Anti–USAG-1 therapy for tooth regeneration through enhanced BMP signaling
通过增强 BMP 信号传导进行牙齿再生的抗 USAG-1 疗法

Phenotypic changes in an Msx1^−/−/USAG-1^−/− mouse generated by mating mouse models of congenital tooth agenesis and supernumerary teeth were investigated. The development of both the maxilla and the mandible was arrested in the early stages. However, a cleft palate was additionally observed in USAG-1^+/+/Msx1^−/− mice (Fig. 1, F and G). Although mouse offspring with a USAG-1^−/−/Msx1^−/− background should have theoretically been obtained with one-sixteenth incidence, only 3 of 151 littermate mice had the USAG-1^−/−/Msx1^−/− genotype (Fig. 1A). Histological evaluation revealed that all USAG-1^−/−/Msx1^−/− mice had normal third maxillary molar teeth (Fig. 1, H and I).
研究了先天性牙齿发育不全和多生牙交配小鼠模型产生的 Msx1^−/−/USAG-1^−/− 小鼠的表型变化。上颌骨和下颌骨的发育在早期阶段都受到阻碍。然而，在 USAG-1^+/+/Msx1^−/− 小鼠中还观察到腭裂（ 图 1，F 和 G）。尽管理论上应该以十六分之一的发生率获得具有 USAG-1^−/−/Msx1^−/− 背景的小鼠后代，但 151 只同窝小鼠中只有 3 只具有 USAG-1^−/−/Msx1^−/− 基因型（ 图 1A）。组织学评估显示，所有 USAG-1^−/−/Msx1^−/− 小鼠的第三上颌磨牙正常（ 图 1，H 和 I）。

Next, we analyzed EDA1^−/−/USAG-1^−/− mice. As EDA1 is located on the X chromosome, female EDA1^−/−/USAG-1^−/− and male EDA1^+/−/USAG-1^−/− mice are null for USAG-1 and EDA1. These double KO mice had normal teeth, hyperdontia, or fused mandibular molars, whereas 75% of the female USAG-1^+/+/EDA1^−/− and male USAG-1^+/+/EDA1^+/− mice had molar hypodontia in the mandible (Fig. 1, J to R′, and fig. S2). Hair loss behind the ear and tail kink, which are the typical phenotypes associated with tabby mice, were present in all USAG-1 and EDA1 double KO mice (Fig. 1V). These results suggest that Usag-1^−/− can rescue congenital tooth agenesis during early tooth development and promote morphogenesis of the whole tooth structure arrested in the late stage.
接下来，我们分析了 EDA1^−/−/USAG-1^−/− 小鼠。由于 EDA1 位于 X 染色体上，雌性 EDA1^−/−/USAG-1^−/− 和雄性 EDA1^+/−/USAG-1^−/− 小鼠对 USAG-1 和 EDA1 无效。这些双 KO 小鼠具有正常的牙齿，牙根过多或下颌磨牙融合，而 75%的雌性 USAG-1^+/+/EDA1^−/− 和雄性 USAG-1^+/+/EDA1^+/− 小鼠的下颌骨有臼齿牙欠位（ 图 1，J 至 R′，图 S2）。耳后脱毛和尾巴扭结是与虎斑小鼠相关的典型表型，存在于所有 USAG-1 和 EDA1 双 KO 小鼠中（ 图 1V）。这些结果表明，Usag-1^−/− 可以挽救早期牙齿发育过程中的先天性牙齿发育不全，促进晚期停滞的整个牙齿结构的形态发生。

To investigate whether inhibition of USAG-1 function rescues congenital tooth agenesis, we purified five mouse USAG-1 monoclonal antibodies (#12, #16, #37, #48, and #57) using a bioactive human USAG-1 recombinant protein derived from Escherichia coli as an antigen and USAG-1^−/− mice. USAG-1 is suggested to inhibit Wnt and BMP signals via direct binding to BMP and the Wnt coreceptor LRP5/6 (28, 29). Therefore, these five antibodies were categorized into three different classes, based on their interfering abilities of the binding to both BMP and Wnt (#57), BMP (#12 and #37), or Wnt (#16 and #48) (Fig. 2, A and B). We confirmed that all antibodies could bind the mouse and human USAG-1 recombinant proteins (Fig. 2C), although #16 and #48 showed low affinity (Fig. 2, D and E). These results enabled the investigation of the function of USAG-1 with respect to BMP and Wnt signaling pathways for the determination of the number of teeth.
为了研究抑制 USAG-1 功能是否能挽救先天性牙齿发育不全，我们使用源自大肠杆菌的生物活性人 USAG-1 重组蛋白作为抗原和 USAG-1^−/− 小鼠纯化了五种小鼠 USAG-1 单克隆抗体（#12、#16、#37、#48 和#57）。建议 USAG-1 通过直接结合 BMP 和 Wnt 辅助受体 LRP5/6 来抑制 Wnt 和 BMP 信号 （28， 29）。因此，根据它们与 BMP 和 Wnt（#57）、BMP（#12 和#37）或 Wnt（#16 和#48）结合的干扰能力，这五种抗体被分为三个不同的类别（ 图 2，A 和 B）。我们证实所有抗体都可以结合小鼠和人 USAG-1 重组蛋白（ 图 2C），尽管#16 和#48 表现出低亲和力（ 图 2，D 和 E）。这些结果使 USAG-1 能够研究 BMP 和 Wnt 信号通路的功能，以确定牙齿数量。

