Apoptosis in <i>oltNH</i> hair follicles.

<p>Apoptosis was investigated by performing the TUNEL assay on paraffin sections. Apoptosis is visualised by Cy3 fluorescence (red signal). DAPI was used as a nuclear counterstain (blue signal). Sections were taken from skin biopsies of <i>oltNH</i> mice (A to G) from postnatal day 2 to 25. A wild-type skin section on postnatal day 13 is shown as control (H). Three biopsies from different animals were used for each time point investigated. Arrows mark the hair bulb (B) or the trailing ends (T) of regressing hair follicles. Arrowheads point at TUNEL positive cells. There are numerous TUNEL positive cells in the matrix of <i>oltNH</i> hair follicles beginning on days 6 and 8 (arrow heads in B and C) and the regressing follicles on day 11 to 13. On day 25, the <i>oltNH</i> hair follicle re-enters anagen and shows no TUNEL positive cells (arrow in G). Throughout this period, there were no TUNEL positive cells in the hair bulbs of wild-type mice (not shown), only some cells in the inner root sheath exhibited a TUNEL positive signal (arrowheads in H), while the medulla showed unspecific autofluorescence. E, epidermis. Bar in E  = 50 µm for all images.</p

<i>Crisp1</i> expression in wild-type and <i>oltNH</i> hair follicles.

<p>In situ hybridisation with a gene-specific probe for <i>Crisp1</i> on skin sections of wild-type (A and C) and <i>oltNH</i> (B and D) mice on postnatal days 6 (A and B) and 8 (C and D). The DIG-labelled probe was visualised using alkaline phosphatase-conjugated anti DIG antibody. Images were taken in brightfield mode with a Nuance VX camera and further processed by spectral analysis using the accompanying software. The specific in situ signal spectrum was pseudo-coloured in red and the eumelanin spectral signal in black. Three biopsies from different animals were used for each time point investigated. Bar  = 100 µm. <i>Crisp1</i> expression is detected in the medulla of wild-type hair follicles (arrows in A and C, red signal), but not in comparable sections of <i>oltNH</i> mice (arrows in B and D).</p

Expression of genes relevant to the developing hair follicle.

<p>Total RNA or mRNA was prepared from dorsal skin biopsies of postnatal day 8 of wild-type (<i>Del(9)olt1Pas</i> heterozygous and <i>Plcd3^mNab</i> heterozygous), <i>oltSH</i> and <i>oltNH</i> mice and gene-specific fragments were amplified by PCR from 1 µg of cDNA. The number of PCR cycles is given below the image. The primers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039203#pone.0039203.s003" target="_blank">Table S2</a>. The first strand was synthesised from mRNA preparations for all RT-PCR experiments with <i>Krtap</i> genes and actin* in the Figure. The limitation of PCR cycles gives a semi-quantitative estimate that <i>Foxn1</i>, <i>Msx2</i>, <i>Hoxc13</i>, <i>Pdgfa</i>, <i>Pdgfb</i>, <i>Shh</i>, <i>Bmp2</i> and <i>Bmp4</i>, as well as the hair keratins Krt35 and Krt86, and the IRS keratin (<i>Krt71</i>) are expressed at comparable levels in wild-type, <i>oltSH</i> and <i>oltNH</i> mutants on postnatal day 8, when the hair shaft in the specimens is being formed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039203#pone-0039203-g004" target="_blank">Figure 4C and 4I</a>). However, <i>Crisp1</i> and <i>Krtap12-1</i>, and possibly also <i>Krtap4-2</i> and <i>Krtap8-2</i> are expressed at lower levels in <i>oltNH</i> mice compared to wild-type and <i>oltSH</i> mutants.</p

Histology of wild-type and <i>oltNH</i> hair follicles during hair follicle morphogenesis and the beginning of the first cycle.

<p>Paraffin and methacrylate (Technovit 7100) sections, HE stain. Postnatal days examined are indicated. In wild-type animals, the hair follicles increase in length during anagen from P2 to P12 and show active melanogenesis in their hair bulbs during this period (arrows in A to E). The diameter of the hair bulb decreased from P6 to P11 and remained like this until catagen sets in on postnatal day 17 (F). In <i>oltNH</i> mice, however, the hair follicles decrease in length after postnatal day 6 (G to K) and exclude the dermal papilla from the bulb on days 11 and 12 (arrows P in J* and K*). Numerous granulocytes (G) are found in the vicinity of the mutant hair bulbs at this time (marked as G in I* and J*). The diameter of the hair bulb decreases remarkably after postnatal day 6 (arrows in G to K). While wild-type mice have entered catagen by day 17 as shown by the long epithelial strand and reduced hair follicle length (arrows in F), <i>oltNH</i> hair follicles re-enter an anagen phase on days 17 to 19 (L and S) exhibiting a broad hair matrix (marked as M in L* and S*), a large dermal papilla (marked as P arrow in S*) and active melanogenesis (L* and S*), which is followed by a regression on postnatal days 22 (T and T*) and 24 (U and U*). While wild-type hair follicles proceed through the first cycle anagen from day 22 to day 37 (arrows in N to R), <i>oltNH</i> hair follicles re-enter anagen on postnatal day 25 (V, marked as P arrow in V*) and show continued increase in hair follicle length by day 30 (W and W*). This growth phase of the mutant follicle ends in a telogen phase on day 37 (X and X*), when wild-type follicles are still in the growth phase (R). Three biopsies from different animals were used for each time point investigated. P, dermal papilla; SG, sebaceous gland; M, matrix; G, neutrophilic granulocyes. Bar  = 100 µm in images A to X, and 50 µm in images G* to L* and S* to X*.</p

The phenotypes of wild-type, <i>Del(9)olt1Pas, oltSH</i> and <i>oltNH</i> mutant mice.

