Abstract
Dominant white phenotype in pigs is considered to be caused by two structural mutations in KIT gene, including a 450-kb duplication encompassing the entire KIT gene, and a splice mutation (G > A) at the first base in intron 17, which leads to the deletion of exon 17 in mature KIT mRNA, and the production of KIT protein lacking a critical catalytic domain of kinase. However, this speculation has not yet been validated by functional studies. Here, by using CRISPR/Cas9 technology, we created two mouse models mimicing the structural mutations of KIT gene in dominant white pigs, including the splice mutation mouse model KIT D17/+ with exon 17 of one allele of KIT gene deleted, and duplication mutation mouse model KIT Dup/+ with one allele of KIT gene coding sequence (CDS) duplicated. We found that each mutation individually can not cause dominant white phenotype. Splice mutation homozygote is lethal and heterozygous mice present piebald coat. Inconsistent with previous speculation, we found KIT gene duplication mutation did not confer the patched phenotype, and had no obvious impact on coat color. Interestingly, combination of these two mutations lead to dominant white phenotype. Further molecular analysis revealed that combination of these two structural mutations could inhibit the kinase activity of the KIT protein, thus reduce the phosphorylation level of PI3K and MAPK pathway associated proteins, which may be related to the observed impaired migration of melanoblasts during embryonic development, and eventually lead to dominant white phenotype. Our study provides a further insight into the underlying genetic mechanisms of porcine dominant white coat colour.
Author summary KIT plays a critical role in control of coat colour in mammals. Two mutation coexistence in KIT are considered to be the cause of the Dominant white phenotype in pigs. One mutation is a 450-kb large duplication encompassing the entire KIT gene, another mutation is a splice mutation causing the skipping of KIT exon 17. The mechanism of these two mutations of KIT on coat color formation has not yet been validated. In this study, by using genome edited mouse models, we found each structural mutation individual does not lead dominant white phenotype, but combination of these two mutations could lead to a nearly complete white coat colour similar to pig dominant white phenotype, possibly due to the inhibition of the kinase activity of the KIT protein, thus its signalling function on PI3K and MAPK pathways, leading to impaired migration of melanoblasts during embryonic development, and eventually lead to dominant white phenotype. Our study provides a further insight into the underlying genetic mechanisms of porcine dominant white coat colour.
Introduction
Due to domestication and long term selection, dominant white is a widespread coat color among domestic pig breeds, such as Landrace and Large White [1]. The dominant white phenotype in domestic pigs is considered to be caused by two structural mutations in the KIT gene, (1) a ∼450-kb tandem duplication that encompasses the entire KIT gene body and ∼150 kb upstream region of KIT gene and (2) a splice mutation at the first nucleotide of intron 17 in one of the KIT copies that leads to the skipping of exon 17, and the production of KIT protein lacking a critical region in kinase catalytic domain. [2–6].
KIT is a class III tyrosine kinase receptor, encoded by the KIT gene. KIT receptor is expressed on several cell types, including mast cells, hematopoietic progenitors, melanoblasts and differentiated melanocytes [7]. The binding of its ligand – stem cell factor (SCF) causes KIT to homodimerize, leading to the activation of its intrinsic kinase activity through autophosphorylation of tyrosine residues. KIT has a number of potential tyrosine phosphorylation sites, which interact with multiple downstream signaling pathways, including the PI3K, MAPK, and Src family kinase pathways [7, 8]. These pathways are involved in the regulation of cells growth, survival, migration and differentiation [9].
The 450-kb large duplication that encompasses the entire KIT gene body previously was speculated to confer the patch phenotype in pigs due to abnormal KIT expression [4]. Based on this, a hypothesis has been proposed that there is an evolutionary scenario whereby the duplication first occurred and resulted in a white-spotted phenotype that was selected by humans. The splice mutation occurred subsequently and resulted in a completely white phenotype, due to the skipping of exon 17 in the mature transcript removes a crucial part of the tyrosine kinase domain, thus enhances the defect in KIT signaling functions [5], and disturbs the migration of melanocyte precursors, leading to dominant white coat colour [2]. This seems reasonable, as normal migration and survival of neural crest-derived melanocyte precursors is dependent on KIT expression and the availability of its ligand [10]. Loss of function mutations in KIT gene could lead to white coat color in mouse, as documented in homozygous KIT K641E mouse [11] and KIT-deficient model Wv/Wv [12]. However, functional analysis of the structural mutations in KIT gene of dominant pigs still need to be carried out to confirm the hypothesis. Here, by using CRISPR/Cas9 technology, we created mouse models mimicking the splice mutation and duplication mutation to investigate the underlying genetic mechanism of dominant white phenotype[13].
