Effect of <i>Tspear</i> down-regulation on murine hair follicles.

<p>(a) Tspear is expressed in mouse hair follicles (HFs) in the hair matrix keratinocytes, outer root sheath, inner root sheath, hair shaft and the infundibulum (scale bar = 50 μm); (b) Back skin tissue strips from <i>K14-H2B-GFP</i> mice were transfected with <i>Tspear</i> siRNA or control siRNA. RNA was extracted from transfected HFs and <i>Tspear</i> RNA expression levels were assessed by qRT-PCR. Results were normalized to <i>Gapdh</i> levels and are expressed as expression levels relative to control samples. Data were pooled from three independent experiments (two sided t-test; **p<0.01); (c-f) Z stacks optical sections of <i>K14-H2B-GFP</i> mouse HFs (c) obtained 24h following transfection with control siRNA (d) or <i>Tspear</i> siRNA (e) were used to calculate average hair bulb diameter. Three measurements were done for each HF in the bulb and proximal hair shaft (c, dashed white lines) and an average diameter was calculated accordingly. Epithelial nuclei are marked with <i>GFP</i> (scale bars = 100 μm). Data was pooled from three independent experiments (F, two sided t-test; **p<0.01); (g-i) Melanin content was assessed by quantitative Masson-Fontana histochemistry in <i>Tspear</i> siRNA treated HFs (h) compared to control (g). Data was pooled from two independent experiments (I, two sided t-test; ***p<0.001) (scale bars = 50 μm); (j-o) Apoptosis was assessed by the TUNEL assay (TUNEL, green; DAPI, blue) at the hair bulb (j-l) and infundibular (m-o) compartments of HFs downregulated for <i>Tspear</i> (k,n) compared to control siRNA treated HFs (j,m) (scale bars = 50 μm). Average number of TUNEL-positive cells in hair follicles in the respective compartments. Data were pooled from two independent experiments (l,o, two sided t-test; ***p<0.001) (scale bars = 50 μm). White dotted lines delineate the outer epidermal surface; (p) RNA was extracted from <i>Tspear</i> siRNA and control siRNA transfected HFs and <i>Notch1</i> RNA expression level was assessed by qRT-PCR. Results were normalized to <i>Rplp0</i> levels and are expressed as expression levels relative to control samples. Data were pooled from three independent experiments (two sided t-test; *p<0.05). E—epidermis; INF–Infundibulum; D—dermis; DP—dermal papilla; IRS—inner root sheath; ORS—outer root sheath; HM—hair matrix; HS—hair shaft; TUNEL—terminal deoxynucleotidyl transferase dUTP nick end labeling.</p

Effect of <i>Tspear</i> down-regulation on murine hair follicles.

<p>(a) Tspear is expressed in mouse hair follicles (HFs) in the hair matrix keratinocytes, outer root sheath, inner root sheath, hair shaft and the infundibulum (scale bar = 50 μm); (b) Back skin tissue strips from <i>K14-H2B-GFP</i> mice were transfected with <i>Tspear</i> siRNA or control siRNA. RNA was extracted from transfected HFs and <i>Tspear</i> RNA expression levels were assessed by qRT-PCR. Results were normalized to <i>Gapdh</i> levels and are expressed as expression levels relative to control samples. Data were pooled from three independent experiments (two sided t-test; **p<0.01); (c-f) Z stacks optical sections of <i>K14-H2B-GFP</i> mouse HFs (c) obtained 24h following transfection with control siRNA (d) or <i>Tspear</i> siRNA (e) were used to calculate average hair bulb diameter. Three measurements were done for each HF in the bulb and proximal hair shaft (c, dashed white lines) and an average diameter was calculated accordingly. Epithelial nuclei are marked with <i>GFP</i> (scale bars = 100 μm). Data was pooled from three independent experiments (F, two sided t-test; **p<0.01); (g-i) Melanin content was assessed by quantitative Masson-Fontana histochemistry in <i>Tspear</i> siRNA treated HFs (h) compared to control (g). Data was pooled from two independent experiments (I, two sided t-test; ***p<0.001) (scale bars = 50 μm); (j-o) Apoptosis was assessed by the TUNEL assay (TUNEL, green; DAPI, blue) at the hair bulb (j-l) and infundibular (m-o) compartments of HFs downregulated for <i>Tspear</i> (k,n) compared to control siRNA treated HFs (j,m) (scale bars = 50 μm). Average number of TUNEL-positive cells in hair follicles in the respective compartments. Data were pooled from two independent experiments (l,o, two sided t-test; ***p<0.001) (scale bars = 50 μm). White dotted lines delineate the outer epidermal surface; (p) RNA was extracted from <i>Tspear</i> siRNA and control siRNA transfected HFs and <i>Notch1</i> RNA expression level was assessed by qRT-PCR. Results were normalized to <i>Rplp0</i> levels and are expressed as expression levels relative to control samples. Data were pooled from three independent experiments (two sided t-test; *p<0.05). E—epidermis; INF–Infundibulum; D—dermis; DP—dermal papilla; IRS—inner root sheath; ORS—outer root sheath; HM—hair matrix; HS—hair shaft; TUNEL—terminal deoxynucleotidyl transferase dUTP nick end labeling.</p

