1. Hair-Cycle-Dependent Expression of Parathyroid Hormone-Related Protein and its Type I Receptor: Evidence for Regulation at the Anagen to Catagen Transition 
Yong Mee Cho, Grant L. Woodard, Maureen Dunbar, Todd Gocken, Juan A. Jimènez, John Foley 
Journal of Investigative Dermatology 
Volume 120, Issue 5, Pages (May 2003)
DOI: /j x
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
PTHrP mRNA expression detected by in situ hybridization in developing mouse skin. Whole body embryos (a–i) or longitudinal sections of trunk skin (j–r) of CD-1 mice taken from E12 (a–c), E14 (d–f), E18 (g–i), 1 wk (j–o), and 3 wk (p–r) were processed for in situ hybridization with 35S-labeled sense and anti-sense probes. The left column has dark field images of sections processed for in situ hybridization with the sense probe as negative control. The middle column has dark field images of serial sections of the left column and was processed for in situ hybridization with the anti-sense probe. The right column has bright field images of the serial sections of the middle column and was counterstained with methylene green. (a–c) At E12, PTHrP mRNA expressed highly in the developing mammary gland (arrow) is not apparent in the periderm (arrowhead). (d–f) At E14 during induction stage, PTHrP mRNA is highly expressed in the epithelial placode (arrow) of developing hair follicle, whereas it is not expressed in the interfollicular epidermis. (g–i) At E18 during morphogenesis stage, the distal portion (arrow) of developing hair follicles express PTHrP mRNA. (j–o) At 1 wk during differentiation stage, full-grown hair follicles express PTHrP mRNA in the isthmus (arrow), ORS, and matrix of bulb. PTHrP transcripts are more abundant in the isthmus and proximal ORS. (m–o) Are the high magnification of distal portion, shaft, and proximal portion of hair follicle, respectively. (p–r) At 3 wk during the first telogen of hair cycle, PTHrP mRNA continues to be expressed exclusively in the isthmus (arrow). Dotted lines delineate the basement membrane. Bar: (a–i,p–r) 100 μm; (j–l) 185 μm; and (m–o) 74 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
PTHrP mRNA expression detected by in situ hybridization in the foot pad and nipple skin of the mouse. Foot pad skin from a 4 mo old mouse and nipples from virgin, pregnant, and lactating mice were processed for in situ hybridization with an 35S-labeled PTHrP sense probe as negative control (left column) or anti-sense probe (middle and right columns). Left and middle columns are dark field images. Right column is bright field images of serial sections hybridized with anti-sense probe and counterstained with methylene green. (a–c) PTHrP mRNA expression is not apparent in the foot pad skin, which has a thick epidermal layer. (d–l) PTHrP mRNA is expressed neither in the epidermis of virgin nipple nor of pregnant and lactating nipples, which undergo dramatic change in its thickness. Arrow indicates hair follicle in (e) and the lactiferous duct in (k). Bar: (a–c,f,i,l) 100 μm; and (d,e,g,h,j,k) 1200 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
PTH1R mRNA expression detected by in situ hybridization in developing mouse skin. Whole body embryos (a–f) or longitudinal sections of trunk skin (g–l) taken from E14 (a–c), E18 (d–f), 1 wk (g–i), and 3 wk (j–l) CD-1 mice were processed for in situ hybridization with an 35S-labeled PTHR1 sense probe as control (left column) or anti-sense probe (middle and right columns). Left and middle columns are dark field images with sense and anti-sense probes, respectively. Right column is bright field images of serial sections of middle column and counterstained with methylene green. (a–f) At E14 and E18, PTH1R mRNA is highly expressed in the upper dermis but not in the epidermis and developing hair follicle. (g–i) At 1 wk, PTH1R mRNA expression is observed in the upper dermis; however, it has decreased in intensity and is more concentrated in the dermis beneath the epidermis and surrounding isthmus (arrow). (j–l) PTH1R mRNA expression is not apparent in 3 wk old mouse skin. Dotted lines delineate the basement membrane. Bar: (a,b,d–f,j–l) 100 μm; (c) 35 μm; (g,h) 250 μm; and (i) 60 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
PTHrP mRNA expression detected by in situ hybridization through the hair cycle and in the vibrissa follicle. After depilation of 7 wk old CD-1 mice to synchronize the hair cycle, longitudinal skin sections taken on the day of depilation (D0, a–c) or 3 d (D3, d–f), 11 d (D11, g–k), and 19 d (D19, l–o) after depilation were processed for in situ hybridization with an 35S-labeled PTHrP sense probe (left column) or anti-sense probe (middle and right columns). An upper lip, including vibrissa hair follicle, from 4 mo old mouse was also prepared by the same method as above. The left and middle columns are dark field images of serial sections. The right column is bright field images of serial sections hybridized with anti-sense probe and counterstained with methylene green. (a–c) During telogen, PTHrP mRNA is weakly expressed in the isthmus (arrow). (d–f) During anagen IIIa, PTHrP mRNA is expressed weakly throughout the hair follicle. (g–k) During anagen VI, PTHrP mRNA is expressed