Results
Splice mutation but not the duplication mutation of KIT gene leads to altered coat color
In pigs, the wild-type KIT allele is recessive and denoted as i. Previous studies considered that two different mutant KIT alleles semidominant IP allele and the dominant I allele confer the patch and dominant white phenotype, respectively (Fig. 1A). I allele presents a 450-kb large duplication (two to three copies) that encompasses the entire KIT gene and at least one of the KIT copies carries a splice mutation (G>A at the first base in intron 17), causing exon skipping and the expression of a KIT protein lacking an essential part of the tyrosine kinase domain. IP allele involves the 450-kb duplication but not the splice mutation[6]. To investigate the effects of KIT gene structural mutations on coat color, we created three genome edited mouse models using C57BL/6 strain. The C57BL/6 mice is dominant black and broadly used in coat color study [14].
To mimic the duplication mutation (IP allele), we knocked in the CDS of KIT gene linked with the enhanced green fluorescent protein (EGFP) reporter via a self-cleaving 2A peptide to facilitate subsequent identification (Fig. 1B). The heterologous of KIT duplication mouse model was denoted as KIT Dup/+. Western blot analysis results demonstrated that EGFP was extensively expressed in the skin of KIT Dup/+ mice, as compared with the wild-type control KIT+/+, which implying the inserted KIT CDS was correctly expressed, as 2A peptide strategy allows the co-expression of KIT proteins and EGFP from the integrated single vector (Fig. 1D). We found duplication of KIT gene did not result in the patch phenotype, as no obvious difference was observed on coat color between KIT Dup/+ and KIT+/+ mice (Fig. 1C).
To mimic the splice mutation, we substitute the first nucleotide (G) of KIT gene intron 17 with A through CRISPR/Cas9 mediated homologous recombination (Fig. 1B). The heterologous of this splice mutation mouse model was denoted as KIT GtoA/+. An extensive screening of the offspring implies that homologous of splice mutation of KIT gene could be lethal, as no survived individual of KIT GtoA/GtoA has been identified. Big white spots appeared on the abdomen of KIT GtoA/+ mice as compared with KIT+/+ (S1 Fig). To determine whether the G to A mutation at the first nucleotide of intron 17 of KIT gene can lead to the skipping of exon 17, RT-PCR was carried out by using the RNA isolated from the skin of KIT GtoA/+ mice. To our surprise, the results showed that exon 17 was not removed from the transcript mRNA, and a small percent of the transcript contained partial region of the intron 17, as determined by Sanger sequencing (Fig. 1F & G). As this model does not mimic the splice mutation well, we did not use it in the subsequent studies.
Therefore, in order to create a mouse model mimicking the skipping of exon 17, we have directly deleted the exon 17 in genomic by using paired sgRNA with one target the intron 16 and another one targets the intron 17 (Fig. 1B). The heterologous of this splice mutation mouse model was denoted as KIT D17/+. An extensive screening of the offspring implies that homologous of splice mutation of KIT gene could be lethal, as no survived individual of KIT D17/D17 has been identified. In addition, IVF experiment by fertilization oocytes from KIT D17/+ females with sperm from KIT D17/+ males resulted in no survived individual of KIT D17/D17 can be obtained (S1 Table). This confirms the previous speculation that IL allele (single copy of KIT gene with the splice mutation) could be lethal in pigs, as this allele was not found among worldwide pig population [6]. RT-PCR analysis of the skin tissue from KIT D17/+ mice indicates that exon 17 is removed from the mature transcript (Fig. 1E), and this was confirmed by Sanger sequencing (Fig. 1G). Interestingly, compared with KIT+/+, KIT D17/+ mice presented a piebald coat colour in head and trunk, a vertical white stripe on the forehead, a half loop of white hair on the shoulder blade area, and dominant white at entire abdominal part. (Fig. 1D & S1 Fig).
Splice mutation but not the duplication mutation of KIT gene significantly reduces melanin accumulation
Histological analysis (Fontana-Masson staining) of the back skin of 5-week old mice, revealed that similar to the KIT +/+ control mice, the hair follicles of both KIT Dup/+and KIT D17/+ mice are long in length, and in the hair bulb, the dermal papilla is completely coated by matrix, the keratogenous zone region is clearly visible and is connected to hair shaft and dermal papilla, and fibrous tract is substantially invisible in the skin (Fig. 2). These results indicate that similar to the KIT +/+ control mice, the hair follicles of 5-week-old KIT Dup/+and KIT D17/+ mice are in the growing stage (anagen V or VI), which is advantageous for the observation of the hair follicle shape, and melanin distribution due to melanin synthesis is more active during this stage [15]. The results of hair follicle shape imply that both the splice mutation and duplication mutation did not impair the hair follicle development significantly.
