Generation of Transgenic Mice
Previous studies on the signaling function of β-catenin employed full-length and NH2
-terminally deleted forms of β-catenin, which show enhanced stability (Munemitsu et al. 1995
). We therefore constructed analogous cDNAs encoding an epitope-tagged full-length (PG) and NH2
-terminally deleted (ΔN80PG) form of plakoglobin and tested if the products of these constructs retained transcriptional activity by performing a luciferase reporter assay (Van de Wetering et al. 1997
). The products of both plakoglobin constructs transactivated ~100–200-fold a Topflash luciferase reporter in the presence of a putative transcriptional partner Lef-1 ( a). This effect was observed in both 293T cells that make few intercellular junctions and HaCaT keratinocytes that contain many desmosomes (Boukamp et al. 1988
). Low levels of transactivation ~10-fold of the Topflash reporter occurred in the absence of Lef-1 and presumably resulted from activation of endogenous Lef/Tcf transcriptional partners. Neither form of plakoglobin transactivated the control Fopflash luciferase reporter construct which is mutated in the Tcf/lef-responsive elements ( a).
To test the potential role of plakoglobin in regulating cell proliferation, in vivo,
we employed a K14 transgene cassette, kindly provided to us by Dr. Elaine Fuchs, which directs expression of transgenes under the control of the K14 promoter to the basal layer of the interfollicular epidermis and the outer root sheath of hair follicles (Guo et al. 1993
; Vassar et al. 1989
). Both full-length K14-PG and NH2
-terminally deleted K14-ΔN80PG ( b) were used to generate transgenic mice in the Swiss Webster strain.
Two mice (nos. 21 and 24) tested positive by Southern analysis of tail genomic DNA for integration of the K14-PG transgene and gave Mendelian transmission to the F1 generation. Seven mice tested positive for integration of the K14-ΔN80PG transgene. Of these, nos. 1, 4, 45, 46 showed Mendelian transmission of the transgene to the F1 generation, nos. 5 and 9 showed <10% transmission, suggesting mosaicism, and no. 31 gave 75% transmission to the F1 progeny, suggesting that the transgene inserted at multiple sites in the genome. Immunofluorescence detection of the epitope tags on frozen sections of tail epidermis indicated that expression of the transgene protein products occurred in one K14-PG founder (no. 21) and three K14-ΔN80PG founders (nos. 4, 9, and 45). Significantly only these four mice displayed hair phenotypes. Their F1 progeny were studied further in the experiments described below.
Expression of the ΔN80PG Transgene
Southern analysis of tail genomic DNA from F1 mice with probes derived from plakoglobin cDNA and a fragment of the engrailed gene showed that lines nos. 21, 4, 9, and 45 carried 50, 40, 10, and 15 copies, respectively, of the transgene, based on normalization and calibration to a series of titrated plasmid controls ( b). Immunofluorescence microscopy with anti-flag and anti-myc antibody on frozen sections of F1 tails detected the epitope tags in transgenic epidermis ( and ) but not normal littermate epidermis showing the strongest expression within the basal layer. Staining was most obvious at the intercellular borders of keratinocytes reflecting the distribution of desmosomes and adherens junctions but was absent from the basal borders where the cells contact the basal lamina via hemidesmosomes (see arrows in and ). In transgenic mice from all four lines, expression was also seen strongly in outer root sheath cells of the hair follicles ( d and 2 c). Staining was also observed in layers of the epidermis and hair follicle, which do not express the K14 promoter. For example, staining was seen in suprabasal epidermal cells ( d and 2, a and c), and in epithelial cells of the hair follicle bulb including those surrounding the dermal papilla ( and ). This likely reflects persistence of the transgene protein product in progeny of previously K14 positive stem cells. Weak staining was seen in the cytoplasm of all positive cells, nuclear localization was not generally observed in the transgenic tissue sections.
