EVA1C is expressed by retinal cell ganglionic axons.

<p>Coronal sections of the developing (<b>A–H</b>) eye and (<b>I–L</b>) optic chiasm at (<b>A–D, I</b>) E14.5, (<b>E–H, J</b>) E17.5, (<b>K</b>) P0 and (<b>L</b>) P10 were immunostained for (<b>A–L</b>) EVA1C and (<b>A–H</b>) GAP43. All sections were counterstained with DAPI. EVA1C was strongly co-expressed with GAP43 by retinal cell ganglionic axons (<b>A–H</b>) as they exit the retina and form the optic nerve (arrowheads) and (<b>I–L</b>) as they cross the midline at the optic chiasm. (<b>K</b>) EVA1C expression was also expressed in the suprachiasmatic nucleus (asterisks) at P0. Scale bars represent 100 µM. Dashed lines demarcate the optic chiasm.</p

EVA1C is expressed by axons projecting through the internal capsule.

<p>Coronal sections of the forebrain at (<b>A</b>) E14.5, (<b>B</b>) E17.5, (<b>C</b>) P0 and (<b>D</b>) P10 were immunostained for EVA1C. All sections were counterstained with DAPI. (<b>A–D</b>) EVA1C was strongly expressed by axons in the internal capsule (arrows). Scale bars represent 100 µM.</p

EVA1C expression in the corpus callsoum.

<p>Coronal sections of the developing cortex were immunostained for (<b>A–L</b>) EVA1C and (<b>E–L</b>) EVA1C and GAP43 at (<b>A</b>) E14.5, (<b>B, E–H</b>) E17.5, (<b>C, I–L</b>) P0 and (<b>D</b>) P10. All sections were counterstained with DAPI. (<b>A</b>) At E14.5, EVA1C was expressed in the cortical plate of the cingulate cortex (arrow) and in axons projecting into fornix (asterisk). EVA1C was not expressed in the underlying deeper layers of the cingulate cortex at this age (diamond). (<b>B</b>) At E17.5, axons arising from the cingulate cortex (asterisk) strongly express EVA1C. Axons form the neocortex (arrows) only weakly expressed EVA1C at this age (arrows). The yellow boxed area is presented in panels E–H. (<b>C</b>) At P0, EVA1C was expressed in the intermediate zone of the cingulate cortex and neocortex (asterisks). Axons entering the ventral corpus callosum (arrows) now express EVA1C. (<b>B, C</b>) EVA1C was highly expressed in the hippocampal commissure (stars). White dashed lines in panels B and C demarcate the intermediate zones and corpus callosum. (<b>D</b>) At P10, EVA1C was strongly expressed in the corpus callosum. (<b>E–H</b>) At E17.5, EVA1C was weakly expressed by callosal axons coursing diffusely (GAP43 staining) in the dorsal region of the corpus callosum (between yellow arrows). EVA1C was also weakly expressed by the more abundant GAP43 positive axons in the ventral corpus callosum (between white arrows). (<b>I–L</b>) At P0, EVA1C and GAP43 were uniformly co-expressed throughout the corpus callosum (between white arrowheads). Scale bars represent (<b>A</b>) 50 µM and (<b>B–L</b>) 100 µM. Dashed lines demarcate the corpus callosum.</p

EVA1C is expressed in the olfactory bulb.

<p>(<b>A–L</b>) Coronal sections of the olfactory bulb at (<b>A</b>) E14.5, (<b>B, E–H</b>) E17.5, (<b>C</b>) P0 and (<b>D, I–L</b>) P10 were immunostained for (<b>A–D</b>) EVA1C and (<b>E–L</b>) EVA1C and GAP43. All sections were counterstained with DAPI. (<b>A–D</b>) EVA1C was strongly expressed in the olfactory nerve (arrows) and in the outer nerve fibre layer (demarcated by dashed line) of the developing and post-natal olfactory bulb. EVA1C was also expressed in the neuropil of the deeper layers of the olfactory bulb (asterisks) at all ages examined. Closer examination of the olfactory nerve layer (yellow boxed region in B and D) at (<b>E–H</b>) E17.5 and (<b>I–L</b>) P14 revealed that it was co-expressed with GAP43, a marker of olfactory sensory axons. (<b>I–L</b>) At P10, EVA1C was expressed at high levels in the olfactory nerve layer (arrows) but comparatively low levels in the glomerular layer (arrowheads). Scale Bars represent 100 µM. Dashed lines delineate: (<b>A–D</b>) the glomerular layer; (<b>E–H</b>) the olfactory nerve layer; (<b>I–L</b>) the glomerular layer.</p

EVA1C expression in the developing spinal cord.

