Journal: Nature Communications

Article Title: Subcellular second messenger networks drive distinct repellent-induced axon behaviors

doi: 10.1038/s41467-023-39516-z

Figure Lengend Snippet: a Lyn-SpiCee- and Lyn-SponGee-expression lead to overshooting axons in the inferior colliculus at P15, by contrast to mRFP-, SpiCee-Kras- and SponGee-Kras-expression. The orange dashed line highlights the position of the posterior end of the superior colliculus (SC). The inferior colliculus (IC) is above this line. The top row images are magnifications of the regions of the bottom row images indicated by the black dashed squares. Scale bars: top row, 250 µm; bottom row, 500 µm. b SpiCee-Kras- and SponGee-Kras-expressing axons (cyan) exit the optic chiasm labeled with TAG1 (magenta), by contrast to the axons of mRFP-, Lyn-SpiCee and Lyn-SponGee-electroporated RGCs. An excess of retino-retinal axons is also detected in SpiCee-Kras- and SponGee-Kras-electroporated animals, as compared to mRFP-, Lyn-SpiCee- and Lyn-SponGee-electroporated RGCs. The top row highlights the mRFP channels in which electroporated axons are seen. Closed arrowheads, axons exiting the optic chiasm; open arrowheads, retino-retinal axons. Scale bar, 200 µm. a , b ** P < 0.01; *** P < 0.001; χ 2 test followed by χ 2 post-hoc tests. Source data are provided as a Source data, number of replicates and P values are provided as a .

Article Snippet: For immunostaining, the samples were incubated with the primary antibodies against TAG1 (1/500, R&D Systems, lot #CDSO0219101) and or DsRed (1/1000, Takara Bio, lot #2103116) in a solution containing 0.5% Triton-X100, 0.2% gelatin, 1XPBS, 0.1 g/L thimerosal, 10 mg/mL saponin.

Techniques: Expressing, Labeling

Journal: Cell reports

Article Title: A tug of war between DCC and ROBO1 signaling during commissural axon guidance

doi: 10.1016/j.celrep.2023.112455

Figure Lengend Snippet: (A) Axon projection and midline crossing in compound Netrin1 and Robo1 KO mutants, examined by anti-ROBO3, anti-TAG1, and anti-NF staining of transverse spinal cord sections at E11.5. Netrin1 −/− null mutants exhibited severe guidance defects in the spinal cord, including misprojections into the roof plate, ventricular zone, and motor columns (highlighted with yellow arrowheads), abnormal exit from the CNS (yellow arrow), and an almost complete absence of the ventral commissure (bracket). Removing Robo1 allowed more axons to cross the midline in Netrin1 −/− null background (the ventral commissure indicated by the bracket is thicker in Netrin1 −/− ; Robo1 −/− embryos), but other guidance errors, indicated by yellow arrowheads and yellow arrows, persisted. (B) Quantification of the ventral commissure size in (A). Anti-ROBO3 was used as it does not label any motor axons. (C and D) Effect of bath-applied SLIT2N (250 ng/mL) on DSC axon outgrowth in the absence and presence of Netrin1, respectively. Explants were cultured in 3D collagen matrix for 40 h without Netrin1 in (C) and for 24 h with 250 ng/mL bath-applied Netrin1 in (D). SLIT2N repressed WT axon outgrowth regardless of whether Netrin1 was present but did not inhibit the growth from Robo1/2 double KO explants. (E and F) Quantification of axon outgrowth in (C) and (D), respectively. (G) DiI tracing of spinal commissural axons in an open-book orientation in compound Netrin1 and Robo1 KO mutants. Most Robo1 −/− axons stalled within and at the contralateral boundary of the FP at E12.5. Introduction of a heterozygous Netrin1 KO allele into Robo1 −/− mutant background allowed more axons to exit. Scale bars, 100 μm. (H) Quantification of midline exit in (G). See for description of quantification. Data were normalized to WT controls and are presented as mean ± SD. One-way ANOVA with a Tukey post hoc test in (B) and (H), two-way ANOVA with a Tukey post hoc test in (E) and (F); p values and animal numbers as indicated; ns, not significant; 5–10 sections, 3–5 explants, and 3–5 DiI injection sites were quantified for each embryo.