It has been argued in other systems that plakoglobin signaling may be an indirect result of competitive displacement of β-catenin from the membrane. To address this possibility we compared the localization of endogenous β-catenin plakoglobin in transgenic (, a–d) and normal skin (, a′–d′). Staining for endogenous plakoglobin with an NH2-terminal antibody ( and ′) and for endogenous β-catenin with a COOH-terminal antibody ( and ′) showed essentially similar patterns in transgenic ( and ) and normal (′ and d′) epidermis and hair follicles.
Figure 2 Expression of the transgene products detected by indirect immunofluorescence. Frozen sections of tail skin from newborn sex-matched ΔN80PG transgenic (a–d) and control (a′ and d′) F1 littermates. Anti-myc antibody detects (more ...)
Examination of mouse skin under the electron microscope showed that transgenic skin contained desmosomes with normal appearance and frequency demonstrating that the transgene expression had no deleterious effect on formation or stability of these cell junctions (, a and b). Immunofluorescent staining of primary keratinocyte cultures from the epidermis of transgenic mice showed strong localization of the epitope tag at cell–cell borders and faint cytoplasmic staining ( c). Strong reaction was also observed in the nucleus of some but not all transgenic cultured keratinocytes ( c). These staining patterns were specific for transgenic keratinocytes and were not observed in cells derived from normal littermates ( c′). As predicted from previous studies, which showed that the first 80 amino acids of plakoglobin are dispensable for its interactions with all known partners (desmoglein, desmocollin, E-cadherin, α-catenin, APC, and Lef/Tcf), no perturbations were seen in the patterns of localization of several major junctional proteins such as desmoplakins, β-catenin, endogenous plakoglobin or E-cadherin (, d–f and d′–f′) (for review, see Cowin and Burke 1996
Electron micrographs show that desmosomes (arrows) occur with normal frequency and structural appearance in the basal layers B, and superficial layers of normal (a) and transgenic (b) epidermis. Primary keratinocytes from newborn transgenic ΔN80PG (c–f) and control (c′–f′) littermates were processed for indirect immunofluorescence microscopy. Anti-myc (myc) (c and c′), anti-plakoglobin (pg) (d and d′), anti–β-catenin (bc) (e and e′), and anti-desmoplakin (dp) (f and f′) were used as primary antibodies. Note that the presence of the transgene product (c–f) causes no alterations in the localization of other junctional components (compare c–f with c′–f′). Bars: (a and b) 0.5 μm; (c–f and c′–f′) 25 μm; (insets) 0.1 μm.
Confirmation that the epitopes recognized by the antibodies in the immunofluorescence assays above represented the PG and Δ80PG products encoded by the transgene was obtained by cross-blotting anti-plakoglobin immunoprecipitates with anti-flag or anti-myc antibody ( a). In each case, a protein of the appropriate mass, ~86 kD for PG and 75 kD for Δ80PG, could be detected in transgenic animals (lanes 2, 4, 6, and 8) but not in their normal littermates (lanes 1, 3, 5, and 7). Primary keratinocytes from normal and transgenic F1 mice were cultured under low Ca2+
conditions, which enrich for basal cells and hence for cells expressing the K14 promoter (Hennings et al. 1980
; Dotto 1998
). After 2–3 d in culture the cells were switched for 12 h to high Ca2+
to permit cell-junction formation (Hennings and Holbrook 1983
; Dotto 1998
). Under these conditions, 100% of the cells express the transgene product as judged by immunofluorescence microscopy. Equal amounts of protein from normal and transgenic keratinocytes, as judged by protein estimation, were processed for immunoblotting. Protein loading was controlled by monitoring the expression level of ribophorin, a resident protein of the endoplasmic reticulum ( b). The level of transgene expression was found to be ~50% that of the endogenous protein in line no. 4. No significant downregulation of the endogenous pool of plakoglobin or β-catenin was observed in the transgenic keratinocytes when compared with normal keratinocytes. However, we observed a slight increase in E-cadherin levels in transgenics ( b). To determine the biochemical partitioning of ΔN80PG protein, keratinocytes were sequentially extracted first in saponin and then in TX-100 containing buffers. GSK-3β and E-cadherin were used, respectively, as markers of cytosolic and membrane-bound fractions ( c). ΔN80PG was found in all three fractions in the same relative proportions as endogenous plakoglobin. No changes were seen in the relative distributions of endogenous plakoglobin or β-catenin among these subcellular fractions when normal mice were compared with transgenics ( c).
