The Impact Of The Keratinocytes-restricted Deletion Of The Cdc42 Gene On The Skin Development And Its Mechanism

Skin is the largest organ in human. It is very important for normal embryonic development and adult continually renewing to provide a barrier between the body and the environment. Skin development and maintenance is a complex biological process, including keratinocytes proliferation, differentiation and apoptosis. The balance between the cellular processes is regulated carefully by many signal transduction pathways, but so far about skin development cascades mechanism is not clarified.Cdc42 is a ubiquitously expressed small GTPase belonging to the Rho family. It exists in an active GTP-bound and an inactive GDP-bound form. Cdc42 is a molecular switch in cell signal transduction pathway. Only in its active form can Cdc42 interact with different effectors, which in turn regulate cell growth, differentiation, apoptosis, cell cycle and a series of other cellular processes. The most important function is regulation of the restructure of the actin cytoskeleton.Recent studies have found that Rho family in small GTPase play an important role in regulating keratinocytes proliferation, differentiation and apoptosis. The previous studies confirmed that the Rho A can regulate keratinocytes proliferation and differentiation and promote skin wound healing through MEKK1/JNK signaling pathway. But, there are few researches about the crucial role of Cdc42 in skin development and self renewal. Recently, our research group generated mice with the keratinocytes-restricted deletion of the Cdc42 gene. Result unexpectedly is mice died within 24 hours after birth. This shows that Cdc42 play a crucial role in skin development. We will analyze the impact of the keratinocytes-restricted deletion of the Cdc42 gene on the skin development and its mechanism through the use of the transgenic mice. This result will provide solid theoretical basis for research and treatment of various skin diseases.1. Generation of the mice with the keratinocytes-restricted deletion of the Cdc42 gene(1) Purpose:To provide effective tools for researching the function of Cdc42 in skin development in vivo.(2) Methods:Using Cre/loxP system, the mice analyzed in this study were generated by crossing Cdc421oxp/loxp mice with K14-cre transgenic mice. The resulting Cdc42loxp/+/K14-Cre (+) mice were then crossed with Cdc421oxp/loxp mice to generate 1/4 KO mice that its genotype is Cdc42loxP/loxP/K14-Cre (+) and 1/2 WT mice that its genotype is Cdc42loxP/+/K14-Cre (-) or Cdc42loxP/loxP/K14-Cre (-). The genotypes are identified by polymerase chain reaction (PCR) technology and immunohistochemical method. According to the accurate gestational age, we obtained various age mice from Embryonic 14.5 days to the first day after birth. The embryos and postnatal mice were fixed in 4% formaldehyde, embedded in paraffin, sectioned and stained.(3) Results:① By PCR genotyping identification, K14-Cre(+) displays 496bp and 200bp two specific bands. Cdc421oxP/loxP only displays a 465bp specific band. KO mice not only have K14-Cre (+) but also have Cdc421oxP/loxP.② By IHC genotyping identification, Cdc42 is expressed only in epidermal basal keratinocytes of KO mice at E15.5. With the development, the expression of Cdc42 is decreased gradually in basal keratinocytes and almost completely disappeared at birth. Results show that Cdc42 gene in epidermal keratinocytes of KO mice is effective knockout.③ KO mice died within 24 hours after birth. Compared with WT mice, the size of KO mice at E17.5, E18.5 and P1 are small and the weight difference has statistics significance. KO mice at P1 are characterized with the glossy and flush skin, like silk.(4) Conclusion:Mice with keratinocytes-restricted deletion of the Cdc42 gene were successfully built.2. Morphological observation of the important organs in KO mice(1) Purpose:To judge whether organs dysplasia cause death of KO mice.(2) Methods:To observe histological structure of liver, kidneys, lungs and heart of KO mice in development by using HE staining; To observe the expression of glycogen of liver cells of KO mice in development by using PAS staining.(3) Results:Except having congestion symptoms in liver, kidney, lung and heart of KO mice at birth and parenchymal cells degeneration caused by hypoxia, the morphological structures of liver, kidney, lung, and heart in KO mice were not seen abnormal in development. The hepatic glycogen synthesis in KO mice is not abnormal in the liver development.(4) Conclusion:The keratinocytes-restricted deletion of the Cdc42 gene does not affect important organs of liver, kidney and lung development.3. The impact of the keratinocytes-restricted deletion of the Cdc42 gene on the skin development and its mechanism.(1) Purpose:To explore the impact of the keratinocytes-restricted deletion of the Cdc42 gene on the skin development and its mechanism.