in the isthmus, ORS, and matrix of bulb. PTHrP transcripts are more abundant in the isthmus (arrow) and proximal ORS (arrowhead). (i,j,k) Are the high magnification of the distal portion, shaft, and proximal portion of a hair follicle, respectively. (l–o) During catagen, PTHrP mRNA continues to be expressed in the isthmus (arrow), whereas its expression is not apparent in the regressing proximal hair follicle (arrowhead). (p–s) In the vibrissa hair follicle, PTHrP transcripts are strongly expressed in the isthmus, bulge, ORS, and matrix. They are especially abundant in the peripheral portion of bulge (arrow) and proximal ORS (arrowhead). (r,s) Are the high magnification of distal and proximal portions of vibrissa hair, respectively. Bar: (a–e,i–k,n,o) 50 μm; (f) 40 μm; (g,h) 130 μm; (l,m) 60 μm; (p,q) 200 μm; and (r,s) 120 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
PTH1R mRNA expression detected by in situ hybridization through the hair cycle. Longitudinal skin sections taken from 7 wk old CD-1 mice on the day of depilation (D0, a–c) and 3 d (D3, d–f) after depilation were processed for in situ hybridization with an 35S-labeled PTH1R sense probe as control (left column) or anti-sense probe (middle and right columns). The left and middle columns are dark field images. Right column is bright field images of serial sections hybridized with anti-sense probe and counterstained with methylene green. (a–c) In the adult mouse skin, PTHR1 mRNA expression is not apparent in the dermis. (d–f) During anagen III, PTH1R transcripts are weakly and transiently expressed in the deep dermis surrounding proximal hair follicle (arrow). Bar: (a–c,f) 50 μm; and (d,e) 81 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Evaluation of relative levels of PTHrP and PTH1R transcript expression in murine skin by RNase protection. (a) RNase protection analysis performed on 40 μg of total RNA derived from individual skin samples of fetal, neonatal, adult, and aged mice. Age of samples is indicated above the figure: E=embryonic, D=day, W=wk, M=mo, Y=year. Rp, R, cyc indicate bands that were protected by mPTHrP, mPTH1R, and mCyclophilin probes, respectively. (b) Densitometric analysis of levels PTHrP (solid bars) and PTH1R transcripts (open bars) relative to cyclophilin as assayed by RNase protection. The data represent the average of two or three samples (some from above, others from assays not shown) and error bars represent the standard deviation when three replicates were available. (c) RNase protection analysis performed on 40 μg of total RNA derived from individual skin samples of mice subjected to depilation. Days after depilation are indicated above the figure. Rp, R, cyc indicate bands that were protected by mPTHrP, mPTH1R, and mCyclophilin probes, respectively.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

8. Figure 7
Transgene-mediated overexpression of PTHrP influences the murine hair cycle. (a) Photomicrographs of major hair types. (WT) indicates hair from 9 wk old male wild-type littermate. K14 indicates hair from 9 wk old male K14-PTHrP mouse. The far left side of the panel shows an annotated view of representative zigzag hairs. Note that the wild type has four major segments, whereas the K14-PTHrP has only three. (b) The panel shows the mean length ± SEM of three types of dorsal hair from the K14-PTHrP mice at 21 d and 9 wk were 32–45% shorter than those of wild-type littermates. p<0.01 for all hair types of K14-PTHrP hair as compared with wild type at both ages. (c) Hematoxylin and eosin-stained sections from formalin-fixed, paraffin-embedded, mid-dorsal skin of age- and sex-matched 12–15 wk old K14-PTHrP mice and their littermates that have been subjected to depilation. Genotype is indicated on the top of the composite (WT=wild type, K14=K14-PTHrP). Days after depilation are indicated along the left side of the figure. Bar: (a) 1 mm; (b) 0.2 mm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

9. Figure 8
Overexpression of PTHrP decreases hair length in FGF-5-null animals. (a) Thirteen week old FGF-5-null mouse. (b) Thirteen week old K14-PTHrP/FGF-5-null littermate. Note the dorsal coat length of the PTHrP/FGF-5-null mouse is discernibly shorter than the FGF-5-null littermate. (c) Ventral surfaces of the male and female K14-PTHrP/FGF-5-null mice. In general there appeared to be slightly more and longer hair on the ventral skin of the transgenic FGF-5 knockouts than the standard K14-PTHrP mice. (d–f) Hematoxylin and eosin-stained sections from formalin-fixed, paraffin-embedded, skin taken from the mice in (a,b). (d) Dorsal skin from the FGF-5-null mouse. (e) Dorsal skin from the K14-PTHrP/FGF-5-null mouse. (f) Ventral skin from the K14-PTHrP/FGF-5-null mouse. The lack of FGF-5 in the skin did not alter the subtle dorsal skin phenotype or the nipple like ventral surface produced by the overexpression of PTHrP. Bar: (d–f) 80 μm. (g) The panel shows the mean length ± SEM of three types of dorsal hair from the K14-PTHrP/FGF-5-null mice at 21 d were 38–49% shorter than those of FGF-5-null littermates. p<0.01 for all hair types of K14-PTHrP/FGF-5-null hair as compared with FGF-5-null.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions