A direct link between Prss53, hair curvature, and skeletal dysplasia

In humans, protease serine S1 family member 53 (Prss53) is highly expressed in the hair follicle, especially the inner root sheath, which is associated with hair shape according to recent genome-wide association study (GWAS) data. However, no animal evidence has indicated a link between Prss53 and hair shape to date. Here, we used CRISPR/Cas9 to generate Prss53-mutated rabbits. The homozygous (Prss53-/-) rabbits exhibited curved hair and skeletal dyskinesia with severe malformation, while the heterozygous (Prss53+/-) rabbits did not exhibit these features. The curvature features of the hair were accompanied by lesions that were generally denser and less well-defined in the cuticular septation of the hair shaft, and the compartments of the hair follicle were incomplete, as evidenced by decreased expression levels of keratinocyte differentiation genes. In addition, skeletal dysplasia, an increased lethality rate and decreased plasma calcium and serum alkaline phosphatase (ALP) levels were determined in the Prss53-/- rabbits. Furthermore, disrupted calcium metabolism, which may play a role in the hair curvature and skeletal dysplasia of Prss53+/- rabbits, was demonstrated by using high-throughput RNA sequencing data. Thus, our study confirmed for the first time that the loss of Prss53 lead to curved hair in animals and provides new insights into the crucial role of Prss53 in calcium metabolism. Author Summary No animal evidence has indicated a link between Prss53 and hair shape to date. The Prss53-/- rabbits exhibited curved hair and skeletal dyskinesia. The disrupted calcium metabolism may play a role in the hair curvature and skeletal dysplasia of Prss53+/- rabbits.


Introduction
Membrane-anchored serine proteases regulate fundamental cellular and developmental processes, including orchestrating neural tube closure, erecting barriers between cells, developing the inner ear, regulating apical sodium entry and fluid volume, controlling the natriuretic peptide system, adjusting cellular iron export to satisfy iron needs, and promoting fertilization 1 . PRSS53, a member of the membrane-anchored serine proteases, hydrolyzes peptide bonds 2 and is highly expressed in the hair follicle IRS, medulla, precortex and some melanocytes 3 .
Previous studies reported that Prss53 is a psoriasis susceptibility locus and is linked to systemic lupus erythematosus, suggesting its potential roles in skin function [4][5][6][7] . The Q30R SNP in Prss53 may influence hair shape and is involved in the human variation of head hair shape among different continental groups 3,8 . In addition, ENaC activity is regulated by the activity of a channel-activating protease of Prss8 (a homologous gene of Prss53), implying the Prss53 may have an indispensable role in ion channel activity 9 .
The triggering of hair curvature is clearly related to symmetry or asymmetry axis formation in follicles and includes Wnt, TGFβ, BMP, Shh and FGFs, etc. 10,11 . In particular, the Wnt/Ca 2+ and Wnt/planar cell polarity (PCP) pathways are additional novel mechanisms that affect hair shape 12 . Furthermore, disruptions in calcium metabolism may contribute to a wide variety of clinical symptoms, including osteoporosis, osteolysis, nephrocalcinosis and adynamie 13 .
Here, we successfully generated Prss53-deficient rabbits. These rabbits exhibited curved hair, severe skeletal malformation and disrupted calcium metabolism.
Altogether, these data suggest that Prss53 may play an important role in calcium metabolism and provide the first direct evidence that Prss53 functions in hair curvature shape and skeletal dysplasia.

Prss53 expression pattern
To explore the function of the Prss53 gene, the expression pattern was determined, as shown in Figure 1. The EST profile data suggest extensive expression of Prss53 in the skin and bone in Homo sapiens . In addition, high Prss53 expression in the developing inner root sheath (IRS), precortex and medulla of the hair follicle has been reported in a previous study 3 , but the Prss53 expression pattern in bone has not been determined.
Generation of Prss53 -/rabbits via the CRISPR/Cas9 system Prss53, a candidate gene for curly hair, was found in a recent GWAS in Latin Americans of mixed European and Native American origin 3 . To disrupt the function of rabbit Prss53, two sgRNAs targeting the third and fourth exon of Prss53 were designed ( Figure 2A) and transferred to embryos. Then, the genomic DNA from each pup was isolated, and Prss53 gene mutations were detected via PCR and Sanger sequencing. As shown in Figure S1B, F0 rabbits carrying Prss53 mutations were obtained and were used for the following study. An off-target assay showed that the CRISPR/Cas9 system did not induce undesirable off-target effects in the Prss53  To obtain a sufficient number of rabbits for a detailed functional study of Prss53, the sexually mature F0 founder was mated with wildtype (WT) female rabbits, and then the heterozygous (Prss53 +/-) rabbits were mated and used to generate homozygous (Prss53 -/-) rabbits ( Figure. S3). These results demonstrated that mutations in Prss53 can be achieved via the CRISPR/Cas9 system with high efficiency in rabbits.

