b3-0129-45b2-b5a6-59ae5b24dc91","decision":"accept","abstract":"We identified Olfml2b as a candidate downstream target of the tendon transcription factor Scleraxis (Scx). Olfml2b is expressed in mouse embryonic tenocytes, colocalizes with a tendon marker Mkx. Luciferase assay identified a Scx-responsive element approximately 5 kb upstream of Olfml2b transcription start site. To investigate its function, we generated an Olfml2b knockout mouse. Despite its tendon-specific expression, loss of Olfml2b did not affect embryonic tendon formation or the gross morphology of adult tendons. These findings indicate that Olfml2b is a possible Scx-regulated tendon gene but is dispensable for tendon development. Further study is required on its physiological role.
","acknowledgements":"We thank Dr. Hiroshi Asahara for kindly providing us Mkx-Ven reporter mouse. We thank Nobuhiro Yoshizawa and Atsumi Sawada for their technical contributions.
","authors":[{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan"],"departments":[""],"credit":["investigation"],"email":"ef231628@meiji.ac.jp","firstName":"Honoka","lastName":"Inoue","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan"],"departments":[""],"credit":["investigation"],"email":"nanami.harry@icloud.com","firstName":"Nanami","lastName":"Ogawa","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan"],"departments":[""],"credit":["investigation"],"email":"cf260415@meiji.ac.jp","firstName":"Rio","lastName":"Nakajima","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan"],"departments":[""],"credit":["investigation"],"email":"arakawa_k@meiji.ac.jp","firstName":"Kohei","lastName":"Arakawa","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3454-2970"},{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan"],"departments":["",""],"credit":["conceptualization","fundingAcquisition","supervision","writing_originalDraft","writing_reviewEditing"],"email":"inui_m@meiji.ac.jp","firstName":"Masafumi","lastName":"Inui","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-4720-007X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This Research is supported by MEXT KAKENHI (19K06697, 22H02636, 24K22257, 26K02842), for M.I., and TERUMO LIFE SCIENCE Foundation for M.I.
","image":{"url":"https://portal.micropublication.org/uploads/660abcdab85de7d578de1b321f8ad765.png"},"imageCaption":"(A) Whole mount in situ hybridization of Olfml2b mRNA (top row) and Tnmd mRNA (bottom row) in E14.5 embryo (n=3). Scale bar: 1 mm. (B) Immunofluorescence of OLFML2B (top row) and Venus (Mkx-Ven) (bottom row) in forelimb section of E15.5 Mkx-Ven embryo (n=3). Scalebar: 250 um (C) Schematic drawing of Olfml2b gene and regulatory region candidates. Black boxes indicate Olfml2b exons. Blue boxes with numbers show the position of regulatory region candidates. Red arrowhead indicates the transcription start site of Olfml2b gene. (D) Left panel, a luciferase assay with pNL1.1-TK (Empty Vec) and pNL1.1-TK-candidate regions. Data is shown with mean + SD and a one-way ANOVA followed by Dunnett post hoc test was used for the statistical analysis. *p < 0.05. Right panel, a luciferase assay with pNL1.1-TK (Empty Vec), pNL1.1-TK-region2 vector with or without the overexpression of Scx and E47. Data is shown with mean + SD and a one-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. ***p < 0.001 (E) The deduced amino acid sequence (upper letters) from wildtype (WT) and mutant (MUT) DNA sequence (lower letters) around 1 base insertion (red letter) in Olfml2b mutant allele. Wildtype (WT) sequences are shown at the top and mutant (MUT) sequences are shown at the bottom. Red letters in DNA sequence and amino acid sequence indicate the inserted base and altered amino acids, respectively. (F) Western blotting of OLFML2B and GAPDH proteins in wildtype (WT) and Olfml2b KO (KO) embryonic limb (n=2). Red arrowhead and asterisk indicate Olfml2b protein and non-specific band, respectively. (G) Whole mount in situ hybridization of Tnmd mRNA in Wildtype (top row) and Olfml2b KO (bottom row) in E14.5 embryo (n=3). Scale bar: 1 mm. (H-I) H&E staining of posterior cruciate ligament (H) and Achilles tendon (I) of 11-weeks-old wildtype mice (top panels) and Olfml2b KO mice (bottom panels) cell density (cells/µm2) within tendon tissue are shown at right (n=2). Scale bar 50 µm.
