A Primary Study On The Function Of Cdc42 In The Vibrissa Follicles During Mouse Embryonic Development

Hair follicle is an important accessory structure which regulate the growth of hair. The distribution of hair follicles is very wide in the individual, almost throughout the all over the body. The hair shaft consists of terminally differentiated keratinocytes that are produced by the hair follicle hair follicle exerts a wide range of functions including physical protection, thermoregulation, excretion and secretion of sweat, sensory activity, and social interactions. Hair follicle development takes place during fetal skin development and relies on tightly regulated ectodermal-mesodermal interactions. After birth, mature and actively growing hair follicles periodically regenerate by spontaneously undergoing repetitive cycles of growth (anagen), apoptosis-driven regression (catagen), and relative quiescence (telogen). However, at the onset of each new follicular cycle in adults the dermal papilla interacts with secondary germ cells in the hair-follicle bulge to regenerate the lower follicle. The signals controlling epidermal-dermal communication include secreted molecules of the Wnt/wingless family, the hedgehog family, and members of the TGF-b/BMP(transforming growth factor-b/bone morphogenetic protein), FGF (fibroblast growth factor) and TNF (tumour necrosis factor) families. However, The regulatory mechanism of hair follicle development and growth cycle is not fully clear, for the clinical treatment of hair loss disease caused the block. So the hair follicle biology and cyclical growth regulatory mechanism become the current hot issue.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, with the role of molecular switch in cell signal transduction process. Cdc42 firstly found in yeast cells, the biological activity is very extensive, which in turn regulate the actin cytoskeleton, microtubule network, cell polarity, proliferation, apoptosis, endocytosis, and secretion. Except for the regulation of cytoskeleton, Cdc42 also has an important roles on cell proliferation and differentiation.Due to the previous study of Cdc42 are mostly limited to cell level, as the development of human genome project and all kinds of genetic engineering technology, conditional knockout technology using Cre/Loxp recombination enzyme system can make people betterto study Cdc42 protein function at the individual level.Mice with a constitutive knockoutof Cdc42 die around implantation, indicating the importance of Cdc42 function in vivo, but preventing the analysis of Cdc42 at later time points of development. Keratin 14/Keratin5 promoter is often used for the purpose gene knockout, which has a certain activity in the inner root sheath and epidermal basal layer. Through K14 specific knockout of Rac1 which also belonging to the small GTPase Rho family, researcher find not only damage hair follicle integrity, but also damage the skin and the sebaceous glands of self-renewal. Through K5 specific knockout of Cdc42, the mouse hair significantly decrease after born two weeks, research consider that Cdc42 controls progenitor cell differentiation and bete-catenin turnover in skin. Our experiment through the K14 as promoter construct keratinocytes-restricted deletion of the Cdc42 gene mouse (Cdc42loxp/loxp Cre+,KO), We found that compared with WT mice of normal control group, P1 day whiskers have been showed abnormal growth, skin surface did not see the growth of hair shaft. This suggests Cdc42 plays an important role in embryonic period of hair follicle development. Thenhow Cdc42 inflnuences cell fate and the mechanism is unknown, therefore we unfold this researching.一、The construction and genotype identification of keratinocytes-restricted deletion of the Cdc42 gene mouse(二) Objective Construct keratinocytes-restricted deletion of the Cdc42 gene mouse, tto lay the foundation for research the function of Cdc42 protein.(三) Methods Using Cre/loxP system, the mice analyzed in this study were generated by crossing Cdc42-Loxp mice with K14-cre transgenic mice. Some are working on machines to extract DNA from the tail of mouse, and using polymerase chain reaction (PCR) to amplify the purpose gene fragment. According to gene fragment length genotype, we selected the genotype of Cdc42loxp/+Cre+mice and crossed back,in order to obtain the genotype of Cdc42loxp/loxpCre+mouse embryos and adult mice.(四) Result1.The inheritance of the transgenic mouse was shown to be in agreement with Mendel’s law of segregation, and the genotype of Cdc42loxp/loxp Cre+had been bred successfully.2. The cross-bred the genetically modified mice reproduced successfully. The time of pregnancy is 19-21 days, the average litter size per labor of male mouse were 6 to 8 respectively, the offspring weighed 0.9g to 1.2g. Genotype of Cdc421oxp/loxp Cre+ KO mouse, the facial whiskers area is smooth, we could only see the heave of hair follicle bulge. There was not the hair shaft, KO mice all died within 24h.