## 3 Results
### Expression Profile of QSOX1 in the Mouse Embryo  
The long and short forms of QSOX1 were detected in the embryo tissue extract, and the short form was the major one (Figure S4). Immunohistochemical staining showed that QSOX1 was widely expressed in various tissues at 15.5 and 17.5 dpc embryos (Figures 4a, S5 and S6a). HistoQuest was applied to quantify levels of QSOX1 protein expressed in each embryonic tissue. We selected an area on each marked tissue (a to v) to include approximately 100 to 600 cells depending on the tissue size. For example, we set a rectangular area in the midbrain and identified 506 cells. The staining intensity of QSOX1 in each cell was shown in the dot blot scattergram with an average intensity of 76 (Figure 4b). Based on this principle, we quantified the expression intensity of QSOX1 in 22 embryonic tissues. We divide the average intensity into three levels; intensity <40 is the weak expression, 40-60 is defined as medium expression, and greater than 60 is the strong expression. As shown in Figure 4c, QSOX1 was significantly expressed in the spinal cord, lens, midbrain, and medulla oblongata. The heart, intercostal muscle, diaphragm, intermediate zone, extrinsic ocular muscle, spine, pons, and epidermis showed moderate expression. The lung, liver, submandibular gland, neopallial cortex, inner retina layer, small intestine, dermis, hair follicle, and pancreas were relatively weakly expressed. Figure 4: The expression level of QSOX1 in various embryonic tissues at 15.5 dpc. (**a**) A representative image of QSOX1 distribution in the 15.5 dpc embryo. The control staining without anti-QSOX1 antiserum is shown in Figure S5. The marks (a to v) indicate the tissues subjected to quantify and their locations. Scale bar = 1 mm. (**b**) The expression intensity of QSOX1 in all cells covered by the midbrain’s rectangular area, region of interest, was quantified using HistoQuest, as shown in the scattergram at the right. (**c**) The scatter plot shows the mean intensity of QSOX1 in cells of each tissue; every small circle indicates the expression intensity of QSOX1 in a single cell. Statistical comparison on expression levels among tissues is shown in the Supplementary Materials, Table S1. QSOX1 was prominently expressed in the brain area at the cerebellum, medulla oblongata, and pons. The midbrain also showed intense staining except for the midbrain roof (Figure 5a). In the striatum, the staining was detected around the neopallial cortex and the intermediate zone (Figure 5b). In the eyes, the lens fiber and epithelium showed more strong staining than the corneal epithelium and retinal pigment (Figure 5c). Significant QSOX1 levels were detected in the spinal cord and observed in the spinal hypertrophic chondrocytes but not in the matured cartilage. Brown fat was weakly stained (Figure 5d). The epidermis showed intense QSOX1 staining, while the dermis was stained relatively weak (Figure 5e). The protein was also expressed in myocardial cells of the heart (Figure 5f), in the lung alveoli and bronchus (Figure 5g), in liver hepatocytes (Figure 5h), in pancreatic secretory cells (Figure 5i), and in mucosal villi of the small intestine (Figure 5j). Although QSOX1 was still distributed in most embryonic tissues at 17.5 dpc, its levels in various tissues appeared to be changed (Figure 5a). QSOX1 was moderately expressed in the heart, liver, spinal cord, and brown fat, followed by the lung, submandibular gland, epidermis, midbrain, and mesentery. The expression levels in the diaphragm, striatum, small intestine, intercostal muscle, and dermis were low (Figure 5b). Compared with 15.5 dpc embryos, QSOX1 levels in the spinal cord, brain regions, epidermis, and diaphragm turned to decrease but increased in the liver and heart. Figure 5: Cellular expression and localization of QSOX1 in tissues of the embryo at 15.5 dpc. The representative longitudinal section of the 15.5 dpc embryo is shown on the top left. The detailed QSOX1 cellular distribution is enlarged from ten subregions (a to f).