Each USAG-1–neutralizing antibody was systemically administered to EDA1 pregnant mice. Low birth and survival rates were observed in mice administered USAG-1–neutralizing antibodies #12, #16, or #48 (Fig. 3A). USAG-1–neutralizing antibodies #16, #37, #48, and #57 rescued molar hypodontia in the mandible of EDA1^−/− mice compared with control mice (Fig. 3, B and C, and fig. S3). USAG-1–neutralizing antibody #37 reversed hypodontia at a high rate and in a dose-dependent manner (Fig. 3B). In addition, USAG-1–neutralizing antibodies #12, #16, #37, and #57 led to the production of supernumerary teeth in the maxillary incisor, mandibular incisor, or molar of EDA1 KO/hetero mice (Fig. 3, B and C, and fig. S3). Unexpectedly, USAG-1–neutralizing antibody #57 induced the formation of supernumerary teeth in the maxillary incisor, mandibular incisor, or molar of wild-type mice at a high rate and a dose-dependent manner (Fig. 3, B and C, and fig. S3). However, fused molars were observed instead of supernumerary teeth in the maxillary molar region (Fig. 3C and fig. S3). Both antibodies neutralized BMP signaling antagonistic function, at least in vitro (Fig. 3, B and C, and fig. S3). These results indicate that BMP signaling is essential for determining the number of teeth in mice. Furthermore, a single systemic administration of a neutralizing antibody can generate a whole tooth.
将每种 USAG-1 中和抗体系统施用于 EDA1 怀孕小鼠。在给予 USAG-1 中和抗体#12，#16 或#48 的小鼠中观察到低出生率和存活率（ 图 3A）。与对照小鼠相比，USAG-1-中和抗体#16，#37，#48 和#57 挽救了 EDA1^−/- 小鼠下颌骨的磨牙牙缺症（ 图 3，B 和 C，以及图 S3）。USAG-1-中和抗体 #37 以剂量依赖性方式高速逆转牙髓不足（ 图 3B）。此外，USAG-1 中和抗体#12，#16，#37 和#57 导致 EDA1 KO /异种小鼠的上颌切牙，下颌切牙或臼齿产生多生牙（ 图 3，B 和 C，以及图 S3）。出乎意料的是，USAG-1-中和抗体#57 以高速和剂量依赖性方式诱导野生型小鼠的上颌切牙、下颌切牙或磨牙中多生牙的形成（ 图 3、B 和 C，以及图 S3）。然而，在上颌磨牙区域观察到融合的臼齿而不是多生牙（ 图 3C 和图 S3）。两种抗体至少在体外中和了 BMP 信号传导拮抗功能（ 图 3，B 和 C，以及图 S3）。这些结果表明，BMP 信号传导对于确定小鼠的牙齿数量至关重要。此外，单次全身给药中和抗体可以产生整颗牙齿。

To determine the epitope of USAG-1–neutralizing antibodies #37 and #57, we performed epitope mapping using 169 linear peptides, including 20 sequential amino acids (Fig. 4, A and D). USAG-1–neutralizing antibody #37 specifically reacted with six overlapping peptides (D16-D21) spanning the region Q¹²⁹EWRCVNDKTRTQRIQLQCQ¹⁴⁸, suggesting that the epitope is localized within the central 10-residue segment containing the sequence VNDKTRTQRI (Fig. 4B). Although the three-dimensional (3D) structure of USAG-1 is unknown, its high sequence homology with sclerostin (SOST) that belongs to the same BMP antagonist DAN family enabled us to build a homology model of mouse USAG-1 using the nuclear magnetic resonance structure of SOST (Fig. 4E) (28). It was revealed that the epitope recognized by antibody #37 lies on the surface-exposed edge strand of the central β sheet of USAG-1, consistent with the ability of #37 to recognize native USAG-1. This region is located far from the NXI motif, which is the binding site for LRP5/6 (Fig. 4E) (29), suggesting that this antibody does not block USAG-1 interaction with the Wnt coreceptor LRP5/6. Antibody #37 did not affect the Wnt1-antagonizing activity of USAG-1 (Fig. 2B). In contrast to #37, antibody #57 did not show reactivity toward any of the USAG-1–derived overlapping peptides (Fig. 4C), indicating that it recognizes a 3D epitope present on the USAG-1 surface.
为了确定 USAG-1 中和抗体#37 和#57 的表位，我们使用 169 个线性肽（包括 20 个连续氨基酸）进行了表位图谱分析（ 图 4，A 和 D）。USAG-1-中和抗体 #37 与横跨 Q¹²⁹EWRCVNDKTRTQRIQQCQ¹⁴⁸ 区域的六个重叠肽 （D16-D21） 特异性反应，表明表位位于包含序列 VNDKTRTQRI 的中央 10 残基片段内（ 图 4B）。尽管 USAG-1 的三维（3D）结构尚不清楚，但其与属于同一 BMP 拮抗剂 DAN 家族的硬化蛋白（SOST）的高序列同源性使我们能够利用 SOST 的核磁共振结构构建小鼠 USAG-1 的同源模型（ 图 4E）（28）。结果表明，抗体 #37 识别的表位位于 USAG-1 中央β片的表面暴露边缘链上，与 #37 识别天然 USAG-1 的能力一致。该区域远离 NXI 基序，NXI 基序是 LRP5/6 的结合位点（ 图 4E）（29），表明该抗体不会阻断 USAG-1 与 Wnt 辅助受体 LRP5/6 的相互作用。抗体#37 不影响 USAG-1 的 Wnt1 拮抗活性（ 图 2B）。与#37 相比，抗体#57 对任何 USAG-1 衍生的重叠肽均未表现出反应性（ 图 4C），表明它识别存在于 USAG-1 表面的 3D 表位。