","imageTitle":"Olfml2b is expressed in embryonic tendon as possible direct target of Scx transcription factor
","methods":"Animals
C57BL/6 mice, ICR mice, and Wistar rats were purchased from Sankyo lab-service (Tokyo, Japan). MkxVen mouse (Ito et al., 2010) is kindly provided by Dr. Asahara. All animal experiments were approved by the animal experiment committees of Meiji University (approval No. MUIACUC2022-04).
Generation of Olfml2b knockout mouse
Olfml2b KO mouse was generated by genome editing (Hashimoto and Takemoto, 2015). Briefly, a crRNA targeting exon3 of the Olfml2b gene (5’- TCGGTTACCACTAAACTCGT -3’), tracrRNA, and S.p.Cas9 protein were purchased from IDT (Coralville, Iowa, USA). The fertilized eggs of C57BL/6 mice purchased from Ark-Resource (Kumamoto, Japan) were mixed with crRNA/tracrRNA/Cas9 protein and electroporation was performed using CUY21EDITⅡ electroporator (BEX, Tokyo, Japan). The electroporation conditions were 30 V (3 msec ON + 97 msec OFF) x 7 times. The surviving 2-cell-stage embryos were transferred to the oviducts of pseudo-pregnant ICR female mice. Genome sequencing of one F1 offspring revealed a 1-bp insertion in exon 3, predicted to cause a frameshift mutation.
Primary tenocyte culture
Primary tenocytes were isolated from 3-weeks-old male Wistar rat and cultured as described previously (Tsuchiya et al., 2025).
Luciferase assay
Genomic sequences from 6 regions neighboring Olfml2b gene were PCR amplified and cloned into pNL1.1 vector (Promega, Wisconsin, USA) together with thymidine kinase (TK) promoter. Primer sequences used to the cloning are listed as below.
Region 1 fw: 5'-CAAATAGGACCCGTGAGAGC-3', rev: 5'-TGGAGCTCGGATTACAGAGG-3'
Region 2 fw: 5'-TTTTCCCGGTCCCTAGTCTC-3', rev: 5'-AGCCTGTTGATTCCATGTCC-3',
Region 3 fw: 5'-GGATGTGAACCCTCAGGAAA-3', rev: 5'-GTGGTGGGTGCTCAGAAGTT-3'
Region 4 fw: 5'-CCGAGGGTCAGTGATGTTTT-3', rev: 5'-CAAATGTTGCTCCCAAGTCA-3'
Region 5 fw: 5'-GCAACCTCCTCCTGGTTGTA-3', rev: 5'-TGGTCTAGCAGTGCAGGATG-3'
Region 6 fw: 5'-CCGATTCCACAGATGATAAGG-3', rev: 5'-TCCAAGATTCCTTTGCGTTC-3'
For overexpression plasmid, mouse Scleraxis coding sequence was PCR amplified and cloned into pcDNA3.1. Expression vector for mE47 was purchased from Addgene (ID34585) (Lee et al., 2011). Tenocytes were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) and extracted with lysis buffer (50 mM HEPES pH 7.8, 200 mM NaCl, 5 mM EDTA, 1%NP40, 5% Glycerol, 1 mM DTT) after 48 hours. Luciferase activities were detected using Nano-Glo Dual-Luciferase Reporter Assay (Promega) as described previously (Saotome et al., 2020). pGL4.53 (Promega) was used as internal control.
Immunofluorescence
Immunofluorescence were performed essentially as described before (Ono et al., 2023; Yamasaki et al., 2026). Briefly, mouse embryos were fixed with 4% paraformaldehyde (PFA) overnight at 4 °C, embedded in optimal cutting temperature (OCT) compound (Sakurafinetek, Tokyo, Japan) and sectioned at 10 µm. Sections were blocked with 10% fetal bovine serum (FBS)/Phosphate buffer saline (PBS) for 1 hour at room temperature and incubated with primary antibodies overnight at 4 °C. The sections were washed with PBST (PBS supplemented with 0.1 % Tween 20), incubated with secondary antibodies for 1 hour at room temperature, washed with PBST and mounted with fluoroshield mounting medium (Immunochemistry Technologies, Davis, CA, USA). α-GFP (Abcam, Cambridge, UK, ab13970, 1:1000), α-olfml2b (Merck HPA054136) were used as primary antibodies.