Hair curvature and skeletal dysplasia in Prss53 -/rabbits
Prss53 encodes a polyserine protease called polyserase-3 (POL3S) and is expressed in the IRS, medulla, and precortex of hair follicles and in melanocytes 3 . In our study, curved hair curvature was present all over the body of Prss53 -/rabbits ( Figure. 2B and S1A). The hair length was not different among the rabbits, but the Prss53 -/rabbits had significantly finer hair than that of the WT rabbits ( Figure. 2C and D). In addition, impaired physical activity was determined in the Prss53 -/rabbits, with a larger knee range of motion compared to the WT rabbits ( Figure.       and H) of the Prss53 -/rabbits. These results confirmed that Prss53 plays an important role in maintaining hair shape and skeletal health.

Abnormal keratinocyte differentiation in Prss53 -/rabbits
A previous study reported that abnormal keratinocyte differentiation negatively affects the hair shaft extension and hair shaft shape 14 . In this study, H&E staining showed no significant pathological changes of the hair follicles in the Prss53 -/rabbits ( Figure. 4A). Next, we examined the hair structure by scanning electron microscopy (SEM). As illustrated in Figure. Table   1). Thus, we suspected that abnormal calcium metabolism is responsible for the hair curvature phenotype of the Prss53 -/rabbits ( Figure. 5D).

Disrupted osteoblast differentiation in Prss53 -/rabbits
Disordered calcium metabolism may induce osteoporosis and osteolysis in clinical settings 13 . To further examine whether disrupted calcium metabolism is responsible for the skeletal dysplasia, the skeletal and a serum biochemical analysis were compared between the Prss53 -/and WT rabbits. As shown in Figure. 6A, the Prss53 -/rabbits exhibited more severe knee abnormalities and were substantially more malformed than WT rabbits by X-ray autoradiograph examination. The serum biochemical analysis demonstrated significantly decreased plasma calcium and ALP levels in the Prss53 -/rabbits ( Figure. 6B and C). In addition, immunohistochemistry and H&E staining confirmed the significantly reduced number of osteoblasts and the complete loss of PRSS53 protein in the Prss53 -/rabbits ( Figure. 6D, E and F) compared to the WT control. Hence, disrupted calcium metabolism is responsible for the skeletal abnormalities in the Prss53 -/rabbits.
In summary, our study provides the first evidence that Prss53 plays an important role in calcium metabolism, which is responsible for keratinocyte and osteoblast differentiation and, thereby, maintains the normal morphology of the hair and skeleton ( Figure. 5D).

Discussion
To date, the function Prss53 was largely unknown, and limited information regarding Prss53 mutant animals was available. In this study, we first generated a stably transmitted Prss53 mutant rabbit via the CRISPR/Cas9 system. Interestingly, the hair curvature phenotype of the Prss53 -/rabbits is consistent with the Latin American population of mixed European and Native American origin with a Q30R substitution in Prss53 3 .
Uncombable hair syndrome is characterized by dry, frizzy, and spangly hair, which has longitudinal grooves and a heart-shaped cross section by SEM 16 .
Consistently, lesions and less well-defined cuticular septation were determined in the Prss53 -/rabbits in our study. In addition, high-throughput RNA sequencing data revealed significantly decreased expression of keratinocyte differentiation genes, including Lats1 17 , Yap1 18 , Adam9 19 , Csta 20 , Krt10 21 , Wnt16 22 and Tp63 23 , in Prss53 -/rabbits compared with WT rabbits, A previous study showed that the hair shaft is derived from the progeny of keratinocyte stem cells in the follicular epithelium, and the growth and differentiation of follicular keratinocytes is guided by a specialized mesenchymal population 14 . Therefore, abnormal keratinocyte differentiation is responsible for disruptions to hair shaft shape, indicating that Prss53 may play an important role in maintaining keratinocyte differentiation.
Unexpectedly, impaired physical activity and decreased plasma calcium may be responsible for the early death of Prss53 -/rabbits. Calcium is essential for bone development by promoting osteogenesis and inhibiting osteoclast activity through increased osteoprotegerin secretion from osteoblasts 24 . Alkaline phosphatase is widely used as a marker of osteoblast differentiation and was significant decreased in the Prss53 -/rabbits when compared with WT rabbits. Previous studies reported that osteoblast differentiation is closely associated with skeletal development, such as in Med23-deficient MSCs or preosteoblasts that display defective bone formation and impaired osteoblast differentiation 25 . Runx2 −/− mice lack both mature osteoblasts and a mineralized skeleton 26,27 . Hence, this study is the first report to demonstrate that disrupted osteoblast differentiation in the Prss53 mutant is responsible for skeletal abnormalities, suggesting that Prss53 may have an indispensable role in skeletal development.
In this study, the significantly reduced number of osteoblasts and disrupted calcium metabolism were determined in the Prss53 -/rabbits by RNA sequencing technology. The G-protein-coupled receptor (CaR) is not limited to the parathyroid gland 28 , and also inkeratinocytes 29 . The calcium-sensing receptor, transient receptor potential channels, and STIM/Orai also function in calcium sensing and calcium entry in keratinocytes 30 . In addition, both extracellular calcium and 1,25(OH) 2 D raise the level of intracellular free calcium as a necessary step toward stimulating keratinocyte differentiation 15 . Accumulating studies have shown that calcium oscillations via restraints of major Ca 2+ entry sources (extracellular Ca 2+ influx and intracellular Ca 2+ release from the endoplasmic reticulum) is required for osteoblast differentiation 31 .
Hence, we hypothesize that disrupted calcium metabolism is responsible for abnormal keratinocyte differentiation in the Prss53 -/rabbits.
To the best of our knowledge, this study is the first animal report on hair curvature and skeletal dysplasia caused by a Prss53 mutation in rabbits and provides evidence that Prss53 is required for keratinocyte and osteoblast differentiation, which is essential for hair and skeletal cell proliferation.