It has been established that the endogenous Wnt pathway inhibitor SOST exerts its inhibitory effect by binding to the “E1” domain of Wnt coreceptor LRP6 (30). As described in the previous section, the conservation of the LRP6-binding motif NXI in USAG-1 strongly suggests that it binds to the same domain of LRP6 as well. We evaluated the USAG-1 binding to the human LRP6 ectodomain fragments of varying lengths. As shown in Fig. 5A, stoichiometric binding of USAG-1 was observed with the E1-E2 domain fragment of LRP6, confirming the prediction that the binding site was located in the E1 domain. In contrast, no binding was observed with E1-E4 or E3-E4 fragments. The lack of binding with E1-containing E1-E4 fragment can be explained by the fact that the NXI-binding surface of E1 is occluded in the context of the whole ectodomain of LRP6, which shows a highly curved “C-shape” in the electron microscopic images (31). We then investigated whether the USAG-1–neutralizing antibodies can interfere with the LRP6–USAG-1 interaction. As shown in Fig. 5B, near-complete inhibition was observed with antibody #16, while #48 exhibited partial inhibition. This finding was consistent with their ability to inhibit the Wnt-modulating activity of USAG-1 (Fig. 2B). Three other antibodies (#12, #37, and #57) did not affect the binding of USAG-1 to LRP6 E1-E2, corroborating their inability to counteract the Wnt-modulating capability of USAG-1 (Fig. 2B). On the basis of these results, we conclude that neutralizing the antagonizing effect of USAG-1 on BMP rather than Wnt signals is more effective in achieving substantial phenotypic changes in mice, i.e., recovering missing teeth or making a whole tooth.
已经确定，内源性 Wnt 通路抑制剂 SOST 通过与 Wnt 辅助受体 LRP6 的“E1”结构域结合来发挥其抑制作用（30）。如上一节所述，USAG-1 中 LRP6 结合基序 NXI 的保守强烈表明它也与 LRP6 的同一结构域结合。我们评估了 USAG-1 与不同长度的人 LRP6 胞外结构域片段的结合。如图 5A 所示，观察到 USAG-1 与 LRP6 的 E1-E2 结构域片段的化学计量结合，证实了结合位点位于 E1 结构域的预测。相比之下，未观察到与 E1-E4 或 E3-E4 片段的结合。与含 E1 的 E1-E4 片段缺乏结合可以通过以下事实来解释：E1 的 NXI 结合表面在 LRP6 的整个胞外结构域的背景下被遮挡，这在电子显微镜图像中显示出高度弯曲的“C 形”（31）。然后，我们研究了 USAG-1 中和抗体是否会干扰 LRP6-USAG-1 相互作用。如图 5B 所示，抗体#16 观察到近乎完全的抑制，而#48 表现出部分抑制。这一发现与它们抑制 USAG-1 的 Wnt 调节活性的能力一致（ 图 2B）。其他三种抗体（#12、#37 和#57）不影响 USAG-1 与 LRP6 E1-E2 的结合，证实了它们无法抵消 USAG-1 的 Wnt 调节能力（ 图 2B）。基于这些结果，我们得出结论，中和 USAG-1 对 BMP 的拮抗作用比 Wnt 信号更有效地实现小鼠的实质性表型变化，即恢复缺失的牙齿或制作整颗牙齿。

To investigate the functional differences between antibodies #37 and #57 with respect to BMP signaling, we analyzed the cross-reactivity of these antibodies with members of the DAN subfamily (Fig. 5C). We detected a faint signal for SOST in transfected human embryonic kidney (HEK) 293 cells using immunohistochemistry with antibody #57 but not with #37 (Fig. 5D and fig. S4). This weak cross-reactivity with SOST is likely due to the similarities in the 3D structures of SOST and USAG-1 (28). Furthermore, systemic administration of an antibody mixture containing antibodies #12, #16, #37, #48, and #57 increased the number of supernumerary teeth and the size of fused teeth in the mandible of USAG-1^−/− mice (Fig. 5, E and F). These results suggest that antibody #57 may inhibit the genetic redundancy responsible for supernumerary tooth formation by affecting SOST, a BMP antagonist.
为了研究抗体#37 和#57 在 BMP 信号传导方面的功能差异，我们分析了这些抗体与 DAN 亚家族成员的交叉反应性（ 图 5C）。我们使用抗体#57 的免疫组织化学在转染的人胚胎肾（HEK）293 细胞中检测到 SOST 的微弱信号，但没有使用#37（ 图 5D 和图 S4）。这种与 SOST 的微弱交叉反应性可能是由于 SOST 和 USAG-1 的 3D 结构相似 （28）。此外，全身施用含有抗体#12，#16，#37，#48 和#57 的抗体混合物增加了 USAG-1^−/− 小鼠下颌骨中多生牙的数量和融合牙齿的大小（ 图 5，E 和 F）。这些结果表明，抗体 #57 可能通过影响 BMP 拮抗剂 SOST 来抑制导致多生牙齿形成的遗传冗余。