<p>(<b>A–F</b>) Transverse sections of the developing spinal cord at (<b>A–C</b>) 10.5 and (<b>D–F</b>) 17.5 dpc were immunostained for EVA1C and countersta. Boxed areas in A and D are represented in B–C and E–F, respectively. EVA1C was strongly expressed by the motor column (MC) and motor neurons (asterisks), and dorsal root ganglionic (DRG) neurons and their respective axons at (<b>A–C</b>) 10.5 dpc but not at (<b>D–F</b>) 17.5 dpc. EVA1C expression was detected in the dorsal root entry zone (arrows) at (<b>A</b>) 10.5 dpc, but not at (<b>D</b>) 17.5 dpc. (<b>A–C</b>) EVA1C expressing commissural axons can be observed coursing along the motor column and across the floor plate at 10.5 dpc (arrowheads). (<b>D–F</b>) EVA1C expression is lost in commissural axons as they cross the floor plate at 17.5 dpc. EVA1C is present in the ventrolateral funiculus (VLF), dorsal columns (DC) and dorsal funiculus (DF) at 17.5 dpc. Scale bar represents (<b>A</b>) 50 µm, (<b>B–C</b>) 25 µm, (<b>D</b>) 25 µm and (<b>E–F</b>) 10 µm.</p

Additional file 3: Figure S1. of Differentially expressed genes from RNA-Seq and functional enrichment results are affected by the choice of single-end versus paired-end reads and stranded versus non-stranded protocols

Venn diagrams of the DEGs comparing 3 biological replicates from the SE data with 2 biological replicates from the PE data. (PPTX 237 kb

Additional file 2: Table S1. of Differentially expressed genes from RNA-Seq and functional enrichment results are affected by the choice of single-end versus paired-end reads and stranded versus non-stranded protocols

Numbers of DEGs found using 2 biological replicates with PE and 3 biological replicates with SE. (XLSX 42 kb

Human BCCs exhibit nuclear YAP and β-catenin in association with ROCK signalling activation and increased ECM collagen deposition.

<p>Representative images of immunohistochemical staining (brown) of Gli2 (A), YAP (B), Thr696-phosphorylated MYPT (C) and β-catenin (D) in normal and human BCCs skin samples. (E) Masson’s trichrome histological staining. IHC, Immunohistochemistry. Scale bars = 20 <b>μ</b>m.</p

Activated ROCK-signalling, increased dermal fibroblast numbers, and dermal fibrosis in the skin of K14-CreER/Rosa-SmoM2 transgenic mice.

<p>(A-G) Immunofluorescence staining and area coverage analysis of dorsal skin tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 transgenic littermate mice detecting Fsp1 (A & B) and Vimentin (C), Phalloidin (G), DIAPH3 (H), Thr696-phosphorylated MYPT1 (I & J), Thr18/Ser19-phosphorylated MLC2 (K & L). (D) Masson’s trichrome histological staining of sections through the dorsal neck skin of tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 mice. (E & J) Dual two-photon SHG and monochromatic transmission (Trans; grayscale in merge) images showing collagen (white in single channel, magenta in merged) in tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 skin sections. Area coverage analysis (5 fields/sample from three mice per genotype) of SHG is quantified. Basement membranes and hair follicles are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.</p

GLI2 activation in the skin of YAP2-5SA-ΔC transgenic mice.

<p>(A) Immunofluorescence staining of dorsal skin sections of YAP2-5SA-ΔC transgenic and wildtype mice detecting GLI2 (green) and YAP (red). Quantification of % YAP-GLI2 co-positive (arrowheads—B), % GLI2 positive (C) and % YAP (D) positive nuclei in the skin sections of YAP2-5SA-ΔC transgenic and wildtype mice. Basement membranes are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.</p