Ethical statement
New Zealand rabbits were obtained from the Laboratory Animal Center of Jilin University. All rabbit experiments were conducted under the approval of the Animal Care Center and Use Committee of Jilin University.

sgRNA design and vector construction
The 3xFLAG-NLS-SpCas9-NLS vector (Addgene ID 48137) was linearized with NotI and in vitro transcribed using a mMessage mMachine SP6 Kit (Ambion) and RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions.

Microinjection and embryo transfer
The procedures of embryo microinjection and embryo transfer were previously described 33,34 . Briefly, female New Zealand white rabbits (6-8 months old) were superovulated with FSH (50 IU) every 12 h for 3 days. After the last injection, the female rabbits were mated with male rabbits and were then injected with 100 IU of human chorionic gonadotrophin (hCG). Rabbit embryos at the pronuclear stage were collected and transferred into oocyte manipulation medium. A mixture of Cas9 and sgRNA mRNA (200 ng/μl and 40 ng/μl, respectively) was microinjected into the embryo cytoplasm. The injected embryos were transferred to EBSS medium for short-term culture at 38.5°C, 5% CO 2 and 100% humidity conditions. Approximately 30-50 injected zygotes were transferred into the oviducts of recipient rabbits.

Mutation detection in pups by PCR
Genomic DNA from WT and Prss53 mutant rabbits was isolated using a TIANamp Genomic DNA Kit (TIANGEN, Beijing, China). The DNA was amplified by 2×Taq Plus MasterMix (TIANGEN), and the PCR primers used to detect mutations were as follows: rabbits-F-5' CAG GAA GTT CCA GTC ACT TGT -3', rabbits-R-5' GGT TGA GAA GGA AGG GAG ATT AG-3'. The PCR products were purified and cloned into the pGM-T vector (TIANGEN, Beijing, China); at least 10 positive plasmid clones were sequenced and analyzed using NCBI BLAST.

Western blot
Western blot analysis was performed as described previously 35 . Samples from the head skin of Prss53 -/and WT rabbits were homogenized and lysed in RIPA buffer supplemented with 2.5 μL/mL protease inhibitor cocktail (Roche) on ice for 30 min. Anti-Prss53 (1:1000, Novus) were used as primary antibody, and β-actin antibody (1:2000, Abcam) was used as loading control.

Histopathology and immunohistochemistry
H&E staining was performed as previously described 33 . Briefly, the tissues were collected from WT and Prss53 -/rabbits, fixed in 4% paraformaldehyde for 48 h, embedded in paraffin wax, sectioned for slides, and stained with H&E.
The immunohistochemistry staining was performed as previously described 36 .

Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) was performed as described previously 37 .
The hair from the heads of Prss53 -/and WT rabbits was attached onto specimen stubs using double-sided conductive tabs and sputter-coated with gold using a Polaron SEM E-1010. Samples were imaged using a S-3400N Scanning Electron Microscope.
Transmission electron microscopy was performed as described previously 38 . The skin from the dorsum of Prss53 -/and WT rabbits was fixed in 3% glutaraldehyde for 4 h at 4°C. Ultrathin longitudinal sections of the skin were cut with an ultramicrotome and a diamond knife and processed for examination by transmission electron microscope (TEM) (H-7640, Hitachi, Japan).

Skeletal histomorphology
X-ray autoradiography pictures of the Prss53 -/and WT rabbits were taken as previously described 33 . Briefly, the YEMA Radiography System with a digital camera attached (Varian, USA) on X-ray film (ROTANODE, Japan) was used in this study. The images were taken at 40 KV with 3 mA exposure.