Last, to confirm that USAG-1–neutralizing activity affects BMP signaling to generate a whole tooth in a nonrodent model, we systemically administered antibody #37 to postnatal ferrets that had both deciduous and permanent teeth. We observed supernumerary tooth formation in maxillary incisor like the third dentition, although a five times higher concentration, three administrations of antibody #37, and immunosuppression were required (Fig. 6, A to D). The supernumerary tooth was likely to have a similar shape to the usual permanent incisor, located to the lingual side of permanent teeth, whereas a shorter root seemed to be growing (Fig. 6, E to G). Therefore, this supernumerary incisor might be categorized as the third dentition (32). Furthermore, phosphorylated Smad-positive cells were observed within pulp of supernumerary tooth (Fig. 6, H and I).
最后，为了确认 USAG-1 中和活性影响 BMP 信号传导以在非啮齿动物模型中产生整颗牙齿，我们系统地将抗体 #37 施用于同时具有乳牙和恒牙的出生后雪貂。我们观察到上颌切牙（如第三牙列）多生牙的形成，尽管需要高出五倍的浓度、三次抗体#37 和免疫抑制（ 图 6，A 至 D）。多生牙的形状可能与通常的恒门牙相似，位于恒牙的舌侧，而较短的牙根似乎正在生长（ 图 6，E 至 G）。因此，该多生切牙可归类为第三牙列 （32）。此外，在多生牙的牙髓内观察到磷酸化的 Smad 阳性细胞（ 图 6，H 和 I）。

This study’s main objectives included the generation and use of a monoclonal anti–USAG-1 antibody to locally arrest and recover tooth development in mice. We also performed experiments to determine whether BMP or Wnt signaling modulated tooth development. This study was approved by the Animal Research Committee of Kyoto University (reference number: Med Kyo 11518), KAC Co. Ltd. (reference number: 19-1103), and the Recombinant DNA Experiment Safety Committee of Kyoto University (reference number: 180211). Experiments were performed in accordance with approved guidelines. All experiments were repeated at least three times. Sample sizes were chosen empirically to ensure adequate statistical power. All valid measurements were included in our analysis. No outliers were excluded. Primary data are provided in the figures or the Supplementary Materials.
本研究的主要目标包括生成和使用单克隆抗 USAG-1 抗体来局部阻止和恢复小鼠的牙齿发育。我们还进行了实验以确定 BMP 或 Wnt 信号是否调节牙齿发育。本研究获得京都大学动物研究委员会（参考编号：Med Kyo 11518）、KAC Co. Ltd.（参考编号：19-1103）和京都大学重组 DNA 实验安全委员会（参考编号：180211）的批准。实验是根据批准的指南进行的。所有实验至少重复三次。样本量是根据经验选择的，以确保足够的统计功效。所有有效的测量值都包含在我们的分析中。没有排除异常值。主要数据见图或补充材料。

USAG-1^−/− mice with a 106-bp deletion in exon 1 were produced using the CRISPR-Cas system with a C57BL/6J genetic background (fig. S1) (Macrogen Co. Ltd., Seoul, South Korea). Dental anomalies similar to those described in previous reports (10), including incisal supernumerary teeth, fused maxillary molars, and supernumerary mandibular molars, were observed in USAG-1^−/− mice. EDA1-deficient mice (Tabby6: C57BL/6J A^w-J-Eda^Ta-6J/J) were obtained from the Jackson Laboratory (JAX stock #000338). Msx1-deficient mice with a 129S4/SvJae genetic background were provided by the Mutant Mouse Resource and Research Centers (MMRRC stock #000068-UCD). We interbred heterozygous USAG-1 and Msx1 mice and analyzed the F₂ generation. To eliminate the influence of the mouse background, only F2 progeny USAG-1^−/−/Msx1^−/− mice were analyzed. Polymerase chain reaction was performed using KOD FX NEO polymerase (KFX-201; TOYOBO, Osaka, Japan) and specific primers. Embryos were obtained by timed mating; day E0 started from midnight, before finding a vaginal plug. Outbred pregnant ferrets were purchased from Marshall BioResources Japan Co. Ltd. A subgroup of the offspring was maintained in immunosuppressive condition, as previously reported (41).
使用具有 C57BL / 6J 遗传背景的 CRISPR-Cas 系统（图 S1）生产外显子 1 中缺失 106-bp 的 USAG-1^−/− 小鼠（Macrogen Co. Ltd.，首尔，韩国）。在 USAG-1^−/− 小鼠中观察到与先前报告（10）中描述的牙齿异常相似，包括切牙多生牙、融合上颌磨牙和多生下颌磨牙。EDA1 缺陷小鼠（Tabby6：C57BL / 6J A^{w-J-Eda Ta-6J} / J）从杰克逊实验室获得（JAX 库存#000338）。具有 129S4/SvJae 遗传背景的 Msx1 缺陷小鼠由突变小鼠资源和研究中心（MMRRC 库存 #000068-UCD）提供。我们杂交了杂合的 USAG-1 和 Msx1 小鼠，并分析了 F₂ 代。为了消除小鼠背景的影响，仅分析了 F2 后代 USAG-1^−/−/Msx1^−/− 小鼠。使用 KOD FX NEO 聚合酶（KFX-201;TOYOBO， Osaka， Japan）和特定引物。胚胎是通过定时交配获得的;E0 天从午夜开始，然后找到阴道塞。近交怀孕雪貂购自 Marshall BioResources Japan Co. Ltd.。正如之前报道的那样，后代的一个亚群维持在免疫抑制状态 （41）。