Whole-mount in situ hybridization (WISH)
WISH analysis was performed as described before (Sagasaki et al., 2026; Tanji et al., 2023). Olfml2b probe sequence were amplified from embryonic limb cDNA using primers below (fw 5’-cagtggagtggacttgctga-3’, rev 5’-cataacgctgcttcaggtca-3’) and cloned into pGEM T-easy vector (Promega). Tnmd probe was described previously (Shukunami et al., 2001).
Histology
Hindlimbs of 11-week-old mice were fixed with 4% PFA overnight at 4 °C, decalcified with 0.5 M EDTA solution, embedded with paraffin wax. Samples were sectioned at 6 µm and hematoxylin and Eosin (H&E) staining was performed as described before (Fukunaga et al., 2021). The cell density of the tendon/ligament tissue were calculated by counting hematoxylin positive nuclei within H&E image and normalized to the tendon area using Image J. Two individuals for each genotype were examined and both data were shown in the panel.
Western blotting
Western blotting was performed as described before (Inui et al., 2018; Saotome et al., 2020). α-Olfml2b (Merck, HPA054136, 1:500) and α-GAPDH (Millipore, MAB374, 1:2000) were used as primary antibodies and α-rabbit IgG-Peroxidase (SIGMA-Aldrich, A0545, 1:2000) and α-mouse IgG-Peroxidase (SIGMA-Aldrich, A2304, 1:2000) were used as secondary antibodies.
","reagents":"","patternDescription":"Tendon is a dense connective tissue that transmits the muscle contractile force to the bones. Scleraxis (Scx) is one of the essential transcription factors for tendon formation and maintenance (Brent et al., 2003; Huang et al., 2015; Liu et al., 2021; Schweitzer et al., 2001; Shukunami et al., 2018; Yoshimoto et al., 2017), however its direct target gene as well as their roles in tendon biology is insufficiently understood (Liu et al., 2021). Previously, we have identified genes that are downregulated in the embryo where Scx-lineage cells were ablated, and thus are possible Scx downstream targets (Ono et al., 2023). Among them, we found that Olfactomedin Like 2B (Olfml2b) gene is expressed in embryonic limb and tail at E14.5, in domains similar to those of Tnmd, a well-known Scx direct target gene (Fig. 1A). Immunofluorescence analysis in embryonic fore limb sections confirmed that OLFML2B protein colocalized with Mkx-Venus (MkxVen) tendon reporter (Ito et al., 2010) (Fig. 1B). Specifically, OLFML2B signals were detected in the flexor and extensor tendons that are positive for MkxVen (Fig. 1B arrowheads). These results indicate that Olfml2b is expressed in embryonic tenocytes. Because Olfml2b encodes a secreted extracellular matrix (ECM) associated protein (Furutani et al., 2005), we hypothesized that it could contribute to the development and function of tendon tissue which is rich in ECMs.
Recent ChIP-seq study identified several Scx binding peaks near Olfml2b gene (Liu et al., 2021). We have examined the transcriptional regulatory activity of sequences around these Scx binding peaks by luciferase assay using primary rat tenocyte (Fig. 1C), and found that 800 bp sequence at 5 kbp upstream of Olfml2b transcription start site (region 2) has positive transcriptional activity (Fig. 1D). Moreover, overexpression of Scx together with E47 increased the luciferase activity, indicating that this sequence could respond to Scx (Fig. 1D).
