Transgenic Research 11: 241–247, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
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Inducible, reversible hair loss in transgenic mice Jingshan Chen1,∗ , Max B. Kelz1 , Guoqi Zeng1 , Cathy Steffen1,2 , Penny E. Shockett3,∗∗ , Gordon Terwilliger4, David G. Schatz3 & Eric J. Nestler1,2,∗ ∗ ∗ 1 Laboratory
of Molecular Psychiatry, Yale University School of Medicine, Connecticut Mental Health Center, 34 Park Street, New Haven, CT 06508, USA 2 Department of Psychiatry and Center for Basic Neuroscience, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA 3 Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06520, USA 4 Department of Comparative Medicine, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06520, USA Received 20 September 2001; revised 11 December 2001; accepted 19 November 2001
Key words: gene expression, luciferase, telogen effuvium, tetracycline inducible system, tTA (tetracycline transactivator)
Abstract Telogen effluvium is a common type of hair loss. Although the morphological changes associated with telogen effluvium have been well characterized, the underlying molecular mechanisms remain unknown, and no animal models have been developed. We report here that inducible transgenic mice expressing high levels of the transcription factor, tTA (tetracycline transactivator), plus a reporter luciferase gene, show a reversible hair loss phenotype. Skin of these mice exhibits an increase in the number of hair follicles at the telogen phase, but a decreased number of follicles at the anagen phase. These changes resemble skin pathology seen in patients with telogen effluvium, which suggests that the inducible transgenic mice may be useful as a model for this disorder. Moreover, since overexpression of several other transgenes failed to cause skin pathology, the present findings also indicate types of molecular abnormalities that may cause reversible hair loss. Introduction Telogen effluvium is a common and distressing syndrome characterized by adult hair loss. Such hair loss, which primarily involves club hairs, can result from perturbation of the hair cycle by several types of drugs (e.g., amphetamine, propanolol, and lithium), crash diets, childbirth, emotional stress, ion deficiency, hair dyes, and systemic illness (Kligman, 1961; Whiting, 1996a, b; Tosti et al., 2001). Although telogen effluvium occurs in males and females, it much more com∗ Present address: Clinical Brain Disorders Branch, National Institute of Mental Health, Bethesda, MD 20892, USA ∗∗ Present address: Department of Biological Sciences, Southeastern Louisiana University, Hammond, LA 70402, USA ∗ ∗ ∗ Author for correspondence: E-mail:
[email protected]
monly reaches a clinical threshold in females because of greater sensitivity to signs of hair loss (Headington, 1993). There are two types of telogen effluvium: acute and chronic (Whiting, 1996a; Rebora, 1997). Acute telogen effluvium lasts for only several weeks; whereas the chronic condition lasts for several months or years. Severe and long-lasting hair loss is often associated with prolonged perturbations such as emotional stress (Rebora, 1997). Chronic telogen effluvium is a common problem in middle-aged females, and is manifest by persistent and severe shedding of hair in a fluctuating course over several years (Whiting, 1996b). One of the major pathological changes seen in scalp biopsies from individuals with telogen effluvium is an increased number of telogen follicles accompanied by a decreased number of anagen follicles
242 (Whiting, 1996a). Most cases are associated with no sign of inflammation in the tissue. Although the morphological changes seen in patients with telogen effluvium have been well characterized, the underlying molecular mechanisms remain unknown. As a result, there is no effective treatment for this syndrome, especially for those cases not associated with inflammation. A major obstacle in studying molecular mechanisms of telogen effluvium is the lack of suitable animal models. Inducible transgenic animal models have been used successfully in studies of the molecular mechanisms underlying such diverse diseases as drug addiction (Kelz et al., 1999), Huntington’s disease (Yamamoto et al., 2000), diabetes (Green et al., 2000), and cardiomyopathy (Baker et al., 2001), as well as the molecular mechanisms of natural processes such as bone formation (Sabatakos et al., 2000) and learning and memory (Malleret et al., 2001). In our studies of inducible transgenic mice (Chen et al., 1998), we serendipitously observed a line with a reversible hair loss phenotype, which we describe here. Expression of an artificial transcription factor, the tetracycline transactivator (tTA), in conjunction with expression of a luciferase reporter gene, caused hair loss in adult animals. Inhibition of gene expression with the tetracycline derivative, doxycycline, completely reversed the hair loss within a week. Histological features of skin in mice showing the hair loss phenotype resembled the pathological changes associated with telogen effluvium in humans. We propose that these mice may serve as a novel animal model of telogen effluvium and may provide insight into the molecular basis of this condition.