Preparation of PA-tagged mouse USAG-1 recombinant protein from mammalian cells was performed as previously reported (42). Other tagged USAG-1 recombinant proteins, derived from E. coli or baculoviral expression systems (R&D systems Inc., MN, USA; MyBiosource, CA, USA), were used for the production of antibodies, as antigens, and in the solid phase and/or sandwich enzyme-linked immunosorbent assay (ELISA). Preparation of the E1-E4 domain of LRP6 was performed as previously reported (30). Expression vectors for mouse DAN family proteins were purchased from OriGene Technologies Inc. (Rockville, MD, USA).
如先前报道的那样，从哺乳动物细胞制备 PA 标记的小鼠 USAG-1 重组蛋白（42）。其他标记的 USAG-1 重组蛋白，源自大肠杆菌或杆状病毒表达系统（R&D systems Inc.，明尼苏达州，美国;MyBiosource， CA， USA）用于生产抗体、抗原以及固相和/或夹心酶联免疫吸附测定 （ELISA）。LRP6 的 E1-E4 结构域的制备如先前报道 （30）。小鼠 DAN 家族蛋白的表达载体购自 OriGene Technologies Inc.（美国马里兰州罗克维尔）。

The anti–USAG-1 monoclonal antibodies were generated by ITM Co. Ltd. (Matsumoto, Japan) as previously described (43). Briefly, USAG-1^−/− mice were immunized with recombinant human USAG-1 protein. Two weeks later, the lymphocytes obtained from iliac lymph nodes were fused with SP2/0 mouse myeloma cells in the presence of 50% polyethylene glycol solution and were selected for 1 week on GIT medium (Wako Pure Chemical Corporation, Osaka, Japan) containing HAT as a supplement. The resultant hybridomas were screened by ELISA, and those secreting anti–USAG-1 monoclonal antibodies were identified. The culture supernatant (10 ml) was loaded onto a Protein G column (GE Healthcare, Chicago, IL, USA), and the antibody was adsorbed onto the column. Bound antibody was eluted using the elution buffer from the MAbTrap Kit (GE Healthcare). The eluted antibody was loaded on a centrifugal filter (Amicon Ultra-15; Millipore, Burlington, MA, USA) for buffer exchange with phosphate-buffered saline (PBS), and concentration was determined. Antibodies were stored at −80°C until use.
如前所述，抗 USAG-1 单克隆抗体由 ITM Co. Ltd.（日本松本）生产 （43）。简而言之，用重组人 USAG-1 蛋白免疫 USAG-1−/⁻ 小鼠。两周后，将从髂淋巴结获得的淋巴细胞在 50%聚乙二醇溶液存在下与 SP2/0 小鼠骨髓瘤细胞融合，并在含有 HAT 的 GIT 培养基（Wako Pure Chemical Corporation，Osaka，Japan）上选择 1 周作为补充剂。通过 ELISA 筛选所得杂交瘤，并鉴定出分泌抗 USAG-1 单克隆抗体的杂交瘤。将培养上清液（10 ml）加载到蛋白 G 色谱柱（GE Healthcare，Chicago，IL，USA）上，并将抗体吸附到色谱柱上。使用 MAbTrap 试剂盒 （GE Healthcare） 中的洗脱缓冲液洗脱结合的抗体。将洗脱的抗体加载到离心过滤器（Amicon Ultra-15;Millipore， Burlington， MA， USA）与磷酸盐缓冲盐水（PBS）进行缓冲液交换，并测定浓度。抗体储存在 -80°C 直至使用。

For determination of alkaline phosphatase (ALP) activity, C2C12 cells were seeded at a density of 6 × 10⁴ cells per well in 96-well plates. After the cells reached confluency, they were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 15% fetal bovine serum (FBS), penicillin G (100 U/ml), streptomycin (100 μg/ml), recombinant mouse BMP7 protein (30 ng/ml or 300 ng/ml) (R&D systems), and recombinant mouse USAG-1 protein (0 to 3000 ng/ml) for 48 hours. Cells were washed twice with PBS and scraped in 0.05% Triton X-100. The cell suspension was sonicated on ice. Aliquots of supernatants were assayed for protein concentration and ALP activity (LabAssay ALP, FUJIFILM Wako Pure Chemical Corporation) as described.
为了测定碱性磷酸酶 （ALP） 活性，将 C2C12 细胞以每孔 6 × 10⁴ 个细胞的密度接种在 96 孔板中。细胞达到汇合后，在 Dulbecco 改良的 Eagle 培养基（DMEM;Sigma-Aldrich，圣路易斯，密苏里州，美国）补充 15%胎牛血清（FBS），青霉素 G（100 U / ml），链霉素（100μg / ml），重组小鼠 BMP7 蛋白（30 ng / ml 或 300 ng / ml）（研发系统）和重组小鼠 USAG-1 蛋白（0 至 3000 ng / ml）48 小时。用 PBS 洗涤细胞两次，并在 0.05%Triton X-100 中刮擦。将细胞悬液在冰上超声处理。如所述，测定上清液等分试样的蛋白质浓度和 ALP 活性（LabAssay ALP，FUJIFILM Wako Pure Chemical Corporation）。