Figure 4 Western blot analysis of PG and ΔN80PG protein expression in skin and keratinocytes. (a) Proteins from tail skin of 25-d sex-matched F1 normal mice (N) nos. 4, 9, 45, 21 (lanes 1, 3, 5, and 7, respectively), and their transgenic littermates (lanes (more ...)
In other studies, NH2
-terminally deleted ΔN89 forms of β-catenin have been shown to have increased stability due to removal of critical phosphoserines that are required for targeting for degradation (Munemitsu et al. 1995
). In transient expression experiments in 293T cells, we consistently saw higher steady-state expression of ΔN80PG than PG, suggesting that removal of this analogous domain may increase the steady-state level of plakoglobin in a similar fashion ( d). We examined the stability of the two forms of plakoglobin by pulse–chase experiments on cultured transgenic keratinocytes. However, in this cell type we saw no obvious difference in the stability of the full-length product as compared with the ΔN80PG product ( e). While ΔN89-β-catenin has been expressed in epidermal keratinocytes, its stability relative to full-length β-catenin has not been assessed in this cell type (Gat et al. 1998
). Thus, at the present time it is not clear if there is a fundamental difference in the mechanisms regulating turnover of plakoglobin and β-catenin, or if keratinocytes differ from colonic and 293T cells in regulating the activity of GSK-3β or other elements in the putative GSK-3β–dependent degradation pathway. Regulated rather than constitutive activity of GSK-3β has recently been reported to occur in Dictyostelium discoideum
(Kim et al. 1999
Transgenic Mice Have Stunted Hair Growth
The striking feature that distinguished transgenic animals of PG line no. 21 and all three ΔN80PG lines from their normal littermates was a pronounced short hair phenotype giving the mice a less fluffy appearance to their coat and a pronounced pink hue (, a and b). Three founders (nos. 21, 4, and 45) and the 50% of their offspring that carried the transgene had dramatically shorter hair uniformly throughout their pelt. Founder no. 9, however, showed both short and long hair in banded and patchy fashion but the 10% of F1 progeny from this line that were transgenic showed the uniform short hair phenotype. These observations are consistent with the presence of the transgene causing the shortening of the hair and with founder no. 9 having mosaic expression of the transgene within its epidermis.
ΔN80PG (a) and 21-d PG (b) transgenic mouse (left) compared with their normal littermates (right). Note the short hair and pink hue of the transgenics. (c) Hair from 25-d normal (N) and ΔN80PG (TG) mice.
Transgenic F1 mice had a taut facial appearance and prominent ears, features resulting in part from the shortness of the hair tufts found abundantly in the cheeks and around the ears of normal mice. It was possible to accurately identify transgenic mice from day 11 on the basis of coat morphology. The short hair phenotype was most obvious once the coat was well formed, between days 15–40, the time span during which the first two highly synchronized hair cycles occur in the mouse. Adult transgenic mice remained distinguishable from wild-type littermates, having a close-cut smooth coat, although the phenotype became less obvious in the older mice probably due to the asynchrony of subsequent hair cycles. Heterozygous and homozygous transgenic mice showed no increase in morbidity or mortality or impairment of fertility up to one year of age. Heterozygous and homozygous pups often looked smaller than their normal littermates, but, as there was no consistent correlation in weight differences between these groups, this observation reflected a difference in coat appearance. The identical phenotype elicited by both the full-length and the NH2-terminally deleted form of plakoglobin argues strongly that the latter acts as an active form of plakoglobin and not by interfering with the function of endogenous plakoglobin.