As Olfml2b is expressed in tenocyte as possible target of Scx transcription factor, we hypothesize that this gene could play some role in tendon formation and homeostasis. In line with this idea, OLFML2B mutation was identified in a family with twin sibling males diagnosed as an anterior cruciate ligament non-contact rupture (Caso et al., 2016). To elucidate the role of Olfml2b in tendon tissue, we have generated Olfml2b knockout (KO) mouse with genome editing technique. Established Olfml2b knockout mouse line has 1 bp insertion mutation in 3rd exon of Olfml2b gene that caused frameshift mutation and premature termination at 178th amino acid to OLFML2B protein (Fig. 1E). Western blotting analysis confirmed that OLFML2B protein is absent in embryonic limb extract of Olfml2b KO mouse (Fig. 1F). Tnmd expression of Olfml2b KO embryo at E14.5 was similar to that of wildtype embryo, indicating that Olfml2b is not essential for the embryonic tendon formation (Fig. 1G). Histological examination of the Achilles tendon and cruciate ligaments in 11-weeks-old Olfml2b KO mice revealed that the gross morphology undistinguishable from those of wildtype mice (Fig. 1H, I). Cell density of cruciate ligaments, but not of Achilles tendon, was slightly lower in Olfml2b KO mice (Fig. 1H, I). Altogether, our results indicated that Olfml2b is expressed in embryonic tendon cell but is not essential for the tendon/ligament tissue formation. We have identified possible regulatory sequence at ~5 kb upstream of Olfml2b gene. Further evaluation such as the narrowing down the crucial sequence and electrophoresis mobility shift assay (EMSA), would elucidate whether it contains direct regulatory element by Scx transcription factor (Shukunami et al., 2018). The role of Olfml2b in tendon tissue remains elusive. Considering the ECM associating nature of Olfml2b, it could be involved in collagen fiber organization in tendon tissue. Given the trend toward reduced cell density in Olfml2b KO cruciate ligament, further analyses with increased sample number and extending to other tendon/ligament tissues may help elucidate the function of Olfml2b. Other members of Olfml gene family could have redundant role that needs to be evaluated in future studies.
","references":[{"reference":"Brent AE, Schweitzer R, Tabin CJ. 2003. A somitic compartment of tendon progenitors. Cell 113(2): 235-48.
","pubmedId":"12705871","doi":""},{"reference":"Caso E, Maestro A, Sabiers CC, Godino M, Caracuel Z, Pons J, et al., Guerado E. 2016. Whole-exome sequencing analysis in twin sibling males with an anterior cruciate ligament rupture. Injury 47 Suppl 3: S41-S50.
","pubmedId":"27692106","doi":""},{"reference":"Fukunaga K, Tanji M, Hanzawa N, Kuroda H, Inui M. 2021. Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation. Biochem Biophys Rep 27: 101047.
","pubmedId":"34189280","doi":""},{"reference":"Furutani Y, Manabe R, Tsutsui K, Yamada T, Sugimoto N, Fukuda S, et al., Sekiguchi K. 2005. Identification and characterization of photomedins: novel olfactomedin-domain-containing proteins with chondroitin sulphate-E-binding activity. Biochem J 389(Pt 3): 675-84.
","pubmedId":"15836428","doi":""},{"reference":"Hashimoto M, Takemoto T. 2015. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci Rep 5: 11315.
","pubmedId":"26066060","doi":""},{"reference":"Huang AH, Lu HH, Schweitzer R. 2015. Molecular regulation of tendon cell fate during development. J Orthop Res 33(6): 800-12.
","pubmedId":"25664867","doi":""},{"reference":"Inui M, Mokuda S, Sato T, Tamano M, Takada S, Asahara H. 2018. Dissecting the roles of miR-140 and its host gene. Nat Cell Biol 20(5): 516-518.
","pubmedId":"29695789","doi":""},{"reference":"Ito Y, Toriuchi N, Yoshitaka T, Ueno-Kudoh H, Sato T, Yokoyama S, et al., Asahara H. 2010. The Mohawk homeobox gene is a critical regulator of tendon differentiation. Proc Natl Acad Sci U S A 107(23): 10538-42.
","pubmedId":"20498044","doi":""},{"reference":"Lee K, Gjorevski N, Boghaert E, Radisky DC, Nelson CM. 2011. Snail1, Snail2, and E47 promote mammary epithelial branching morphogenesis. EMBO J 30(13): 2662-74.
","pubmedId":"21610693","doi":""},{"reference":"Liu H, Xu J, Lan Y, Lim HW, Jiang R. 2021. The Scleraxis Transcription Factor Directly Regulates Multiple Distinct Molecular and Cellular Processes During Early Tendon Cell Differentiation. Front Cell Dev Biol 9: 654397.
","pubmedId":"34150754","doi":""},{"reference":"Ono Y, Schlesinger S, Fukunaga K, Yambe S, Sato T, Sasaki T, et al., Inui M. 2023. Scleraxis-lineage cells are required for correct muscle patterning. Development 150(10): pii: dev201101. 10.1242/dev.201101.
","pubmedId":"37246520","doi":""},{"reference":"Sagasaki R, Taniguchi A, Oka S, Mishima M, Yamasaki Y, Kawa Y, et al., Inui M. 2026. Three-dimensional observation of the muscle-tendon integration process in mouse embryos. Dev Dyn: 10.1002/dvdy.70167.