To assess the neutralizing effects of the anti–USAG-1 antibodies on Wnt/β-catenin signaling modulated by recombinant mouse USAG-1, we used the TOP reporter system based on the dual-luciferase reporter assay system (Promega, Madison, WI, USA). Briefly, HEK293 cells (1.0 × 10⁴ cells per well in a 48-well plate) were transiently transfected with constitutively active herpes simplex virus thymidine kinase promoter-driven Renilla luciferase (20 ng per well) as an internal control, a β-catenin–responsive firefly luciferase reporter plasmid TopFlash (50 ng per well) (Millipore), and Wnt1 expression plasmid (1 ng per well) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). After 4-hour incubation, the plasmids and the transfection reagent in DMEM supplemented with 10% FBS were replaced with a fresh medium containing recombinant mouse USAG-1 protein (1 μg/ml). Cells were harvested after 20 to 24 hours, and both firefly and Renilla luciferase activity were measured in duplicate or triplicate according to the manufacturer’s instructions. The firefly luciferase activity was normalized against the Renilla luciferase activity.
为了评估抗 USAG-1 抗体对重组小鼠 USAG-1 调节的 Wnt/β-catenin 信号传导的中和作用，我们使用了基于双荧光素酶报告基因测定系统的 TOP 报告基因系统（Promega，Madison，WI，USA）。简而言之，使用 Lipofectamine 3000 瞬时转染组成型活性单纯疱疹病毒胸苷激酶启动子驱动的海肾荧光素酶（每孔 20 ng）、一种 β-连环蛋白反应性萤火虫荧光素酶报告质粒 TopFlash（每孔 50 ng）（Millipore）和 Wnt1 表达质粒（每孔 1 ng）瞬时转染 HEK293 细胞×（48 孔板中 10 ng^{4 个细胞} ）作为内部对照、连环蛋白反应萤火虫荧光素酶报告质粒 TopFlash（每孔 50 ng）（Millipore）和 Wnt1 表达质粒（每孔 1 ng）（Thermo Fisher Scientific， 美国马萨诸塞州沃尔瑟姆）。孵育 4 小时后，将补充有 10%FBS 的 DMEM 中的质粒和转染试剂替换为含有重组小鼠 USAG-1 蛋白（1μg/ml）的新鲜培养基。20 至 24 小时后收获细胞，并根据制造商的说明一式两份或一式三份测量萤火虫和海肾荧光素酶活性。萤火虫荧光素酶活性与海肾荧光素酶活性标准化。

Epitope mapping was performed by Kinexus Co Ltd. (Vancouver, Canada). Briefly, SPOT synthesis of two copies of a peptide array (15-mer peptide scan of a protein with 183 amino acids; human Sostdc1 without signal peptide) was performed on a cellulose membrane. Two of the synthesized copies of the peptide array were incubated with primary mouse USAG-1 antibodies (0.3 g/ml), and the bound antibody was detected by incubating the arrays with the detection reagent (1:25,000 dilution; HRPalpaca anti-mouse antibody) and subsequent treatment with electrochemiluminescence reagent.
表位图谱由 Kinexus Co Ltd.（加拿大温哥华）进行。简而言之，在纤维素膜上进行肽阵列的两个拷贝的 SPOT 合成（具有 183 个氨基酸的蛋白质的 15 聚体肽扫描;没有信号肽的人 Sostdc1）。将两个合成的肽阵列拷贝与原代小鼠 USAG-1 抗体（0.3 g/ml）一起孵育，并通过将阵列与检测试剂（1：25,000 稀释;HRPalpaca 抗小鼠抗体）和随后用电化学发光试剂处理。

Reactivity of each monoclonal antibody (mAb) with native USAG-1 in solution was evaluated by immunoprecipitation. Briefly, 5 μg of purified anti–USAG-1 mAbs was incubated with 15 μl of Protein A-Sepharose (GE Healthcare) for 2.5 hours at 15° to 25°C, followed by a brief wash with PBS. The beads were incubated with the culture supernatants of the Expi293F cells transiently transfected with either mouse or human USAG-1 containing N-terminal PA tag (42). After extensive washing with PBS, the bound proteins were eluted from the beads by adding SDS sample buffer and then analyzed by SDS–polyacrylamide gel electrophoresis (PAGE) using 5 to 20% gradient gel under nonreducing conditions.
通过免疫沉淀评估每种单克隆抗体 （mAb） 与溶液中天然 USAG-1 的反应性。简而言之，将 5 μg 纯化的抗 USAG-1 mAb 与 15 μl 蛋白 A-琼脂糖 （GE Healthcare） 在 15° 至 25°C 下孵育 2.5 小时，然后用 PBS 短暂洗涤。将珠子与瞬时转染含有 N 端 PA 标签的小鼠或人 USAG-1 的 Expi293F 细胞的培养上清液一起孵育 （42）。用 PBS 广泛洗涤后，通过添加 SDS 样品缓冲液从珠子中洗脱结合的蛋白质，然后在非还原条件下使用 5%至 20%梯度凝胶通过 SDS-聚丙烯酰胺凝胶电泳（PAGE）进行分析。

Binding kinetics of anti–USAG-1 antibodies were analyzed using bio-layer interferometry with Octet RED system (ForteBio, Fremont, CA, USA). Binding assays were performed in 96-well microtiter plates at 25°C with orbital sensor agitation at 1000 rpm. Amine reactive (AR2G) sensors were immobilized with each antibody dissolved at 10 to 20 μg/ml in 10 mM sodium acetate buffer (pH 6.0) followed by quenching with 1 M ethanolamine (pH 8.5). Purified mouse USAG-1 was serially diluted in a running buffer [20 mM Hepes and 150 mM NaCl (pH 7.2) containing 0.005% Tween 20] and added to different wells (final volume: 200 μl). The binding was monitored by dipping the sensors into the wells for 120 s, followed by dissociation in the running buffer for 120 s. After each binding experiment cycle, antibody-immobilized biosensors were regenerated by dipping in a regeneration buffer [10 mM glycine-HCl (pH 3.0)]. The K_D values were determined using Octet Data Analysis Software 7.1 (ForteBio) using a 1:1 global fitting model.
使用生物层干涉测量法和 Octet RED 系统（ForteBio，Fremont，CA，USA）分析抗 USAG-1 抗体的结合动力学。在 25°C 的 96 孔微量滴定板中进行结合测定，并以 1000 rpm 的速度搅拌轨道传感器。将胺反应性（AR2G）传感器固定化，将每种抗体以 10 至 20μg/ml 溶解在 10 mM 乙酸钠缓冲液（pH 6.0）中，然后用 1 M 乙醇胺（pH 8.5）淬灭。将纯化的小鼠 USAG-1 在运行缓冲液[含有 0.005%吐温 20]的 20mM Hepes 和 150mM NaCl（pH 7.2）中连续稀释，并加入不同的孔中（最终体积：200μl）。通过将传感器浸入孔中 120 秒，然后在运行缓冲液中解离 120 秒来监测结合。在每个结合实验周期之后，通过浸入再生缓冲液 [10 mM 甘氨酸-盐酸 （pH 3.0）] 中再生抗体固定化生物传感器。K_D 值是使用 Octet Data Analysis Software 7.1 （ForteBio） 使用 1：1 全局拟合模型确定的。

Binding between USAG-1 and LRP6 ectodomain was evaluated as follows. The soluble human LRP6 ectodomain fragments containing different regions (E1-E4, residues 1 to 1244; E1-E2, residues 1 to 629; E3-E4, residues 630 to 1244) were C-terminally His-tagged and transiently expressed in Expi293F cells as described previously (44). After immobilizing onto Ni-NTA beads, they were further incubated with the culture supernatants of the Expi293F cells stably expressing mouse USAG-1 established previously (42). The bound USAG-1 was eluted together with the LRP6 fragments by SDS and analyzed by nonreducing SDS-PAGE. For the assessment of the ability of anti–USAG-1 antibodies to compete with LRP6 binding, Protein A beads were incubated with each antibody (step 1), followed by incubation with USAG-1 (step 2), and lastly with LRP6 E1-E2 fragment (step 3) to allow the formation of a ternary complex. The bound proteins were analyzed by nonreducing SDS-PAGE. The diminished intensity of the signal corresponding to the LRP6 E1-E2 fragment indicated the overlap of the binding sites for the antibody and LRP6.
USAG-1 和 LRP6 胞外结构域之间的结合评估如下。含有不同区域（E1-E4，残基 1-1244;E1-E2，残基 1 至 629;E3-E4，残基 630 至 1244）在 C 端 His 标记并在 Expi293F 细胞中瞬时表达，如前所述 （44）。固定在 Ni-NTA 珠子上后，将它们与先前建立的稳定表达小鼠 USAG-1 的 Expi293F 细胞的培养上清液进一步孵育（42）。将结合的 USAG-1 与 LRP6 片段一起通过 SDS 洗脱，并通过非还原 SDS-PAGE 进行分析。为了评估抗 USAG-1 抗体与 LRP6 结合竞争的能力，将蛋白 A 珠与每种抗体一起孵育（步骤 1），然后与 USAG-1 孵育（步骤 2），最后与 LRP6 E1-E2 片段孵育（步骤 3）以形成三元复合物。通过非还原 SDS-PAGE 分析结合蛋白。与 LRP6 E1-E2 片段相对应的信号强度降低表明抗体和 LRP6 的结合位点重叠。

Pregnant EDA1 mice at E13 of gestation (4 to 6 weeks) were intraperitoneally injected with anti–USAG-1 antibodies (16 μg/g). Offspring were analyzed at 5 weeks of age. After removing the skin, dissected maxillae and mandibles from the heads of the offspring were soaked in 0.02% proteinase K prepared in PBS at 37°C for 4 days and cleaned with 5% H₂O₂ at 15° to 25°C for 5 min. Last, they were rinsed in H₂O and air-dried. Neonates were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (7 mm) were cut and stained with hematoxylin and eosin. Offspring of ferrets at 1 and 3 weeks after birth or 1, 3, and 5 weeks were intraperitoneally injected with anti–USAG-1 antibodies (16 or 80 μg/g). They were analyzed by taking photographs and micro-computed tomography (micro-CT).
在妊娠 E13（4 至 6 周）的怀孕 EDA1 小鼠腹腔注射抗 USAG-1 抗体（16μg/g）。在 5 周龄时对后代进行分析。去除皮肤后，将从后代头部解剖的上颌骨和下颌骨浸泡在 PBS 中制备的 0.02%蛋白酶 K 中，在 37°C 下浸泡 4 天，并用 5%H₂O₂ 在 15°至 25°C 下清洁 5 分钟。最后，将它们在 H₂O 中冲洗并风干。新生儿固定在 4%多聚甲醛中并包埋在石蜡中。切片（7 mm）被切割并用苏木精和伊红染色。在出生后 1 周和 3 周或 1、3 和 5 周时，腹腔注射抗 USAG-1 抗体（16 或 80 μg/g）的雪貂后代。通过拍照和显微计算机断层扫描 （micro-CT） 对其进行分析。