Analysis of Hair
Hairs plucked from the heads of 25-d sex-matched F1 transgenic and control littermates were examined under a dissecting microscope. We first asked whether the appearance of the transgenic mice resulted from lack of the longer outer coat hairs. The percentage of each of three major types of hair (guard, awl, and zigzag), however, were represented to the same degree in both the transgenic animals and their normal littermates. All hair types of the transgenic mice showed a striking reduction in size, being 30–40% shorter than hairs of normal littermates of the same age ( c and ). The root and tapered ends of the hairs were clearly visible, ruling out the possibility that the hairs were shorter due to fragility and breakage. In zigzag hairs, all four segments were present but each segment was shorter in the transgenic than in the wild-type hairs. Moreover, the segments became progressively more affected, with segments that formed first being reduced in length by ~20% and those formed last being reduced by ~50%. By scanning electron microscopy, the structure of the cuticle of the coat hairs appeared normal. However, transgenic hairs were composed of ~20% fewer cells than normal hairs and the cells were 14% smaller in diameter. No significant differences were found between the length of hairs in the different lines, suggesting that the transgene dose was irrelevant beyond a critical threshold. This was also suggested by the fact that homozygotes could not be phenotypically distinguished from heterozygotes.
Lengths (mm) ± Standard Deviations of Hairs
Mechanistic Studies: PG and ΔN80PG Expression Results in Early Withdrawal from the Growing Phase (Anagen) of the Hair Follicle Cycle, Reduction in Cycling Cells, Premature Apoptosis, and Follicle Degeneration but Not from Changes in the Differentiation Program
To search for a basis for the unusual length of hairs from the transgenic mice, we examined skin from the mid-back region of two to three pairs of normal and transgenic littermates from each line at multiple time intervals throughout the first 41 d encompassing the first two hair cycles. Up to day 11 there was no discernible difference in the histological appearance of the transgenic epidermis or primordial follicles from those of normal littermates. The length and width of the follicular bulb, number, spacing, and angling of the hair follicles was similar and in cross-sections of epidermis the follicle density appeared identical in both groups. However, remarkable differences were observed during later stages of anagen. In this strain of mice (Swiss Webster) anagen begins late in gestation ~E17 and peaks around day 14 as estimated by follicle length (see below). At day 13 normal mouse skin shows the classical features of the anagen phase of the hair cycle with the epithelial component of the follicle bulb containing large numbers of cells in mitotis (see arrows in a). In contrast, by day 13 transgenic hair follicles have already entered into catagen and display large numbers of involuting follicles trailing compact dermal papilla ( b). As a result the dermis of the transgenic skin becomes much thinner than the normal. Hair follicles from normal skin do not enter catagen until day 15 ( c) at which time the follicles of the transgenic skin have already fully regressed and entered the quiescent telogen phase ( d). Follicle lengths were measured, from the base of the follicle bulb to the epidermis, to quantify the first two hair cycles (). A similar but more attenuated scenario is seen in the second cycle. Transgenic mice enter the second cycle slightly ahead of their normal counterparts but again leave earlier (). All transgenic lines showed this trend.
Figure 6 Histopathology of transgenic mice. 5-μm paraffin-embedded sections of backskin from sex-matched control and ΔN80PG transgenic F1 littermates were stained with hematoxylin and eosin (a–d). (a) 13-d control hair follicles show the (more ...)
Figure 7 Comparison of the first two hair follicle growth cycles in K14-PG and K14-ΔN80PG transgenic mice and their normal littermates. Hair follicle length corresponds to the length in μm from the base of the hair follicle bulb to the epidermal (more ...) Proliferation.
To determine the effects of ΔN80PG expression on cellular proliferation in epidermis and hair follicles we undertook a double (pulse) labeling procedure. Double (pulse) labeling gives information both on the number of cells cycling at any one time and the number of cells that have undergone two rounds of DNA synthesis (Lehrer et al. 1998
). Mice were first injected with BrdU then 24 h later injected with 3
H-TdR. A statistically significant decrease was observed in the number of labeled cells in the transgenic group of mice (8.9 ± 4.5) as compared with their wild-type littermates (11.6 ± 3.1; P
< 0.001 where the number [n] of mice in each group = 10). Moreover a highly significant difference was seen in the number of cells undergoing two S phases within a 24-h period (). In transgenic epidermis, 8.5 ± 4.5% of cells initially labeled with BrdU were also labeled with 3
H-TdR indicating they had undergone two rounds of DNA synthesis as compared with 13.4 ± 4.8% of cells in normal epidermis (P
< 0.001 where n
= 10 mice in each group). Thus, a significant decrease in cycling cells results from ΔN80PG expression.
Figure 8 Comparison of BrdU- and 3H-TdR–labeled nuclei of ΔN80PG transgenic and normal epidermis. 4-d ΔN80PG transgenic (left) and normal littermates (right) were subjected to subcutaneous injection of BrdU followed 24 h later by a similar (more ...) Apoptosis.
The factors governing the extremely rapid and synchronous involution of hair follicles during catagen are poorly understood, but are thought to involve an apoptotic pathway (Lindner et al. 1997
). We therefore investigated whether the early involution observed by day 13 in the hair follicles of mice expressing ΔN80PG results from or is associated with premature onset of such a mechanism. TUNEL analysis was performed to detect DNA fragmentation as a marker of apoptotic nuclei in sections of three pairs of transgenic and normal littermate epidermis. Representative pictures are shown (). In normal epidermis, no apoptotic nuclei were observed until day 13 and at this stage they were restricted to a few clusters of cells within the inner root sheath of the hair follicle. Apoptotic nuclei were not observed in normal matrix keratinocytes of the hair follicle until day 15 when the earliest signs of catagen became obvious. TUNEL positive nuclei reached a maximum at day 17 and were clustered around the dermal papilla cells next to the hair matrix and in the bulge isthmus. These results are similar to previous observations on normal epidermis (Lindner et al. 1997
). In contrast we observed a small number of apoptotic nuclei at day 11 in the inner root sheath of the transgenic hair follicles, a few apoptotic nuclei were seen in the matrix keratinocytes at day 13 reaching a peak at day 15. Apoptotic changes occur two days earlier in the transgenic than in normal hair follicles, with a time course in both groups that reflects the stage of involution. In both groups apoptotic nuclei first appear in the early stages of catagen as the lower portion of the follicles begin to degenerate and reach a maximum at mid catagen. Thus, in both groups apoptotic changes detected by TUNEL accompany but do not precede the changes observed histologically in the hair follicles.
Figure 9 Apoptotic changes detected by TUNEL staining occur earlier in the matrix cells of transgenic hair follicles. Paraffin embedded sections from the backskin of K14-ΔN80PG and control littermates were subjected to TUNEL staining to determine DNA fragmentation (more ...) Differentiation.
Changes in K10 and K16 expression have recently been reported to lead to altered proliferative potential of keratinocytes and hair follicles in vivo and in vitro (Paladini and Coulombe 1998
; Paramio et al. 1999
). To determine whether the transgenic mice exhibited changes in the normal differentiation program of the epidermis or hair follicles frozen sections of epidermis were stained with a panel of antibodies. These included antibodies against: keratins 5 and 14, which are synthesized in the basal layers; keratin 1, which is expressed in the suprabasal layers; keratin 6, which is expressed under conditions of hyperproliferation; high sulfur hair proteins, which are restricted to cells of the hair cuticle and loricrin which is expressed in the granular layer. No detectable changes were observed in response to transgene expression ().
Figure 10 Expression of differentiation-specific markers in epidermis of transgenic compared with normal mice. 5-μm frozen sections of newborn tail skin (a, b, a,′ and b′) or 7-d backskin (c–e and c′–e′) from (more ...)