<p>The phenotype of wild-type (Wt), <i>Del(9)olt1Pas</i> (<i>olt</i>) homozygotes, <i>oltSH</i> and <i>oltNH</i> mice during hair follicle morphogenesis (P11 and P14) and the first hair cycle (P24 and P28). The + and – indicate the wild-type and mutant allele, respectively. The genotypes given below the images were determined by genomic PCR assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039203#pone-0039203-g002" target="_blank">Figure 2</a> B. Mutants of the <i>oltSH</i> phenotype have a reduced dorsal pelage on day P11 and P14 (A and B), which becomes very sparse during the first hair cycle (C and D). Ventrally, <i>oltSH</i> mutants show total alopecia (E to H) compared to homozygous <i>Del(9)olt1Pas</i> mice (F to H), in which the coat is predominantly reduced in the medial femoral and inguinal region (arrows in F, G, and H). <i>oltNH</i> mutants have no pelage.</p

Histology of the infundibular region and distorted hair shafts.

<p>Methacrylate (Technovit 7100) sections of representative areas in the dorsal skin on postnatal day 9, HE stain. The phenotype (Wt, <i>olt</i> (i.e. <i>Del(9)olt1Pas</i>), <i>oltSH</i> or <i>oltNH</i>, respectively) and genotype with respect to the <i>Plcd1</i> (<i>Plcd1*</i> “-” refers to the <i>Del(9)olt1Pas</i> mutation) and <i>Plcd3</i> (Plcd3** “-” refers to the <i>Plcd3</i>^mNab allele) gene is indicated for each image. At least 4 biopsies of each genotype have been investigated. In E, Bar  = 25 µm. These is no hair loss and are no distorted hair shafts neither in wild-type mice heterozygous for both mutant alleles (A), nor those heterozygous for the <i>Del(9)olt1Pas</i> mutation and homozygous for the <i>Plcd3</i>^mNab allele (B), nor others being wild-type for <i>Plcd1</i> and homozygous for the <i>Plcd3</i>^mNab mutant allele (C). Arrows indicate normal hair shafts. Arrowheads mark distorted hair shafts in <i>Del(9)olt1Pas</i> (<i>olt</i>), <i>oltSH</i> and <i>oltNH</i> mice. The alterations of the hair shafts appear histologically similar in all three mutant specimens.</p

Histological aspects of <i>oltSH</i> mutants on postnatal day 12.

<p>Methacrylate (Technovit 7100) sections of representative areas in the dorsal skin on postnatal day 12 <i>oltSH</i> mice, HE stain, bar  = 100 µm in A, bar  = 25 µm in B to D. Boxed areas in A are shown at higher magnification B, C, and D respectively. Four different animals were investigated. A. The overview shows hair follicles in anagen (box B) and catagen (box C) side by side. B. The hair follicle shows active melanogenesis as a hallmark of anagen (arrowhead). C. The hair follicle is in catagen and has excluded the dermal papilla (arrowhead). D. Accumulations of neutrophilic granulocytes (arrowhead) are seen in the vicinity of follicles in catagen.</p

Histomorphometric analysis of hair follicle length and hair bulb diameter.

<p>Measurements were taken from a sample of 150 hair follicles in three different biopsies using the Image J software. Statistical analysis using the paired t-Test was performed employing the GraphPad Prism4 software. Data are expressed as mean ± SEM. *** signifies p<0.001. White columns depict data from wild-type mice and chequered columns those from <i>oltNH</i> mice. The age of the mice is given on the X axis. Hair follicle length represents the distance from the infundibulum to the most distal part of the hair follicle. The widest diameter of the hair bulb or distal end of the hair follicle in catagen and telogen stages is shown as “hair bulb diameter”. Both parameters indicate that <i>oltNH</i> mice terminate their first postnatal anagen by day 14 and re-initiate a growth phase thereafter, which in turn ends by day 24. The second cyclic growth phase of <i>oltNH</i> hair follicles ends by day 37.</p

Proliferation in wild-type and <i>oltNH</i> hair follicles.

<p>Proliferation in the hair follicles of wild-type (A, B, C, D, E) and <i>oltNH</i> (<i>del(9)olt1Pas</i> −/−, <i>Plcd3^mNab</i> −/−) (F, G, H, I, J) dorsal skin on postnatal day 2 (A, F), day 6 (B, G), day 8 (C, H), day 11 (D, I), and day 12 (E, J) is visualised by PCNA immunoreactivity (red signal). DAPI was used as a nuclear counter stain (blue signal). Paraffin sections. Bar  = 50 µm for all images is given in A. White arrowheads mark the hair bulb. Three biopsies from different animals were used for each time point investigated. In wild-type mice, PCNA immunoreactivity is detected in the nuclei of the matrix cells of the hair bulb throughout the period examined (arrowheads in A to E). In the <i>oltNH</i> mutant, the PCNA immunoreactivity is prominent in the matrix on days 2 and 6 (arrowheads in F and G), while already on day 8 some hair bulbs show much fainter immunoreactivity (right arrowhead in H). In the trailing ends of the hair follicles on days 11 the immunoreactivity is very faint (arrowheads in I), and on day 12 undetectable (arrowhead in J). Note that there is no PCNA immunoreactivity in the cells of the dermal papilla.</p