We performed 3D micro-CT scans (inspeXio SMX-100CT; Shimadzu, Kyoto, Japan) on the maxillary incisors of ferrets, 13 weeks after birth. We converted CB files [512 × 512 pixels, 8 bits; voxel size, x:y:z = 1:1:1 (~0.06 mm per side)] to TIFF files, and 3D images were reconstructed and analyzed using computer imaging software (VGSTUDIO MAX; Volume Graphics GmbH., Heidelberg, Germany).
我们进行了 3D 显微 CT 扫描（inspeXio SMX-100CT;Shimadzu， Kyoto， Japan）在雪貂的上颌切牙上，出生后 13 周。我们将 CB 文件[512 × 512 像素，8 位;体素大小，x：y：z = 1：1：1（每边~0.06 mm）]转换为 TIFF 文件，并使用计算机成像软件（VGSTUDIO MAX;Volume Graphics GmbH.，德国海德堡）。

Immunocytochemistry was performed using standard techniques. Briefly, HEK293 cells were seeded on poly-l-lysine–coated coverslips (Matsunami Glass Ind. Ltd., Osaka, Japan). FLAG-tagged DAN family protein expression plasmids were transfected (1 μg per well) into the cells using Lipofectamine 3000. After transfection (24 hours), the cells were fixed with 4% paraformaldehyde/PBS (Sigma-Aldrich) for 30 min. Next, the cells were washed with PBS three times and incubated in blocking buffer (10% bovine serum albumin/PBS) for 1 hour, followed by incubation in the mouse monoclonal anti–USAG-1 antibody or anti-FLAG antibody (4 ng/ml) (Sigma-Aldrich) in the blocking buffer overnight at 4°C. To visualize the immunoreactivity, the cells were incubated with Cy3-labeled secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA)/PBS (1:400) after being washed three times with PBS. Nuclear staining was performed using 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific).
使用标准技术进行免疫细胞化学。简而言之，将 HEK293 细胞接种在聚左旋赖氨酸涂层的盖玻片上（Matsunami Glass Ind. Ltd.，大阪，日本）。使用 Lipofectamine 3000 将 FLAG 标记的 DAN 家族蛋白表达质粒转染（每孔 1 μg）到细胞中。转染后（24 小时），用 4%多聚甲醛/ PBS（Sigma-Aldrich）固定细胞 30 分钟。接下来，用 PBS 洗涤细胞 3 次，并在封闭缓冲液（10%牛血清白蛋白/ PBS）中孵育 1 小时，然后在小鼠单克隆抗 USAG-1 抗体或抗 FLAG 抗体（4 ng / ml）中孵育在封闭缓冲液中，在 4°C 下过夜。 为了可视化免疫反应性，在用 PBS 洗涤 3 次后，将细胞与 Cy3 标记的二抗（Jackson ImmunoResearch Laboratories，West Grove，PA，USA）/PBS（1：400）一起孵育。使用 4'，6-二脒基-2-苯基吲哚（Thermo Fisher Scientific）进行核染色。

Paraffin-embedded sections of ferret was immunostained with primary rabbit polyclonal antibodies against phosphorylated Smad 1/5/8 (1:50; Merck KGaA, Darmstadt, Germany) and secondary biotinylated anti-rabbit/mouse antibodies (Nichirei Bioscience, Tokyo, Japan), as previously described (11, 32). Sections were then counter-stained with hematoxylin, dehydrated in a graded series of ethanol and xylene, and covered with coverslips.
用针对磷酸化 Smad 的兔一级多克隆抗体对雪貂的石蜡包埋切片进行免疫染色 1/5/8 （1：50;如前所述，Merck KGaA，达姆施塔特，德国）和二级生物素化抗兔/小鼠抗体（Nichirei Bioscience，东京，日本）（11,32）。 然后用苏木精复染切片，在一系列分级乙醇和二甲苯中脱水，并用盖玻片覆盖。

Data are shown as means ± SEs. For comparing multiple conditions, a one-way analysis of variance (ANOVA) was performed, followed by two-tailed Dunnett’s multiple comparisons test. Statistical significance of differences was assessed as follows: *P < 0.05, **P < 0.01. Statistical analyses were performed using the SAS statistical software, version 9.4 (SAS Institute, Cary, NC).
数据显示为均值± SE。为了比较多种条件，进行了单因素方差分析（ANOVA），然后进行了双尾 Dunnett 多重比较检验。差异的统计学显着性评估如下：*P < 0.05，**P < 0.01。使用 SAS 统计软件 9.4 版（SAS Institute，Cary，NC）进行统计分析。