(四) Conclusion:1. We had succeed in building keratinocytes-restricted deletion of the Cdc 42 gene mouse.2. Cdc42 gene deletion causes the growth and development of hair follicl e abnormal in embryo mice.二、Vibrissa follicles morphogenesis during mouse embryonic development(一) Objective Observation of the hair follicles morphogenesis during mouse embryonic development, to provide a basis for follicular developmental biology research and molecular regulation mechanism.(二) Methods We took mouse embryo aged from embryo 10.5(E10.5) day to postnatal 1(P1) day for laboratory experiments. In this paper, the histological structure of hair follicles were observed on routine paraffin sections that were stained with HE stain.(三) Result1. At E10.5 day, the skin of mouse was extremely thin. The cells arranged well which were simple squamous epithelium. However, we have found no phenomenon of local thicken and number of cells enhance. We observed the characteristic as an accumulation of nuclei,which is virtually impossible to recognize with routine histologic techniques. This is the first stage of HF development, also described as pregerm stage.2. At E12.5 day, the epidermis was composed of one or two layer keratinocytes, the epidermis cells formed round or cube, the same size cells and lined up tightly, the same size cells and lined up tightly. We could see the epidermis local thicken and arranged in columned. Long axis of the cell was perpendicular to the basal layer the number mesenchymal cells was increased and speedily gathered. There was slightly change of direction, in contrast to regular epidermal keratinocytes, HF keratinocytes display a vertically polarized orientation, these formed symmetric structure of placode.3. At E13.5 day, the hair germ developed into a more prominent and enlarged, broad column of epidermal keratinocytes, and presenced some angle to the epidermis. Basal cells were arranged by shuttle, the cells unregularly or irregularly arranged which former hair germ. Dermal papilla cells began to little gather under the bottom of hair germs. The arrangement direction of dermal papilla cells changed little, which were precursor cells of hair papilla. This was stage 2 of HF morphogenesis, called hair germs.4. At E14.5 day, epidermal basal layer cells developed into a more prominent, and had became a solid, elongated column of epithelial cells, which consists of multiple layers of keratinocytes that became arranged with a lot of cube shape cells in its center and columnar cells outside. They were palisade-like keratinocytes which had little cytoplasm but hyperchromatic nuclei. The cylindrical structures within cells have developed a prototype of inner root sheath, the hair follicles of connective tissue has been clearly distinguishable. The hair peg has become more elongated and displays the first characteristics of adolescent HF:a bulb-like thickening of its proximal portion, and a prominent, wide DP cavity. We has described this stage as the hair peg stage and early hair follicular stage.5. At E15.5-E16.5 days, the hair follicles developed into hair formation stage,the characteristic structure of Hair follicle had appeared. At E15.5 day, the endof the hair follicle primordium expanded and were very small and round, which become concentrically arranged around the follicular axis. Dermal papilla cells invaginated into the hair matrix, surrounding with columnar cells or cubical cells which had large central nucleus. They were hair matrix cells, hair bulb was composed of hair matrix cells surrounding the dermal papilla cells. At E16.5 day, displayed an enlarged future bulge cone-shaped structure above the DP, the HP further developed through the follicle, some of which lies as far as subcutaneous tissue. At this time, the pale epithelial layer or Henle’s layer of theinner root sheath (IRS) starts to develop, long ovoid cells which superposed with each other resided internally. Its long axis paralleled to the direction of hair growth, it was cuticle of internal root sheath. Henle’s layer consisted of one layer of closely arranged and small cells with flat nucleus membrane. Outer root sheath were connected to the epidermal basal layer, we could see the typical structure of hair follicles.6.At E17.5-P1 days, the HF acquires its maximal length and reaches the subcutaneous muscle layer (panniculus carnosus), and a prominent shaft emerges through the epidermis. Morphologically, the HF now resembles a mature anagen VI HF.(四) Conclusion1. The morphogenesis of hair follicles during mouse embryonic development agrees with stages of development and regeneration of hair follicles according to the standards of Paus R. There are five stages:pregerm stage, placode stage, hair germ stage, hair peg stage, hair follicular stage.2. The morphogenesis of hair follicles during mouse embryonic development take place at E12.5 day, earlier than the skin of abdominal and back. The typical structure of hair follicles has formed at E16.5 day. The hair follicles further mature, and into the hair follicle growth period at E17.5-P1 days.3. The morphogenesis of hair follicles during mouse embryonic development are asynchronous way, at the same time can see different stages of hair follicles.三、The effects of Cdc42 on hair follicle development of embryonic mouse(一) Objective To primary study on the function of Cdc42 in the vibrissa follicles during mouse embryonic development, provide experimental bases for the mechanism of hair follicle growth and regeneration cycle.(二) Methods Using Cre/loxP system, We constructed the keratinocytes-restricted deletion of the Cdc42 gene mice. Littermate mice were selected,which aged from E15.5、E17.5 and P1, and identificated genotype of mice then allocated into WT group and KO group. On this basis, this study used HE staining to observe the morphological structure。number。length of hair follicles, and immunohistochemical (IHC) method to detect the expression of hair follicle specific keratin and related protein, also with 5-bromodeoxyuridine (Brdu) immunohistochemical staining to research the function of Cdc42 GTPase in the process of the proliferation role.(三) Result1.At E15.5 day, Cdc42-positive cells expressed in the hair germ which fromed from invagination of basal layer cells in WT group. Compared with the control group in WT mice, Cdc42 expression decreased significantly in hair germ of KO mice. The results suggest the activity of Cdc42 GTPase in hair follicles of transgenic mice effectively down-regulation, the build is successful in mice.2. At P1 day, there was transparent white hair shaft in WT mice under the stereo microscope. However, KO groups had only small heave of structure but did not see growth of hair shaft.3. At P1 day, both WT and KO groups could see outer root sheath, inner root sheath, hair bulb was composed of hair matrix cells surrounding the dermal papilla cells, the typical structure of hair follicles formed.. The morphology had no significant difference between the two groups. The number of hair follicles had no significant difference between the two groups(P=0.35). The length of hair follicles had significant difference between the two groups (P>0.05), the average length of the hair follicles was (218.6±25.μm)1in WT group, and (152.1±18.7μm) in KO group.4. At E17.5 day, in WT group, K15 positive expression mainly located in outer root sheath of hair follicle, the positive cells expressed less in KO mice (P< 0.01). At P1 day, K15 positive expression mainly located in outer root sheath of hair follicle in WT group. Compared to the WT group, there was no positive expression in outer root sheath of KO mice (P<0.01). At E17.5 day, K19 strongly expressed in outer root sheath of hair follicle, there was no significant difference between WT group and KO group. At P1 day, in WT group,K19 expressed in outer root sheath of hair follicle and the hair matrix, but the positive cells expressed less in outer root sheath of KO mice, hair matrix cells hardly expressed. The result suggests that the number of hairfollicle stem cells significantly reduced in KO mice.5. At E17.5 day, in WT group, K6 positive expression mainly located in outer root sheath of hair follicle, but outer root sheath and epidermis cells did not have. However, not only outer root sheath but also outer root sheath and epidermis cells were positive expressed K6. At P1 day, in WT group, K14 positive expression mainly located in outer root sheath of hair follicle and the basal layer of epidermal, stratum spinosum cells didn’t express. And KO group, except the surface of base layer, also see the K14 positive cells increasing, expression in stratum spinosum layer and the outer root sheath. At P1 day, K1 expressed in epidermal basal, there was no significant difference between WT group and KO group. The result suggests that hair follicles showed excessive proliferation, differentiate more to the epidermal layer,but the terminal differentiation unaffected.6. At E17.5 day, the positive cells of beta-catenin distributed mainly over membranes of outer root sheath cells and hair bulb cells in WT group. In hair follicle of KO mice, beta-catenin positive expression in the cytoplasm and nucleus of the inner root sheath、outer root sheath and hair bulb of the hair follicles, which expression was stronger than the WT group (P＜0.01). The trends of expression of E-cadherin in hair follicles were consistent with beta-catenin. The result suggests that Cdc42 knockout of KO mice caused beta-catenin and E-cadherin abnormal expression, affected the cell adhesion and cell proliferation and differentiation.(四) ConclusionCdc42 play an important role in the development of embryonic mouse hair follicle, the following three aspects:1) Cdc42 is an essential prerequisite to maintain stability of the hair follicle stem cells.2) Cdc42 plays a key role in regulating cell adhesion.3) Cdc42 regulate the proliferation and differentiation of hair follicle cells through E-cadherin and beta-catenin molecule signal to regulate the proliferation and differentiation of hair follicle stem cells.