","pubmedId":"42383507","doi":""},{"reference":"Saotome H, Ito A, Kubo A, Inui M. 2020. Generation of a Quantitative Luciferase Reporter for Sox9 SUMOylation. Int J Mol Sci 21(4): 10.3390/ijms21041274.
","pubmedId":"32070068","doi":""},{"reference":"Schweitzer R, Chyung JH, Murtaugh LC, Brent AE, Rosen V, Olson EN, Lassar A, Tabin CJ. 2001. Analysis of the tendon cell fate using Scleraxis, a specific marker for tendons and ligaments. Development 128(19): 3855-66.
","pubmedId":"11585810","doi":""},{"reference":"Shukunami C, Oshima Y, Hiraki Y. 2001. Molecular cloning of tenomodulin, a novel chondromodulin-I related gene. Biochem Biophys Res Commun 280(5): 1323-7.
","pubmedId":"11162673","doi":""},{"reference":"Shukunami C, Takimoto A, Nishizaki Y, Yoshimoto Y, Tanaka S, Miura S, et al., Hiraki Y. 2018. Scleraxis is a transcriptional activator that regulates the expression of Tenomodulin, a marker of mature tenocytes and ligamentocytes. Sci Rep 8(1): 3155.
","pubmedId":"29453333","doi":""},{"reference":"Tanji M, Wada K, Sakamoto K, Ono Y, Inui M. 2023. Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization. Dev Growth Differ 65(1): 48-55.
","pubmedId":"36517457","doi":""},{"reference":"Tsuchiya Y, Matsuo H, Asahara H, Inui M. 2025. Matrix stiffness regulates Mkx expression in rat tenocyte through TRPM7. Biochem Biophys Rep 43: 102178.
","pubmedId":"40740470","doi":""},{"reference":"Yamasaki Y, Sakamoto K, Yashiro S, Kawa Y, Kondow A, Kubo A, Hitachi K, Inui M. 2026. SMAD2 ubiquitination through PY motif regulates skeletal muscle mass and fibrotic degeneration. Sci Rep 16(1): 6666.
","pubmedId":"41617874","doi":""},{"reference":"Yoshimoto Y, Takimoto A, Watanabe H, Hiraki Y, Kondoh G, Shukunami C. 2017. Scleraxis is required for maturation of tissue domains for proper integration of the musculoskeletal system. Sci Rep 7: 45010.
","pubmedId":"28327634","doi":""}],"title":"Embryonic tenocytes express Olfml2b, a potential direct target of Scleraxis
","reviews":[],"curatorReviews":[]},{"id":"fc1881c1-0c3c-433e-9e2e-3f9de29f4fa7","decision":"publish","abstract":"We identified Olfml2b as a candidate downstream target of the tendon transcription factor Scleraxis (Scx). Olfml2b is expressed in mouse embryonic tenocytes, colocalizes with a tendon marker Mkx. Luciferase assay identified a Scx-responsive element approximately 5 kb upstream of Olfml2b transcription start site. To investigate its function, we generated an Olfml2b knockout mouse. Despite its tendon-specific expression, loss of Olfml2b did not affect embryonic tendon formation or the gross morphology of adult tendons. These findings indicate that Olfml2b is a possible Scx-regulated tendon gene but is dispensable for tendon development. Further study is required on its physiological role.
","acknowledgements":"We thank Dr. Hiroshi Asahara for kindly providing us Mkx-Ven reporter mouse. We thank Nobuhiro Yoshizawa and Atsumi Sawada for their technical contributions.
","authors":[{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan"],"departments":[""],"credit":["investigation"],"email":"ef231628@meiji.ac.jp","firstName":"Honoka","lastName":"Inoue","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan"],"departments":[""],"credit":["investigation"],"email":"nanami.harry@icloud.com","firstName":"Nanami","lastName":"Ogawa","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan"],"departments":[""],"credit":["investigation"],"email":"cf260415@meiji.ac.jp","firstName":"Rio","lastName":"Nakajima","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan"],"departments":[""],"credit":["investigation"],"email":"arakawa_k@meiji.ac.jp","firstName":"Kohei","lastName":"Arakawa","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3454-2970"},{"affiliations":["Laboratory of Animal regeneration systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan"],"departments":["",""],"credit":["conceptualization","fundingAcquisition","supervision","writing_originalDraft","writing_reviewEditing"],"email":"inui_m@meiji.ac.jp","firstName":"Masafumi","lastName":"Inui","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-4720-007X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This Research is supported by MEXT KAKENHI (19K06697, 22H02636, 24K22257, 26K02842), for M.I., and TERUMO LIFE SCIENCE Foundation for M.I.
