Journal: Open Biology

Article Title: Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

doi: 10.1098/rsob.160091

Figure Lengend Snippet: ATF3 regulates injury-related expression of neuropeptides and other genes. ( a–i ) Unlesioned and injured FN of Atf3 +/+ and Atf3 −/− mice were subjected to qPCR analysis after 3 days of injury to quantify mRNA abundance of primers indicated. In wt mice (grey bars), facial nerve injury resulted in induction of Sprr2j ( a ), Vip ( b ), Ngf ( c ), Wnt2b ( d ), Galanin ( e ), Grp ( f ), Timp1 ( g ) and the known ATF3 target gene Hsp27 ( h ). In contrast to wt mice, induction of Sprr2j, Vip, Ngf, Wnt2b, Galanin, Grp and Hsp27 mRNA abundance was reduced upon facial nerve lesion in Atf3 mutant mice (white bars). Timp1 mRNA induction was more pronounced upon ATF3 loss-of-function ( g ). The Vip2 receptor ( Vipr2 ) was downregulated by facial nerve injury in wt and ATF3-deficient mice ( i ). Numbers in bars in ( b ) reflect independent biological replicates for experiments in ( a–i ). ( j–l ) Confirmation of reduced galanin expression in ATF3-deficient mice upon facial nerve injury. Deafferented wt ( j ) and ATF3-deficient ( k ) FN were stained with anti-galanin directed antibodies. In wt mice ( j ), galanin localized in secretory vesicle-like structures (see inset) of FMNs (some labelled with an arrow). The number of galanin immunoreactive FMNs was reduced in Atf3 mutant mice ( k ). ( l ) Quantification of galanin positive neurons without and 3 and 12 days after lesion. Data are presented as mean ± s.d. ** p ≤ 0.01. Scale bar ( j,k ) = 50 µm; inset = 5 µm.

Article Snippet: Camptothecin was added at 2 μM for 24 h. Recombinant peptides for mouse VIP (Tocris; no.1911), human gastrin releasing peptide (GRP) (Tocris; no.1789), mouse galanin (Tocris; no.2696) and mouse Wnt2b (R&D systems; no.3900-WN-025) were added at 1 nM (VIP, GRP, galanin) and 1 μg ml −1 (Wn2tb) for 24 h to the cultures.

Techniques: Expressing, Mutagenesis, Staining

Journal: Open Biology

Article Title: Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

doi: 10.1098/rsob.160091

Figure Lengend Snippet: ATF3 mediates neuropeptide and Wnt2b expression in primary PNS neurons. ( a–d ) Adult wt or ATF3-deficient mouse DRG neurons were infected with adenoviral (AV) particles resulting in GFP (control) or ATF3 expression. mRNA levels of Atf3 ( a ), Wnt2b ( b ), Galanin ( c ) and Grp ( d ) were analysed by qPCR. Viral infection strongly enhanced Atf3 mRNA abundance in wt and Atf3 mutant neurons ( a ). Wnt2b ( b ), Galanin ( c ) and Grp ( d ) mRNA levels were augmented upon viral ATF3 overexpression in wt and Atf3 mutant DRG neurons. ( e,f ) Primary wt neurons overexpressing GFP or ATF3 were subjected to ChIP analysis with anti-ATF3 or IgG (control) antibodies. ATF3 occupancy at potential ATF3 binding sites of the Galanin ( e ) and Grp ( f ) promoter was tested with qPCR. ATF3 promoter occupancy was observed in ATF3-overexpressing samples only in the presence of anti-ATF3 but not IgG antibodies suggesting ATF3 binding at the Galanin ( e ) and Grp ( f ) promoter. Numbers in bars reflect independent numbers of experiments. Data are presented as mean ± s.d. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Camptothecin was added at 2 μM for 24 h. Recombinant peptides for mouse VIP (Tocris; no.1911), human gastrin releasing peptide (GRP) (Tocris; no.1789), mouse galanin (Tocris; no.2696) and mouse Wnt2b (R&D systems; no.3900-WN-025) were added at 1 nM (VIP, GRP, galanin) and 1 μg ml −1 (Wn2tb) for 24 h to the cultures.

Techniques: Expressing, Infection, Control, Mutagenesis, Over Expression, Binding Assay

Journal: Development (Cambridge, England)

Article Title: RPE specification in the chick is mediated by surface ectoderm-derived BMP and Wnt signalling.

doi: 10.1242/dev.096990

Figure Lengend Snippet: Fig. 1. Signals involved in early optic vesicle patterning in the chick. (A) At stage HH 9, MITF protein is not detected in the ov (arrows). (B) At stage 10, MITF protein is present throughout the distal ov (arrow). Mesenchymal cells (arrowheads) are located dorsally. (C,C′) At stage 10, pSmad1/5 is detected in the entire distal ov (arrow). (D,D′) Higher magnification of C, showing pSmad1/5 labelling in neuroepithelial cells (arrow) and in the surface ectoderm (arrowhead). (E,E′) Stronger accumulation of pSmad1/5 is observed in the dorsal ov (arrows) compared with the ventral region (asterisks). (F,F′) Parallel section of E. Strong β- catenin labelling in the entire surface ectoderm/prospective lens ectoderm (arrow). Nuclear β-catenin is not observed in the ov (arrowhead). (G-I) Wnt2b expression during the initial stages of eye development. Wnt2b expression is restricted to the dorsal surface ectoderm (arrow), dorsal ov (arrowhead) and dorsal prosencephalon (white asterisks). Wnt2b transcripts are not detected in the ventral surface ectoderm and ov (asterisks). (J,K) Frontal and lateral view of a stage 10/11 chick head. Wnt2b transcripts in the ectoderm (arrows) covering the dorsal ov (white dashed line). Expression is not detected ventrally (asterisks). In all panels, dorsal is up.

Article Snippet: The DNA/RNA constructs used were Wnt2b siRNA and siRNA-A (both Santa Cruz), pC S2 hSmad1WT (Addgene plasmid 22989) and pC S2 hSmad1-GM (Addgene plasmid 22992; Edward de Robertis; Pera et al., 2003; Fuentealba et al., 2007).

Techniques: Expressing