(2) Methods:To detect the barrier function of the skin by performing Skin permeability assay and dehydration test assay. To observe dynamically the histological structures of the KO mice skin in development by HE staining and PAS staining. To observe the ultrastructure of the KO mice skin by scanning electron microscopy and transmission electron microscope. To detect the expression of K19, K15, K14, K1, K6, Loricrin and Involucrin in the epidermal keratinocytes of the KO mice by immunohistochemistry method, the purpose is to observe the keratinocytes differentiation in development. To detect the expression of beta-catenin, E Cadherin, Desmoplakin, ZO-1 and a6-intergrin in the epidermal keratinocytes of the KO mice by immunohistochemistry method, the purpose is to observe the changes of the cell junction between the keratinocytes in development. To detect the expression of p-ERK、p-JNK、p-p38 and p-c-Jun by immunohistochemistry method.(3) Results:① Epiderm barrier impairment of KO mice:By Skin permeability analysis, Hematoxylin do not penetrated the stratum corneum of KO mice similar with WT mice, but the color of stratum corneum of KO mice is deeper than that of WT mice. After skin dehydration test, the body weight of WT mice fell by about 1.5% during the survival time of KO mice, while the body weight of KO mice fell about 5%. The results confirm KO mice underwent dehydration due to the impairment of the epidermal barrier.② Epidermal histological structure abnormalities of KO mice:Results of Hematoxylin-eosin-staining show deletion of Cdc42 results in epidermal histological structure abnormalities. The epidermis of KO mice began to exhibit microstructure abnormalities at E15.5, which the surface of the skin being flat and epidermal ridge being not obvious. At P1, the epiderm of KO mice becomes slightly thicker than that in WT mice; the gap between the basal cells and the spine cells and the gap between the spine cells become wider than that of WT mice, some spine cell shrink, intercellular bridges are clear; the epidermal basal cells of local epiderm in KO mice appear irregular arrangement and changed shape; the stratum corneum of epiderm in WT mice that is loose appear to be stripped, but the stratum corneum of epiderm in KO mice become dense. Results of PAS staining show glycogen content and distribution of the epidermal keratinocytes are not obvious abnormality between KO mice and WT mice during development. By observation of scanning electron microscopy, the surface of the epiderm in WT mice appears obvious eminences which are full and deep ditches, the small granular swells are distributed uniformly on the surface of eminences at E17.5. Compared with WT mice, the eminences are not full and a large number of fissure appear on the surface of the epiderm in KO mice and the number of the small granular swells are fewer than that in WT mice. At P1, the surface of the skin in WT mice is uneven, like the wave; there are many regular small swells on the surface of the skin. Compared with WT mice, the surface of the skin in KO mice is relatively flat; the swells on the surface of the skin are less and larger than that in WT mice and some swells have stripped. By observation of transmission electron microscope, the gap between the basal cells and the spine cells and the gap between the spine cells in KO mice become wider than that in WT mice at P1. Although intercellular bridges stretch, the number of intercellular bridge is not obviously less than that in WT mice. Some basal cells and spine cells shrink and the spine cells become relatively flat.③ Proliferation increase and apoptosis decrease of the epidermal keratinocytes in KO mice:Results of BrdU show the positive cells in both WT mice and KO mice were mainly distributed in the epithelial basal layer at E15.5, E17.5 and P1. With development, the number of the positive cells gradually decreases. However, the number of the positive cells in KO mice are more than that in WT mice, the differences are significant (P<0.05). Results of TUNEL show the keratinocytes of epidermis in both KO mice and WT mice began to appear apoptosis at E17.5. With development, the number of the apoptosis cells between WT and KO mouse gradually increased. However, the number and distribution of the positive cells in KO mice were changed. The number of the positive cells became obviously decrease. The positive cells in WT mice are distributed in the stratum granulosum and the whole stratum spinosum, even stratum basale while the positive cells in KO mice are only distributed in the stratum granulosum and the superficial stratum spinosum.④ Epidermal basal cells can be terminal differentiation in KO mice, but its differentiation form was changed:K14, K6, K1, Loricrin and Incolucrin were tested at E15.5, E17.5 and P1. K14 are produced in the basal cells of stratified epithelia. The expression of K14 is correlated with the mitotic activity and the degree of pluripotency of the basal cells in stratified epithelia. K1 is produced in the suprabasal cells of the epidermis and is therefore regarded as important for postmitotic differentiation in stratified keratinizing and cornifying epithelia. Loricrin and Incolucrin are produced in granular layer. They are also important maker of the suprabasal cells which has been completed postmitotic differentiation and ongoing keratinization. K6 is produced in the hyperproliferative epithelial cells. K6 is expressed in a variety of internal stratified epithelia. However, the keratinocytes of the interfollicular epidermis do not express K6 in general.Results of k14 expression show K14 is produced in the cells of the stratum basale and the deep stratum spinosum in WT mice, while K14 is produced in all of the cells of the stratum basale and the stratum spinosum in KO mice. Results of k6 expression show the epidermal cells can not produce K6 at any time in WT mice while some cells of the deep stratum spinosum began to produce K1 at E17.5 and all of the cells of the stratum spinosum express K1 at P1 in KO mice. The abnormal expression of K14 and K6 indicated that Cdc42 knockout changed the form of the keratinocytes differentiation. K14 and K6 are both related to the mitotic activity. The abnormal expression of K6 and K14 also indicated Cdc42 knockout can enhance the proliferation ability of the keratinocytes. This result is consistent with that of BrdU. The expression of K1 and Loricrin and Involucrin in both WT mice and KO mice were normal in the aspect of number and position, this demonstrated Cdc42 knockout affected the form of keratinocytes differentiation but the formation did not affect the terminal differentiation of keratinocytes.⑤ Number of epidermal stem cells in the interfollicular epidermis of KO mice decreases:K19 and K15 are the sign of epidermal stem cells. Compared with WT mice, the distribution of K19 and K15 is not abnormal in KO mice; they are produced in the basal cells of interfollicular epidermis. However, the number of the positive cells and the expressing intensity of K19 and K15 significantly decrease in KO mice. Results show that the number of epidermal stem cells in the interfollicular epidermis of KO mice decrease.⑥ Tight junctiong and intermediate junction between the keratinocytes in KO mice are destroyed:the structural proteins of the different types of cell junction were tested at E15.5, E17.5 and P1. ZO-1 is a structural protein of the tight junction. Results of ZO-1 show that the expression of ZO-1 in both WT mice and KO mice are distributed in the stratum basale and the stratum spinosum. The expressing intensity of ZO-1 at E15.5 and E17.5 is stronger than that at P1 in WT mice, especially at E17.5. Contrast with the same age WT mice, the expressing intensity of ZO-1 decreases significantly in KO mice. These results indicate Cdc42 knockout destroys the tight junctiong between the keratinocytes. E-Cadherin andβ-catenin are the structural proteins of the intermediate junction. Results of E-Cadherin show that the expression of E-Cadherin in both WT mice and KO mice are distributed in the stratum basale and the stratum spinosum. With development, the expressing intensity of E-Cadherin in WT mice increases gradually. The expressing intensity of E-Cadherin in KO mice is stronger than that of WT mice at E15.5 and E17.5, especially at E17.5, but is weaker than that of WT mice at P1. The expression of β-catenin in both WT mice and KO mice is consistent with the expression of E-Cadherin. These results indicate that Cdc42 knockout destroys the intermediate junctiong between the keratinocytes. Desmoplakin and a6-intergrin are the structural protein of desmosome and hemidesmosome respectively. Results show that the expression of desmoplakin and a6-intergrin in both WT mice and KO mice is not abnormal. These results indicate Cdc42 knockout do not destroy desmosome and hemidesmosome.⑦ Results of the expression of p-JNK, p-p38, p-ERK and p-c-Jun:The expression of p-JNK in both WT mice and KO mice is consistent with the expression of p-p38. Results show that the expression of p-JNK and p-p38 in KO mice is not different with that in WT mice. All of the epidermal cells express p-JNK and p-p38 at E15.5, E17.5 and P1. The expression of p-ERK in WT mice is dynamic in the development. The expression of p-ERK in the superbasal cells is strong at E15.5 and E16.5, the expression of p-ERK diminishes significantly at E17.5 and disappears at P1. Compared with WT mice, there are only a few scattered expression of p-ERK in the superbasal cells in KO mice at E16.5, the rest of the time are not expression of p-ERK. The expression of p-c-Jun in WT mice is dynamic in the development. The position cells are scattered in the epidermal at E15.5. The number of the positive cells in the epidermis increased obviously at E17.5, the expressing intensity of p-c-Jun in the stratum granulosum is stronger than that in other layer of epidermis. The position cells are very few which scattered in the stratum granulosum at P1. Compared with WT mice, the expression of p-c-Jun in KO mice is not different at E15.5. The number of the positive cells that distributed in the superficial stratum spinosum and the stratum granulosum in KO mice is significantly few than that in WT mice at E17.5. However, the number of the positive cells that are distributed in the stratum granulosum in KO mice is more than that in WT mice at P1.(4) Conclusion:① Cdc42 plays an important role in maintaining tight junctions and intermediate junctions between the keratinocytes.② Cdc42 knockout cause the number of interfollicular epidermal stem cells decrease which damaged skin self-renew.③ Cdc42 knockout inhibits the apoptosis of keratinocytes.④ Cdc42 may regulate biological functions of keratinocytes by activating ERK/MAPK signal pathway.