Western blotting Western blotting for FosB was performed exactly as described previously (Hope et al., 1994) using an antibody that recognizes all known Fos family proteins. Levels of FosB immunoreactivity were quantified by measuring the optical density of specific bands using an image analysis system with NIH image software and gray-scale calibration. Luciferase assay Skin was obtained from several areas of the body, dissected, and homogenized in lysis buffer (purchased from Roche Molecular Biochemicals) by use of a polytron. Twenty microliters of the lysate were mixed with 100 µl of luciferase reagents, and luciferase activity was measured in a luminometer as described previously (Chen et al., 1998). Luciferase activity was then normalized for total protein measured by the Lowry method, and expressed as activity per mg total protein. Histological analysis A 2 × 2 cm piece of skin was dissected from dorsal thorax of each transgenic mouse. The skin was fixed in 10% buffered formalin for 24 h, and then embedded in parafin. The embedded tissue block was sectioned at a thickness of 5 µm. Sections of skin tissue were then stained with hematoxylin and eosin, according to standard protocols, and examined by light microscopy.
Results Materials and methods
Development of inducible transgenic mice with hair loss phenotype
Transgenic mice Tetracycline-regulated gene expression was used in the development of inducible transgenic mice carrying the neuron-specific enolase (NSE)-tTA, TetOpluciferase, and TetOp-tTA genes (Chen et al., 1998). Presence of the TetOp-tTA gene provides a positive feedback auto-regulatory system (Shockett et al., 1995), which can be used to increase the expression level of inducible transgenes (Figure 1). We also used another line of mouse containing the TetOpluciferase gene without a TetOp-tTA gene (Hasan et al., 2001).
In our initial experiments with the tetracycline gene regulation system, we generated several founder lines of mice expressing the NSE-tTA gene (Chen et al., 1998). We used the NSE promoter in an attempt to limit transgene expression to the brain. We next bred several lines of NSE-tTA mice with a TetOp-luciferase reporter mouse in order to identify regions of brain targeted by the system. The TetOp-luciferase mouse also contained a copy of the TetOp-tTA gene, which, through a positive feedback loop, had been shown to dramatically increase levels of inducible transgene expression (Shockett & Schatz, 1996).
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Figure 1. Schematic diagram of the modified tetracycline-regulated gene expression system. This system involves three genes. (a) Gene 1 encodes the tetracycline transactivator (tTA). Expression of tTA is controlled by the neuron-specific enolase (NSE) promoter, which directs tissue-specific expression of the tTA. (b) Gene 2 encodes a tTA gene under the control of the tetracycline-responsive promoter (Tetop), which contains a minimal CMV promoter and seven copies of tetracycline operators. This construct forms a positive feedback loop. Since the Tetop promoter is tightly regulated, Gene 2 needs a small amount of tTA from Gene 1 to initiate the positive feedback loop. (c) Gene 3 encodes a target gene such as luciferase under the control of the Tetop promoter. The level of target gene expression is correlated with the level of tTA expression, and is regulated by tetracycline.
To our surprise, one line of NSE-tTA mice (line A), when bred to contain the TetOp-tTA and TetOpluciferase genes, developed total hair loss shortly after weaning (Figure 2(a)). This complete hair loss occurred gradually over a period of 2 weeks. Seven other lines of NSE-tTA mice, crossed with TetOptTA × TetOp-luciferase mice, showed no hair loss. The selective appearance of hair loss in the one line of NSE-tTA mice correlated with luciferase expression in skin. Thus, the mice that showed hair loss exhibited high levels of luciferase activity in the skin (Figure 2(b)); whereas the other lines of tritransgenic mice that did not show hair loss showed no detectable luciferase activity in skin (not shown). Importantly, high levels of tTA, but not of luciferase, may be responsible for the hair loss, because no hair loss was observed when the NSE-tTA mice were crossed with TetOp-luciferase mice that do not contain a TetOp-tTA gene (see Discussion).