No obvious difference was observed in the content of melanin contained in hair follicles between KIT Dup/+ and KIT +/+ mice (Fig. 2). While almost no melanin is observed in the hair follicles within the white coat area, and the melanin level of hair follicles in the black coat area of KIT D17/+ is significantly lower (indicated by yellow arrow head). Therefore, the KIT duplication mutation did not impair the melanin accumulation, whereas the splice mutation significantly impaired melanin accumulation in hair follicle.
The piebald coat colour of KIT D17/+ mice is caused by the reduction of melanocytes
The reduction of melanin content in hair follicles may be due to reduced melanocytes, or reduced ability of melanocytes on melanin synthesis. To determine the piebald coat of KIT D17/+ mice is caused by which factor, we used KIT protein as the marker to detect the distribution and amounts of melanocytes in the hair follicle of 5-week-old mice. Compared with the KIT +/+ control mice, we found that the immunohistochemical staining of KIT decreased in the hair follicles of black coat area of KIT D17/+ mice, while the staining decreased more significantly in the white coat region (Fig. 3A). This was confirmed by qPCR and Western blot analysis of the skin tissues (Fig. 3B and 3C). In theory, the deletion of exon 17 should not affect the expression level of KIT gene, thus the reduced KIT expression level in skin tissue should be due to reduced melanocyte quantity. qPCR analysis of the isolated peritoneal cell derived mast cells indicated that deletion of the exon 17 did not affect the expression level of KIT gene (Fig. 3B & S2 Fig). The reduced expression of KIT gene together with another two marker genes (DCT and Melan A) of melanocytes in KIT D17/+ mice (Fig. 3C) suggested that the splice mutation of KIT gene could lead to reduced number of melanocytes in mice hair follicles.
Unlike the KIT D17/+mice, the KIT Dup/+ mice contained an additional copy of KIT CDS, which in theory could lead to increased expression of KIT gene. qPCR analysis of the isolated mast cells confirmed that the expression level of KIT gene was improved in KIT Dup/+ mice as compared with the KIT +/+ control mice (Fig. 3B). However, in mouse skin, the expression level of KIT in KITDup/+ mice was not significantly different from the KIT +/+ control mice as revealed by immunohistochemical and Western blot analysis (Fig. 3A & C). in addition, the expression level of melanocyte marker gene DCT was not affected, but another melanocyte marker gene MelanA was significantly improved in the skin of KIT Dup/+ mice (Fig. 3C). These results suggested that the duplication mutation of KIT gene may not affect the number of melanocytes in the skin, but may affect melanin synthesis.
Interestingly, we observed that the distribution of melanocytes in hair bulb is broader in KIT D17/+mice as compared with the KIT +/+ control mice. This phenomenon was more obvious in white coat area than in the black coat area (Fig. 3A). In addition, we found the distribution of melanocytes in the hair bulb of KIT Dup/+ mice was relatively broader than that in the KIT +/+ control mice (Fig. 3A). Previous studies considered that only melanocytes that are close to the dermal papilla can secrete and provide melanin to the hair [16], we speculate the altered distribution of melanocyte in both KIT splice mutation and duplication mutation mice may have certain impact on melanin accumulation.
Splice mutation of KIT gene impairs the kinase activity of the KIT protein and affects embryonic melanoblast migration
Previous studies speculated that KIT mutations in dominant white pigs could disturb the migration of melanocyte precursors melanoblasts during the embryonic period [2]. In order to determine whether the splice mutation or the duplication mutation of KIT gene impairs the migration of melanoblasts during embryonic period, we stained the KIT protein as a marker to detect the distribution of melanoblasts in the transverse section mice at E14.5. We found no obvious changes in the location of melanoblasts in KIT Dup/+compared to the KIT +/+ control mice (S3 Fig). Though the distribution of melanoblasts in KIT D17/+ near the neural tube is not different between in KIT D17/+ and the KIT +/+ mice, the number of melanoblasts in the dorsolateral migration pathway, near the forelimb and the abdomen epidermis is significantly reduced (Fig. 4A). This indicates that duplication mutation of KIT gene alone does not impair the migration of melanoblasts, in contrast, the splice mutation could significantly impair the migration of melanoblasts at embryonic stage, however, it does not completely block the migration process, a certain number of melanoblasts could migrate to the corresponding destination positions, and leads to the piebald phenotype.
We observed that, compared with the KIT +/+ control mice, the colour of black hair of KIT D17/+ mice became significantly lighter as the mice grew older. The determination of the blackness of mouse hair showed that the blackness of the black hair of KIT D17/+ mice was comparable to that of KIT +/+mice at 2 W, however, the blackness of the black hair of KIT D17/+ mice decreased dramatically at 14 W, and was close to that of white hair of the KIT D17/+ mice (Fig. 4B). Interestingly, we found the blackness of the hair of KIT Dup/+mice was relatively lower than that of KIT +/+mice at 2 W, but became comparable to that of KIT +/+mice at 14 W (Fig. 4B). Thus, we speculate that the splice mutation of KIT gene may affect the renewal or melanin synthesis function of melanocytes in mice, which in turn causes the blackness of hair to decrease rapidly with age. To examine whether the impaired melanoblast migration and melanocyte renewal is caused by altered kinase activity of the KIT protein receptor, Western blot analysis of the skin tissue was carried out. We found splice mutation significantly reduced the phosphorylation level of KIT protein (Fig. 3C & D), indicating this mutation could lead to impaired autophorylation ability of KIT protein. However, both the expression level and phosphorylation level of AKT, a key protein of the PI3K pathway, was not impaired by the splice mutation of KIT gene. Also, the expression of ERK1/2, key proteins of the MAPK pathway, was not affected by the splice mutation, but the phosphorylation level of ERK1/2 was slightly increased (Fig. 3C & D). This result looks confusing, therefore, we further analyzed the expression and phosphorylation levels of AKT and ERK1/2 in follicles by immunohistochemical (IHC) analysis. The amount of target protein was determined by using Combined Positive Score (CPS), which is the number of target protein staining cells divided by the total number of viable cells, multiplied by 100. The results revealed that the expression level of both AKT and ERK1/2 in hair follicles was not affected by the splice mutation of KIT gene (Fig. 4C), however, the phosphorylation levels of these proteins decreased significantly in the follicle of black coat region of KIT D17/+mice, and phosphorylated AKT and ERK1/2 barely can be detected in the follicle of white coat region of KIT D17/+mice (Fig. 4C). Western blot results indicate that the duplication mutation of KIT gene increased the phosphorylation level of KIT protein (Fig. 3C & D). This is probably due to the increased expression level of KIT protein. Similar to the splice mutation, the duplication mutation did not affect both the expression level and phosphorylation level of AKT. It also did not affect the expression level of ERK1/2, but slightly increased the phosphorylation level of ERK1/2 (Fig. 3C & D). IHC analysis revealed that both the expression level and the phosphorylation levels of AKT and ERK1/2 in the follicle of KIT Dup/+mice was not significantly affected (Fig. 4C). As AKT and ERK1/2 are respectively involved in the PI3K and MAPK pathways, which are responsible for melanoblast migration and differentiation, and melanin synthesis in melanocyte, therefore, the impaired melanoblast migration and accelerated hair greying in KIT D17/+mice should be related to impaired function of KIT kinase caused by the splice mutation of KIT gene.
Combination of the splice mutation and duplication mutation of KIT gene caused severely impaired melanoblast migration during embryonic stage, and dominant white phenotype
As the splice mutation and duplication mutation of KIT gene individually did not lead to dominant white phenotype, we are curious whether the combination of these two mutations (denoted as compound mutations) can lead to dominant white phenotype. Therefore, the KIT Dup/+male and the KIT D17/+female was crossed to produce the KIT Dup/D17 offspring as determined by PCR analysis of the deleted exon 17 and integrated EGFP reporter (Fig. 5A). Interestingly, the KIT Dup/D17 mice presented a coat colour resembling the porcine dominant white phenotype: except for few gray hairs appearing near the eyelids and hip, the whole body was covered with white hairs (Fig. 5B). With the increase of age, the gray hairs of the eyelids and hips of KIT Dup/D17 mice gradually disappeared (S4 Fig). Through histological observation of the back skin of KIT Dup/D17 mice, we found that melanin is hardly visible in the hair follicles (Fig. 5C). However, no apparent morphological difference of the follicle was observed between KIT Dup/D17 mice and the KIT +/+ control mice. The hair follicles of 5-week-old KIT Dup/D17mice showed a typical characteristics of the growing stage (anagen V or VI) (Fig. 5C). This indicates that the compound mutation did not affect the development of hair follicle, but severely impaired the accumulation of melanin in hair follicles.
We suspected that similar to KIT D17/+mice, the decreased melanin accumulation in the hair follicles of KIT Dup/D17 mice may be caused by reduced number of melanocytes in the hair follicles. qPCR analysis of the isolated mast cells from KIT Dup/D17 mice showed that compound mutations lead to improved expression level of KIT gene (Fig. 5D), which was mainly due to integrated additional copy of KIT CDS. In contrast, the expression level of KIT gene decreased significantly in skin tissue of KIT Dup/D17 mice as determined by qPCR analysis (Fig. 5D), this was further confirmed by Western blot analysis (Fig. 5E). The reduced expression of KIT gene together with another three marker genes (DCT, MelanA and S100) of melanocyte in skin tissue of KIT Dup/D17 mice (Fig. 5E) suggested that compound mutations of KIT gene could lead to reduced number of melanocytes in mice hair follicles, which may contribute to the decreased melanin accumulation in the hair follicles of KIT Dup/D17 mice.
IHC analysis of the skin tissue confirmed that the number of melanocytes in the hair follicles of KIT Dup/D17 mice decreased significantly as compared with the KIT +/+ control mice, with only a few layers of melanocytes in close proximity to the dermal papilla visible (Fig. 5C).
Few gray hairs presented in the whole white background of KIT Dup/D17 mice at young age, and they disappear gradually as determined by the hair blackness analysis (Fig. 5G & S4 Fig). This implies that the compound mutations of KIT gene may affect the renewal of melanocytes in hair follicles.
To investigate the underlying molecular mechanism of the compound mutations of KIT gene on coat colour changing, the kinase function of KIT protein was determined by Western blot analysis of the skin tissue of KIT Dup/D17 mice. The results showed that the phosphorylation level of KIT in the skin of KIT Dup/D17 mice is significantly lower than that of the KIT +/+ control mice (Fig. 5E). Both the expression level and phosphorylation level of AKT, a key protein of the PI3K pathway, decreased significantly. Though the expression of ERK1/2, key proteins of the MAPK pathway, was not affected, but the phosphorylation level of ERK1/2 decreased dramatically (Fig. 5E). These results indicate that the compound mutations of KIT gene substantially impaired the signaling function of KIT protein receptor on PI3K and MAPK pathways. However, to our surprise, further IHC analysis of the hair follicle showed that phosphorylation of AKT and ERK1/2 in KIT Dup/D17seems not affected by the compound mutations as determined by CPS (Fig. 5F). This may imply a complex interaction between the splice mutation and duplication mutation of the KIT gene.
The compound mutations of KIT gene impaired the signaling function of KIT protein, which in turn could affect melanoblast migration at embryonic stage. Therefore, IHC analysis of the distribution of melanoblasts in the transverse section of KIT Dup/D17 mice at E14.5 by staining the marker protein KIT. The results showed that compared with the KIT +/+control mice, the number of melanblast in the embryo of KIT Dup/D17 mice increased significantly in the neural tube, and the number of melanoblasts in the dorsolateral migration pathway, the forelimb epidermis and the abdomen epidermis decreased significantly (Fig. 5H). This result indicates that the compound mutations of KIT gene severely blocked melanoblast migration at embryonic stage, leading to increased accumulation of melanoblasts in the neural tube of the KIT Dup/D17 mice, and a coat colour resembling porcine dominant white phenotype.
Discussion
In our study, we created mice models by using CRISPR/Cas9 technology to mimic the structural mutations of KIT gene in dominant white pigs. We used KIT D17/+ mice model to research the effect of coat colour on KIT exon 17 deletion and explored the impact mechanism of KIT duplication on coat colour by KIT Dup/+ mice model. We found that the KIT duplication did not influence mouse coat colour but KIT exon 17 deletion turns black hair of mouse into piebald colour. The experimental results prove that the KIT exon 17 deletion reduced the kinase function of KIT and impair it signaling transduction on PI3K and MAPK pathways, which are involved in melanoblast migration, leading to certain percent of melanoblast blocked in migration from dorsal to ventral region during embryo development, resulting in a piebald coat of the mouse. Interestingly, combination of these two mutations lead to dominant white phenotype. In mutation KIT Dup/D17 mouse embryo, melanoblasts severe blocked in the neural tube that could not migration. Those make KIT Dup/D17 mouse displaying domiant white phenotype (Fig 6).
KIT plays key roles in driving the melanocyte migration from the neural crest along the dorsolateral pathway to colonize the final destination in the skin[17]. Mutations at the KIT gene is associated with the Dominant White coat colour of several important commercial breeds, like Large White and Landrace. The Dominant White coat colour is determined by the duplication of about 450-kb region encompassing the entire KIT gene (copy number variation, CNV) and by the presence of a splice mutation in intron 17 in one of the duplicated copies, that causes the skipping of exon 17[3]. KIT allele with two normal KIT copies has been considered to cause the presence of pigmented regions (patches) in white pigs[3]. In addition, a hypothesis has been proposed that the KIT allele carries a single copy of a mutated KIT gene (with splice mutation) that should be lethal if homozygous [3, 18]. These perspectives have not yet been validated by functional studies in the past decades. Our functional study confirmed that homozygous of the splice mutation in KIT gene is lethal, as KIT D17/D17 mouse could not be obtained. However, whether the lethal is due to anemia in embryonic stage as previously found in KIT defect mouse model need to be further validated. We also found that duplication of the KIT gene may not contribute to the patch phenotype found in pigs, as both KIT Dup/+ and KIT Dup/Dup mice did not present the patch coat like that on Pietrain pig, but a coat colour basically indistinguishable from the KIT+/+ control mice (Fig.1C & S1 Fig). Previous study has proposed that increased KIT expression from pig IP may affect ligand availability, which in turn disturbs the migration of melanocyte precursors, resulting in the patch phenotype [2]. This dosage effect may not be true, as our results showed that increased expression level of KIT protein from additional copy of KIT gene (KIT CDS in our mouse model) seems to have minimal effect on signaling function of KIT protein receptor on PI3K and MAPK pathways (Fig. 3A & 3C), and thus no obvious effect on melanoblast migration, melanocyte and follicle development, and melanin synthesis. In different to KIT duplication presented in IP allele in the pig, in our KIT duplication mouse model, only KIT CDS was inserted, the large fragment of regulatory regions was not included. The duplicated copy in IP allele may lack some regulatory elements located more than 150 kb upstream of KIT gene body[4], this regulatory mutation may lead to dysregulated expression of one or both copies of KIT, and thus contribute to the patch phenotype. Mutations in other genes responsible for pigmentation in pigs may be associated with the patch phenotype could not be ruled out.
The exon 17 of KIT gene encodes the 790-831 amino acids of KIT protein receptor, a highly conserved region of tyrosine kinase domain, which contains Tyr 823 residue that that is conserved in almost all tyrosine kinases, which is phosphorylated during KIT activation and is thought to act to stabilize the stability of KIT tyrosine kinase activity [8]. The splice mutation leading to the lacking of this region is previous considered to be responsible for the impaired KIT signal transduction, and thus the severe defect in the migration and survival of melanocyte precursors. Our IHC analysis of the skin tissue confirmed that the splice mutation can impair KIT signal transduction on PI3K and MAPK pathways (Fig. 4C). PI3K pathway regulates cell growth, proliferation, differentiation and survival [24], and MAPK regulates cell proliferation and apoptosis [25]. In addition, the MAPK pathway is also responsible for phosphorylating and activating MITF, which in turn activates the transcription of mRNAs of several important proteins involved in melanin synthesis, such as Tyrosinase, TRP and TRP2 [19]. The impaired melanoblast migration during embryonic stage (Fig. 4A), and reduced number of melanocyte and melanin accumulation in hair follicle (Fig. 3A) in KIT D17/+ mice, could be attributed to the impaired PI3K and MAPK signaling induced by the splice mutation.
Previous study proposed an evolutionary scenario whereby KIT duplication occurred first and resulted in a white-spotted phenotype, and the splice mutation occurred subsequently and resulted in a completely white phenotype. The presence of one normal KIT copy in I ensures that white pigs have a sufficient amount of KIT signaling to avoid severe pleiotropic effects on hematopoiesis and germ-cell development [5]. Thus the KIT duplication mutation seems to exhibit a rescue function to the splice mutation. Therefore, at first, we expected that the KIT duplication could restore the splice mutation in KIT Dup/D17 mice. However, combination of these two mutations lead to more severely impaired signaling on PI3K and MAPK pathways (Fig.5E & 5F), melanblast migration (Fig. 5H), melanocyte number reduction and melanin accumulation (Fig.5 H), resulting in nearly completely white coat colour (Fig. 5B). Thus the KIT duplication mutation does not seems to play a rescue role to the splice mutation. The underlying mechanism of the interaction between these two mutations could be very complicated. As the activation of intrinsic kinase activity of KIT receptor dependents on the binding of SCF ligand to form homodimer. Thus we speculated that improved expression of normal form of KIT protein in KIT Dup/D17 mice may increase the chance of formation of KIT/KIT D17 dimer upon the binding of SCF as compared with that in KIT D17/+ mice. Given that the amount of SCF ligand is limited, more KIT/KIT D17 dimer presented on the cell surface of melanoblast may significantly reduce the activation of subsequent PI3K and MAPK signaling pathways, resulting in more severely impaired melanoblast migration, and a more pronounced phenotype change in coat colour (Fig. 6).
Through observation of the light and electron micrograph of a section of skin, a previous concluded that melanocytes and their precursors were absent in the hair bulb of the dominant white (I) pigs, and the dominant white color in the pig is due to a defect in the development of melanocytes [20]. However, we found that the combination of KIT duplication mutation and splice mutation did not completely block the melanoblast migration (Fig. 5H), and few melanocytes or their precursors can be detected in the skin hair follicles of KIT Dup/D17 mice through immunostaining of marker protein of melanocytes (Fig. 5C). This was confirmed by q-PCR and Western blot analysis of additional melanocytes marker proteins in the skin of KIT Dup/D17 mice (Fig. 5D). In addition, in our unpublished experiments, expression of several melanocyte marker proteins was detected by q-PCR and Western blot in skin tissue of Large White pigs, implying the exist of melanocyte or its precursors in dominant white pigs. These results indicate that the combination of KIT duplication mutation and splice mutation although impair the development of melanocyte severely, but still few melanocyte precursors can migrate to destination.