Expression analysis.

<p>(a) A comparative analysis of gene expression profiles of primary keratinocytes transfected with <i>TSPEAR</i> specific siRNA or control siRNA (left panel) revealed a number of differentially expressed genes including <i>NOTCH1</i> and additional genes encoding elements of the <i>NOTCH1</i> regulatory network and/or known to be involved in hair and tooth development. Pathway analysis (IPA software, see details in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006369#sec007" target="_blank">Materials and Methods</a>, right panel) revealed that <i>TSPEAR</i> down-regulation affects a <i>NOTCH</i>-associated regulatory network; (b) Gene expression following siRNA-mediated <i>TSPEAR</i> down-regulation was assessed using qRT-PCR. Results are expressed as percentage of gene expression in cells transfected with <i>TSPEAR</i>-specific siRNA relative to gene expression in siRNA control-transfected cells ± standard error (two sided t-test; *p<0.05, **p<0.01). Results are normalized to <i>GAPDH</i> RNA levels; (c,d) NOTCH1 expression was assessed by immunostaining (c) in skin biopsies obtained from an affected individual (IV-4, family A; right panel) and from a healthy individual (left panel). Immunostaining was significantly reduced in affected vs. normal skin (d) (scale bars = 25 μm; (e) HaCaT cells were co-transfected with a NOTCH1-responsive luciferase reporter gene and <i>TSPEAR</i>-specific siRNA or control siRNA. Luciferase activity was measured after 48 hours and normalized to Renilla luciferase. Results represent the mean of three independent experiments ± standard error (two sided t-test; ***p<0.001).</p

Clinical and pathological features.

<p>(a) Family pedigrees are presented in the upper panels. Black symbols denote affected individuals. PCR-RFLP assays, performed as detailed in materials and methods, were used in each family to confirm co-segregation of the mutation with the disease phenotype (lower panels). Mutation c.1728delC is associated with the presence of a 108 bp fragment in families A and B, while mutation c.454_457delCTGG results in a 514 and 256 bp fragments in family B; in addition, both mutations c.1852T>A and c.1915G>A are associated with the presence of a 180 bp fragment in family C; (b-d) Clinical features displayed by the patients include (b) hypodontia with conical teeth, (c) anterior scalp hypotrichosis and (d) follicular accentuation; (e) A skin biopsy obtained from scalp skin of individual IV-4 of family A and stained for hematoxylin and eosin, demonstrates paucity of rudimentary hair follicles; (f-g) Scanning electron microscopy (SEM) analysis of hair shafts obtained from the scalp demonstrates flattened and partially absent cuticular scales (arrows) in the patient hair (f) as compared with a healthy individual (g) (scale bar = 100 μm).</p

Mutation analysis.

<p>(a) Direct sequencing of <i>TSPEAR</i> revealed a homozygous missense transversion c.1726G>T and a homozygous c.1728delC deletion in family A patients; heterozygous c.454_457delCTGG, c.1726G>T and c.1728delC mutations in family B patient; and heterozygous c.1852T>A and c.1915G>A missense mutations in family C patient. Wildtype sequences are given below the mutant sequence for comparison; (b) The predicted consequences of the 4 mutations are depicted along a schematic representation of the TSPEAR protein structure with its different domains.</p