Since tTA is a transcription factor, these results raise the possibility that abnormal transcription in skin causes the hair loss phenotype. To test whether expression of any transcription factor in skin can cause hair loss, we crossed the same line of NSE-tTA mice with mice carrying a TetOp-FosB gene. (FosB is a member of the Fos family of transcription factors.) Expression of high levels of FosB was seen in the skin of the resulting bitransgenic mice (Figure 3(b)), but no hair loss was observed (Figure 3(a)). Hair loss also was not seen in bitransgenic mice, derived from the same NSE-tTA line, that express several other transcription factors (i.e., TetOp-CREB, TetOp-mCREB, or TetOp-cJun) (not shown). Hair loss in the transgenic mice is fully reversible The tetracycline system provides for inducible transgene expression, since tTA can be inhibited by low
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Figure 2. Hair loss phenotype in transgenic mice overexpressing the TetOp-tTA and TetOp-luciferase genes. (a) The bitransgenic mouse to the left carries the TetOp-tTA and TetOp-luciferase genes. The tritransgenic mouse to the right carries the NSE-tTA (line A), TetOptTA and TetOp-luciferase genes. The two mice shown in the figure are littermates. Only the tritransgenic mice exhibit the hair loss phenotype. (b) Bar graph shows the mean ± s.e.m. luciferase activity in skin extractss of bitransgenic and tritransgenic mice (n = 3).
concentrations of tetracycline or the tetracycline derivative, doxycycline, which has a higher affinity for tTA. It was, therefore, of interest to determine whether hair loss in the NSE-tTA × TetOp-luciferase × TetOp-tTA tritransgenic mice could be regulated by doxycycline exposure. In the first series of experiments, mice conceived and raised off doxycycline developed complete hair loss by 6 weeks of age. When mice were then treated with different concentrations of doxycycline in the drinking water, they grew back hair within 1 week (Figure 4(a)). The reversal of the hair loss phenotype was dose-dependent, and closely paralleled levels of transgene expression (Figure 4(b)). At the higher concentrations of doxycycline used, reversal of hair loss appeared complete, with the mice indistinguishable from wildtype controls. In a second series of experiments, tritransgenic mice were raised from conception on doxycycline (200 µl/ml in drinking water) and showed no detectable hair loss or any other skin abnormality throughout
Figure 3. Normal hair phenotype in transgenic mice overexpressing the transcription factor FosB. (a) The single transgenic mouse to the right carries the NSE-tTA (line A) gene. The bitransgenic mouse to the left carries the NSE-tTA (line A) and TetOp-FosB genes. The two mice shown in the figure are littermates; neither of these genotypes exhibit hair loss. (b) Western blot showing high levels of expression of FosB in the skin of bitransgenic (NSE-tTA × TetOp-FosB) mice, with undetectable levels in NSE-tTA mice.
adulthood. When 8–12-week-old mice were then removed from doxycycline, hair loss began to appear 2–3 weeks later and became complete within another week. These data show that the hair loss phenotype is fully reversible and is not due to a developmental abnormality. Rather, it is caused by transgene expression in fully developed mice. Pathological changes in the transgenic mice resemble those seen in patients with telogen effluvium To better understand the nature of the hair loss seen in the tritransgenic mice, we analyzed sections of skin from tritransgenic mice with the hair loss phenotype and from littermate controls without the phenotype. Mice carrying only the NSE-tTA gene, and not the TetOp-luciferase or TetOp-tTA genes, showed normal skin histology, including normal-appearing hair follicles, which was indistinguishable from wildtype animals. In contrast, dramatic abnormalities were
245 evident in the skin of the tritransgenic animals with hair loss. The number of hair follicles at the telogen stage was increased and the number of hair follicles at the anagen phase was decreased in these mice (Figure 5). No cellular infiltrates were observed in the tissue, which suggests that the hair loss does not involve an inflammatory process. These changes are reminiscent of pathological abnormalities seen in patients with non-inflammatory subtypes of telogen effluvium (Whiting, 1996a,b). Strikingly, tritransgenic mice that had developed hair loss but were then treated with doxycyline to restore their coats, showed an increased number of anagen hair follicles and a decreased number of telogen hair follicles compared with untreated tritransgenic mice (Figure 5). Moreover, tritransgenic mice raised on doxycycline, and which therefore never developed hair loss, exhibited skin histology that was indistiguishable from controls. Discussion The inducible transgenic mouse developed in this study is the first transgenic animal model, to our knowledge, with a reversible hair loss phenotype. The pathological changes in the skin of the inducible transgenic mice resemble those seen in patients with telogen effluvium, which suggests that the inducible transgenic mouse could be used as an animal model for this disorder. The mechanism underlying the hair loss phenotype is unknown. One possibility is that high levels of expression of the artificial transcription factor, tTA, in the skin may be responsible for the phenotype. In contrast, it does not appear that high levels of luciferase expression in the skin is responsible, since expression of luciferase (in the absence of the TetOptTA transgene) failed to produce hair loss. Although speculative, these findings raise the possibility that a perturbation of transcription may be involved in the pathophysiology of telogen effluvium. However, it is also possible that the hair loss phenotype results from the disruption of a gene by the insertion of the TetOp-tTA and TetOp-luciferase transgenes (which are linked). In this scenario, expression of that endogenous gene would be impaired only when the transgenes are expressed (i.e., in the absence of doxycycline). Identification of the insertion site of these transgenes is now needed to further explore this possibility. The NSE promoter is often used to direct gene expression to neurons, however, the 1.8 kb fragment
used in most studies is known to be active in several peripheral tissues as well (Sabatakos et al., 2000; Tanaka et al., 2001). For example, the same NSEtTA line used in the present study, which directs expression in skin, also directs expression in bone, specifically within osteoblasts (Sabatakos et al., 2000), and in retina and lens (Kelz et al., 2000). Gene expression directed in peripheral tissues by the NSE promoter appears to be in mesenchymal precursor cells. Melanocytes in the germinative layers of hair follicles are of neural crest origin. During embryonic development, melanocyte precursor cells migrate from the neural crest and enter the developing epidermis. Therefore, the NSE promoter could direct expression of tTA in melanocytes and other precursor cells present in hair follicles to cause the hair loss phenotype observed in the present study. However, further experiments are needed to definitively identify the cell types within skin that express tTA in the inducible transgenic mice. Nevertheless, the present results indicate that NSE-tTA mice can be used as genetic tools to direct expression of a variety of genes under control of the TetOp promoter to skin. Our hypothesis that high levels of tTA expression in the skin may result in hair loss suggests that perturbation of transcription in skin tissue may be the molecular mechanism of hair loss in telogen effluvium. Indirect support for this notion comes from the fact that gonadal steroid hormones, which have been related to telogen effluvium and other forms of hair loss such as alopecia, bind to ligand-gated transcription factors that regulate the expression of other genes (Sonoda et al., 1999). However, our results also show that expression of not any transcription factor in skin can cause hair loss, because no hair loss was observed in inducible transgenic mice that express FosB or several other transcription factors in this tissue. tTA is a fusion protein that consists of the tetracycline-binding domain of the tet repressor from bacteria fused to the transactivation domain of VP16 (a viral transcription factor) (Gossen & Bujard, 1992). Consequently, tTA could disrupt normal transcriptional regulation in skin, possibly by transcriptional squelching, and thereby promote apoptosis-driven hair follicle regression mediated by the transcription factor p53 (Botchkarev et al., 2001). However, this is speculative, since the specific changes in gene expression caused by tTA are difficult to predict a priori. Therefore, an important goal of future studies is to analyze gene expression profiles from the inducible transgenic mice in the presence or
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Figure 4. Correlation between the hair loss phenotype and the inducible transgene expression. (a) Bar graph shows the level of luciferase expression in the skin of tritransgenic mice (NSE-tTA [line A] × TetOp-tTA × TetOp-luciferase) drinking different concentrations of doxycycline (from 0.25 to 200 µg/ml) in water. A positive control (+) is the tritransgenic mice drinking regular water, and a negative control is bitransgenic mice (TetOp-tTA × TetOp-luciferase) drinking regular water. (b) Reversal of hair loss phenotype by turning off expression of the TetOp-tTA and TetOp-luciferase genes with doxycycline. Tritransgenic mice, which showed a severe hair loss phenotype at weaning, were given doxycycline in the drinking water after weaning. The hair loss phenotype was completely reversed by doxycycline in a dose-dependent manner. A positive control (+) is a tritransgenic mouse drinking regular water, and a negative control is a bitransgenic mouse drinking regular water.
Figure 5. Reversible pathological changes in hair follicles in the skin of tritransgenic mice. Skin specimens were obtained from bitransgenic mice (TetOp-tTA × TetOp-luciferase, left panels) fed with regular water, tritransgenic mice (NSE-tTA [line A] × TetOp-tTA × TetOp-luciferase) fed with regular water (middle panels), and tritransgenic mice fed with doxycycline (200 µg/ml) in the drinking water (right panels). Sections of fixed skin were stained with hematoxylin and eosin, and examined by light microscopy. The results shown in the figure are representative of the analysis of at least four animals in each group.
247 absence of doxycycline over a fine time course to identify those genes whose altered expression is associated with the onset (and reversal) of hair loss. Such studies, and other investigations of the inducible transgenic mice described here, could provide important clues to the molecular mechanisms of telogen effluvium and the regulation of hair loss in general.
Acknowledgements This work was supported by grants from the National Institute on Drug Abuse and the National Institute of Mental Health.
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