In coclusion, our study provides a further insight into the underlying genetic mechanisms of porcine dominant white coat colour.
Materials and Methods
Establishment of mouse models
All mouse models are established on the C57BL/6 background by Model Animal Research Center of Nanjing University (China) as described in previous report [21], with minor modifications. Briefly, C57BL/6 mice were kept under a 12/12 h light/dark cycle. To produce zygotes for pronuclear injection, female mice were injected with 5 IU pregnant mare’s serum gonadotropin (PMSG), and 46–48 h later injected with 5 IU hCG to induce ovulation 10-12 h later. Following the hCG injection, put the females together with male mice in single cages overnight. Fertilized oocytes were isolated from the oviducts for pronuclear injection. To generate KIT Dup/+ and KIT GtoA/+mouse model, Cas9 mRNA, sgRNA and the according donor plasmid (Fig. 1B) were injected into the pronuclei of zygotes. To generate KIT D17/+mouse model, Cas9 mRNA and a pair of sgRNA (Fig. 1B) were injected. All sgRNA sequences are listed in S2 table. Injected zygotes were transferred into the oviducts of surrogate recipient female mice to deliver genome-edited pups. KIT Dup/D17 mice were obtained by mating KIT D17/+females with KIT Dup/+ males because KIT D17/+males are infertile.
All procedures were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC), Sun Yat-sen University (Approval Number: IACUC-DD-16-0901).
Mouse genotyping
Mice genotypes are identified by PCR. The tail of 1week old mice are cut off to extract DNA by using a tissue DNA extraction kit (OMEGA). Primers used in PCR are summarized in S2 table.
Mice skin RNAs were prepared using TRIzol (Invitrogen) extraction followed by DNase (Ambion) treatment, and reverse transcription was carried out using the instructions of Reverse Transcription System (Promega).
Primers for mouse genotype identification please refer to S2 table. Polymerase chain reaction (PCR) was carried out using the GeneStarTM PCR Mix system. Each PCR reaction mix contained 1× GeneStarTM PCR Mix buffer, 1.0µM of each primer and about 100 ng DNA template. The procedure in the thermal cycling was an initial 5 min hold at 95 °C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 60°C, and 30 sec at 72°C, and finishing with 10 min incubation at 72°C.
In order to determine the mutant sequences, the PCR products were recovered by OMEGA DNA Gel Recovery Kit, cloned into pMD-18T vector (TAKARA) and transformed into DH5α competent cells. Plasmids then were purified from E. coli cells for Sanger sequencing.
Isolation and culture of peritoneal cell derived mast cells
Isolation and culture of peritoneal cell derived mast cells was performed as previous described protocol [22] with minor modifications. 5 ml of PBS and 2 ml of air was injected into peritoneal cavity of 14 weeks old mice. Then injected mice were carefully shaken in the palm for 5 mins. Subsequently, the cell-containing fluid of the peritoneal cavity is gently collected in a plastic Pasteur pipette. After centrifugation, cells were resuspended, and cultured in DMEM medium supplemented with serum, cytokines IL-3 (CP39; novoprotein) and SCF (C775; novoprotein). After 10 days culture, CD117 and FceR1 makers were used to determine whether cultured cells are mast cell by flow cytometry analysis using the Beckman Coulter Gallios™ Flow Cytometer. For surface staining, cells were stained with APC anti-Mouse CD117 (17-1171; affymetrix) and FITC anti-mouse Fc epsilon receptor I alpha (FceR1) (11-5898; affymetrix) at room temperature for 30 minutes, then washed with PBS and then re-suspended in PBS.
Histological and immunohistochemical analysis of tissue sections
Skin tissues and embryos were fixed overnight in 10% (w/v) paraformaldehyde with 0.02 MPBS (pH 7.2) at 4 °C, processed and mounted in paraffin, then serially cut into 5μm-thick sections by Rotary Microtome (MICROM). Histological sections were stained with hematoxylin and eosin (H&E), observed and photographed under a fluorescent microscopy (Zeiss). For immunohistochemistry experiments, sections were treated with 3% H2O2 to quench endogenous peroxidase activity, then treated with 5% bovine serum albumin to block nonspecific protein binding sites. Sections were incubated with primary antibody at 4 ◦C overnight, and then stained by using anti-Rabbit HRP-DAB Cell and tissue staining kit (R&D, CTS005). Detection was followed by TSA plus Fliorescein (Perkinelemer, NEL741001KT). All antibodies including KIT (ab47587; abcam), Phospho-KIT (Try719) (#3391; Cell Signaling), Green Fluorescent Protein (AB3080P; merk), DCT (ab74073; abcam), Erk1/2 (#4695; Cell Signaling), Akt (#9272; Cell Signaling) Phospho-Akt (#4060; Cell Signaling) and Phospho-Erk1/2 (#8544; Cell Signaling) were diluting in 1:200 with PBS.