","image":{"url":"https://portal.micropublication.org/uploads/660abcdab85de7d578de1b321f8ad765.png"},"imageCaption":"(A) Whole mount in situ hybridization of Olfml2b mRNA (top row) and Tnmd mRNA (bottom row) in E14.5 embryo (n=3). Scale bar: 1 mm. (B) Immunofluorescence of OLFML2B (top row) and Venus (Mkx-Ven) (bottom row) in forelimb section of E15.5 Mkx-Ven embryo (n=3). Scalebar: 250 µm (C) Schematic drawing of Olfml2b gene and regulatory region candidates. Black boxes indicate Olfml2b exons. Blue boxes with numbers show the position of regulatory region candidates. Red arrowhead indicates the transcription start site of Olfml2b gene. (D) Left panel, a luciferase assay with pNL1.1-TK (Empty Vec) and pNL1.1-TK-candidate regions. Data is shown with mean + SD and a one-way ANOVA followed by Dunnett post hoc test was used for the statistical analysis. *p < 0.05. Right panel, a luciferase assay with pNL1.1-TK (Empty Vec), pNL1.1-TK-region2 vector with or without the overexpression of Scx and E47. Data is shown with mean + SD and a one-way ANOVA followed by Tukey’s post hoc test was used for the statistical analysis. ***p < 0.001 (E) The deduced amino acid sequence (upper letters) from wildtype (WT) and mutant (MUT) DNA sequence (lower letters) around 1 base insertion (red letter) in Olfml2b mutant allele. Wildtype (WT) sequences are shown at the top and mutant (MUT) sequences are shown at the bottom. Red letters in DNA sequence and amino acid sequence indicate the inserted base and altered amino acids, respectively. (F) Western blotting of OLFML2B and GAPDH proteins in wildtype (WT) and Olfml2b KO (KO) embryonic limb (n=2). Red arrowhead and asterisk indicate OLFML2B protein and non-specific band, respectively. (G) Whole mount in situ hybridization of Tnmd mRNA in Wildtype (top row) and Olfml2b KO (bottom row) in E14.5 embryo (n=3). Scale bar: 1 mm. (H-I) H&E staining of posterior cruciate ligament (H) and Achilles tendon (I) of 11-weeks-old wildtype mice (top panels) and Olfml2b KO mice (bottom panels) cell density (cells/µm2) within tendon tissue are shown at right (n=2). Scale bar 50 µm.
","imageTitle":"Olfml2b is expressed in embryonic tendon as possible direct target of Scx transcription factor
","methods":"Animals
C57BL/6 mice, ICR mice, and Wistar rats were purchased from Sankyo lab-service (Tokyo, Japan). MkxVen mouse (Ito et al., 2010) is kindly provided by Dr. Asahara. All animal experiments were approved by the animal experiment committees of Meiji University (approval No. MUIACUC2022-04).
Generation of Olfml2b knockout mouse
Olfml2b KO mouse was generated by genome editing (Hashimoto and Takemoto, 2015). Briefly, a crRNA targeting exon 3 of the Olfml2b gene (5’-TCGGTTACCACTAAACTCGT-3’), tracrRNA, and S.p.Cas9 protein were purchased from IDT (Coralville, Iowa, USA). The fertilized eggs of C57BL/6 mice purchased from Ark-Resource (Kumamoto, Japan) were mixed with crRNA/tracrRNA/Cas9 protein and electroporation was performed using CUY21EDITⅡ electroporator (BEX, Tokyo, Japan). The electroporation conditions were 30 V (3 msec ON + 97 msec OFF) x 7 times. The surviving 2-cell-stage embryos were transferred to the oviducts of pseudo-pregnant ICR female mice. Genome sequencing of one F1 offspring revealed a 1-bp insertion in exon 3, predicted to cause a frameshift mutation.
Primary tenocyte culture
Primary tenocytes were isolated from 3-weeks-old male Wistar rat and cultured as described previously (Tsuchiya et al., 2025).
Luciferase assay
Genomic sequences from 6 regions neighboring Olfml2b gene were PCR amplified and cloned into pNL1.1 vector (Promega, Wisconsin, USA) together with thymidine kinase (TK) promoter. Primer sequences used to the cloning are listed as below.
Region 1 fw: 5'-CAAATAGGACCCGTGAGAGC-3', rev: 5'-TGGAGCTCGGATTACAGAGG-3'
Region 2 fw: 5'-TTTTCCCGGTCCCTAGTCTC-3', rev: 5'-AGCCTGTTGATTCCATGTCC-3',
Region 3 fw: 5'-GGATGTGAACCCTCAGGAAA-3', rev: 5'-GTGGTGGGTGCTCAGAAGTT-3'
Region 4 fw: 5'-CCGAGGGTCAGTGATGTTTT-3', rev: 5'-CAAATGTTGCTCCCAAGTCA-3'
Region 5 fw: 5'-GCAACCTCCTCCTGGTTGTA-3', rev: 5'-TGGTCTAGCAGTGCAGGATG-3'
Region 6 fw: 5'-CCGATTCCACAGATGATAAGG-3', rev: 5'-TCCAAGATTCCTTTGCGTTC-3'
For overexpression plasmid, mouse Scleraxis coding sequence was PCR amplified and cloned into pcDNA3.1. Expression vector for mE47 was purchased from Addgene (ID34585) (Lee et al., 2011). Tenocytes were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) and extracted with lysis buffer (50 mM HEPES pH 7.8, 200 mM NaCl, 5 mM EDTA, 1% NP40, 5% Glycerol, 1 mM DTT) after 48 hours. Luciferase activities were detected using Nano-Glo Dual-Luciferase Reporter Assay (Promega) as described previously (Saotome et al., 2020). pGL4.53 (Promega) was used as internal control.
Immunofluorescence
Immunofluorescence were performed essentially as described before (Ono et al., 2023; Yamasaki et al., 2026). Briefly, mouse embryos were fixed with 4% paraformaldehyde (PFA) overnight at 4 °C, embedded in optimal cutting temperature (OCT) compound (Sakurafinetek, Tokyo, Japan) and sectioned at 10 µm. Sections were blocked with 10% fetal bovine serum (FBS)/Phosphate buffer saline (PBS) for 1 hour at room temperature and incubated with primary antibodies overnight at 4 °C. The sections were washed with PBST (PBS supplemented with 0.1 % Tween 20), incubated with secondary antibodies for 1 hour at room temperature, washed with PBST and mounted with fluoroshield mounting medium (Immunochemistry Technologies, Davis, CA, USA). α-GFP (Abcam, Cambridge, UK, ab13970, 1:1000), α-Olfml2b (Merck HPA054136) were used as primary antibodies.
Whole-mount in situ hybridization (WISH)
WISH analysis was performed as described before (Sagasaki et al., 2026; Tanji et al., 2023). Olfml2b probe sequence were amplified from embryonic limb cDNA using primers below (fw 5’-cagtggagtggacttgctga-3’, rev 5’-cataacgctgcttcaggtca-3’) and cloned into pGEM T-easy vector (Promega). Tnmd probe was described previously (Shukunami et al., 2001).
Histology
Hindlimbs of 11-week-old mice were fixed with 4% PFA overnight at 4 °C, decalcified with 0.5 M EDTA solution, embedded with paraffin wax. Samples were sectioned at 6 µm and hematoxylin and Eosin (H&E) staining was performed as described before (Fukunaga et al., 2021). The cell density of the tendon/ligament tissue were calculated by counting hematoxylin positive nuclei within H&E image and normalized to the tendon area using Image J. Two individuals for each genotype were examined and both data were shown in the panel.
Western blotting
Western blotting was performed as described before (Inui et al., 2018; Saotome et al., 2020). α-Olfml2b (Merck, HPA054136, 1:500) and α-GAPDH (Millipore, MAB374, 1:2000) were used as primary antibodies and α-rabbit IgG-Peroxidase (SIGMA-Aldrich, A0545, 1:2000) and α-mouse IgG-Peroxidase (SIGMA-Aldrich, A2304, 1:2000) were used as secondary antibodies.
","reagents":"","patternDescription":"Tendon is a dense connective tissue that transmits the muscle contractile force to the bones. Scleraxis (Scx) is one of the essential transcription factors for tendon formation and maintenance (Brent et al., 2003; Huang et al., 2015; Liu et al., 2021; Schweitzer et al., 2001; Shukunami et al., 2018; Yoshimoto et al., 2017), however its direct target gene as well as their roles in tendon biology is insufficiently understood (Liu et al., 2021). Previously, we have identified genes that are downregulated in the embryo where Scx-lineage cells were ablated, and thus are possible Scx downstream targets (Ono et al., 2023). Among them, we found that Olfactomedin Like 2B (Olfml2b) gene is expressed in embryonic limb and tail at E14.5, in domains similar to those of Tnmd, a well-known Scx direct target gene (Fig. 1A). Immunofluorescence analysis in embryonic fore limb sections confirmed that OLFML2B protein colocalized with Mkx-Venus (MkxVen) tendon reporter (Ito et al., 2010) (Fig. 1B). Specifically, OLFML2B signals were detected in the flexor and extensor tendons that are positive for MkxVen (Fig. 1B arrowheads). These results indicate that Olfml2b is expressed in embryonic tenocytes. Because Olfml2b encodes a secreted extracellular matrix (ECM) associated protein (Furutani et al., 2005), we hypothesized that it could contribute to the development and function of tendon tissue which is rich in ECMs.
Recent ChIP-seq study identified several Scx binding peaks near Olfml2b gene (Liu et al., 2021). We have examined the transcriptional regulatory activity of sequences around these Scx binding peaks by luciferase assay using primary rat tenocyte (Fig. 1C), and found that 800 bp sequence at 5 kbp upstream of Olfml2b transcription start site (region 2) has positive transcriptional activity (Fig. 1D). Moreover, overexpression of Scx together with E47 increased the luciferase activity, indicating that this sequence could respond to Scx (Fig. 1D).
As Olfml2b is expressed in tenocyte as possible target of Scx transcription factor, we hypothesize that this gene could play some role in tendon formation and homeostasis. In line with this idea, OLFML2B mutation was identified in a family with twin sibling males diagnosed as an anterior cruciate ligament non-contact rupture (Caso et al., 2016). To elucidate the role of Olfml2b in tendon tissue, we have generated Olfml2b knockout (KO) mouse with genome editing technique. Established Olfml2b knockout mouse line has 1 bp insertion mutation in 3rd exon of Olfml2b gene that caused frameshift mutation and premature termination at 178th amino acid to OLFML2B protein (Fig. 1E). Western blotting analysis confirmed that OLFML2B protein is absent in embryonic limb extract of Olfml2b KO mouse (Fig. 1F). Tnmd expression of Olfml2b KO embryo at E14.5 was similar to that of wildtype embryo, indicating that Olfml2b is not essential for the embryonic tendon formation (Fig. 1G). Histological examination of the Achilles tendon and cruciate ligaments in 11-weeks-old Olfml2b KO mice revealed that the gross morphology undistinguishable from those of wildtype mice (Fig. 1H, I). Cell density of cruciate ligaments, but not of Achilles tendon, was slightly lower in Olfml2b KO mice (Fig. 1H, I). Altogether, our results indicated that Olfml2b is expressed in embryonic tendon cell but is not essential for the tendon/ligament tissue formation. We have identified possible regulatory sequence at ~5 kb upstream of Olfml2b gene. Further evaluation such as the narrowing down the crucial sequence and electrophoresis mobility shift assay (EMSA), would elucidate whether it contains direct regulatory element by Scx transcription factor (Shukunami et al., 2018). The role of Olfml2b in tendon tissue remains elusive. Considering the ECM associating nature of Olfml2b, it could be involved in collagen fiber organization in tendon tissue. Given the trend toward reduced cell density in Olfml2b KO cruciate ligament, further analyses with increased sample number and extending to other tendon/ligament tissues may help elucidate the function of Olfml2b. Other members of Olfml gene family could have redundant role that needs to be evaluated in future studies.
","references":[{"reference":"Brent AE, Schweitzer R, Tabin CJ. 2003. A somitic compartment of tendon progenitors. Cell 113(2): 235-48.
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","pubmedId":"41617874","doi":""},{"reference":"Yoshimoto Y, Takimoto A, Watanabe H, Hiraki Y, Kondoh G, Shukunami C. 2017. Scleraxis is required for maturation of tissue domains for proper integration of the musculoskeletal system. Sci Rep 7: 45010.
","pubmedId":"28327634","doi":""}],"title":"Embryonic tenocytes express Olfml2b, a potential direct target of Scleraxis
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