qPCR
For all gene expression level detection, total RNAs were prepared using TRIzol (Invitrogen) extraction followed by DNase (Ambion) treatment, and reverse transcription was carried out following the instructions of Reverse Transcription System (Promega). The resulting total cDNAs were analyzed quantitatively using FastStart Universal SYBR Green Master kit (Roche) with primers for KIT, ERK, AKT, PLCG and DCT. Expression profiles were tested in triplicate on at least two mice on an LC480 instrument (Roche). Data were analyzed using the comparative Ct (ΔΔCt) method and one-tail, unpaired student T test (significance cutoff p<0.01). Gene expression levels were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Western blot analysis
Proteins were extracted using Lysis Buffer (Key GEN), and proteins concentration was determined by using PierceTM BCA Protein Assay Kit (Thermo). 300 ng protein was subjected to 10% SDS gel and electrotransferred onto PVDF membrane (Roche). After blocking for 1h with 3% BSA in PBS, the membrane was incubated with primary antibodies at 4 ◦C overnight.
The rabbit anti-KIT antibody (ab47587; abcam), rabbit anti-Phospho-KIT (Try719) antibody (#3391; Cell Signaling), rabbit anti-Green Fluorescent Protein antibody (AB3080P; merk), rabbit anti-DCT antibody (ab74073; abcam), rabbit anti-Erk1/2 antibody (#4695; Cell Signaling) and rabbit anti-Akt antibody (#9272; Cell Signaling) were diluting in 1:1000, rabbit anti-Phospho-Akt antibody (#4060; Cell Signaling) and rabbit anti-Phospho-Erk1/2 antibody (#8544; Cell Signaling) were diluting in 1:2000, rabbit anti-GAPDH antibody (AP0063; biogot) was diluting in 1:5000 with PBS. Following by 10 min three times washing with TBST, the membrane was then incubated with 1:5000 goat anti-rabbit secondary antibodies (Abcam, ab6721) for 1h at room temperature. Protein bands were visualised using Kodak image station 4000MM/Pro (Kodak), according to the manufacturer’s instructions, and exposed to FD bio-Dura ECL (FD, FD8020). Protein levels were standardized by comparison with GAPDH.
Statistical analysis
All data were analyzed by using EXCEL (version 2016). The data were expressed as the means ± SEM. Only values with p < 0.05 were accepted as significance.
Supporting information
S1 Fig. KIT GtoA/+, KIT D17/+ and KIT Dup/Dup mice phenotype. (A) White spots appeared on the abdomen of KIT GtoA/+ mice as compared with KIT+/+. KIT D17/+ mice presented a piebald coat colour in head and trunk, a vertical white stripe on the forehead, a half loop of white hair on the shoulder blade area, and dominant white at entire abdominal region. There was no difference between the coat colour of KIT+/+ and KIT Dup/Dup mice at 14 W old. (B) Schematic diagram of primers designed for identification of KIT D17/+ and KIT Dup/Dup mice identification. (C) PCR identification of the KIT D17/+ and KIT Dup/Dup mice.
S2 Fig. Identification of mouse peritoneal mast cells through flow cytometry analysis. KIT (stained by anti-Mouse CD117 APC) and FcεRI (stained by anti-mouse Fc epsilon receptor I alpha) were used as markers of mast cell.
S3 Fig. Using KIT as marker to detect melanoblast migration in KIT +/+ and KIT Dup/+ mice embryo. KIT is used as marker to detect melanoblast migration in KIT +/+ and KIT Dup/+ mice embryo (E14.5).
S4 Fig. Coat colour changing of KIT Dup/D17 mice during growth up. With the increase of age, the gray hairs of the eyelids and hips gradually disappeared.
S1 Table. Homologous of splice mutation of KIT gene could be lethal. Oocytes from superovulated KIT D17/+ females were in vitro fertilized with sperms collected from 5 KIT D17/+ males, and transferred to 10 surrogate females to generate offspring. No KIT D17/D17 pups were born but 13 KIT +/+ and 24 KIT D17/+ pups were obtained.
S2 Table. Oligos and primers used in this study.
Acknowledgement
This work was jointly supported by National Transgenic Major Program (2016ZX08006003-006), National Key R&D Program of China (2018YFD0501200), and Key R&D Program of Guangdong Province (2018B020203003).