Article Snippet: Antibodies used for IHC are as follows: anti-DCC (AF844, R&D), anti-ROBO1 (AF1749, R&D), anti-ROBO2 (E4M6D, Cell Signaling Technology), anti-ROBO3, anti-ROBO3.1, anti-TAG1 (AF4439, R&D), anti-NF (2H3, DSHB), and Alexa Fluor 594-conjugated secondary antibodies (Jackson ImmunoResearch).

Techniques: Staining, Cell Culture, Mutagenesis, Injection

Journal: Cell reports

Article Title: A tug of war between DCC and ROBO1 signaling during commissural axon guidance

doi: 10.1016/j.celrep.2023.112455

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used for IHC are as follows: anti-DCC (AF844, R&D), anti-ROBO1 (AF1749, R&D), anti-ROBO2 (E4M6D, Cell Signaling Technology), anti-ROBO3, anti-ROBO3.1, anti-TAG1 (AF4439, R&D), anti-NF (2H3, DSHB), and Alexa Fluor 594-conjugated secondary antibodies (Jackson ImmunoResearch).

Techniques: Recombinant, Injection, Knock-Out, Sequencing, Software

Journal: JCI Insight

Article Title: Defective jagged-1 signaling affects GnRH development and contributes to congenital hypogonadotropic hypogonadism

doi: 10.1172/jci.insight.161998

Figure Lengend Snippet: ( A – D ) Representative confocal images of the nasal septum of a GW 9.5 fetus immunostained for GnRH1 (white), JAG1 (red), and TAG1 (green). JAG1 (arrows, B ) is expressed by GnRH1-expressing neurons (arrows, A ), as well as by other neurons belonging to the MM and by vomeronasal/terminal fibers expressing TAG1. ( E – H ) GnRH1 (white), DLL1 (red), and TAG1 (green) expression in a coronal section of the nasal region of a GW 9.5 fetus. DLL1 is expressed by GnRH neurons (arrows, E and F ), by other neurons belonging to the MM, and by vomeronasal/terminal fibers. ( I ) Schematic representation of a GW 11.5 human fetus head (coronal view). The black box indicates the forebrain region of immunofluorescence analysis displayed in J – L . ( J – L ) DLL1 is expressed by a GnRH neuron that entered the forebrain. Scale bars: 20 μm. The experiments were replicated 3 times with similar results in n = 2 samples (GW 9.5 and GW 11.5). HF, human fetus.

Article Snippet: Slides were washed 3 times in PBS 1× and incubated for 3 days in the primary antibody solution (PBS 1×, 0.3% Triton X-100, 2% normal donkey serum) at 4°C. the primary antibodies used were anti-DLL1 rabbit 1:100 (Abcam, ab10554), anti-Jag1 rabbit 1:100 (Cell Signaling Technology, 2155), anti-TAG1 goat 1:500 (R&D Systems, AF4439), and anti-GnRH guinea pig 1018, 1:10,000 (gift by Erik Hrabovszky, Laboratory of Endocrine Neurobiology, Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest, Hungary).

Techniques: Expressing, Immunofluorescence

Journal: Autophagy

Article Title: The autophagy-inducing kinases, ULK1 and ULK2, regulate axon guidance in the developing mouse forebrain via a noncanonical pathway

doi: 10.1080/15548627.2017.1386820

Figure Lengend Snippet: Abnormal axonal fasciculation in the Ulk1/2-deficient animals is associated with mislocalization of CNTN2 in the projection neurons. (A) Neuronal cell adhesion molecule distribution was unaltered in the ulk1/2-DKO brains compared to that in the controls. (B) The intensity of CNTN2 immunostaining (red) was dramatically decreased in distal CTAs of the ulk1/2-DKO brain at E18.5. All of the sections were counterstained with CHL1 cell adhesion molecule (green). (C) Western blot analyses of the extracts prepared from the cortex (proximal CTAs) and striatum (distal CTAs) confirmed normal expression of neuronal cell adhesion molecule but significantly decreased CNTN2 levels in the striatum. (D and E) Quantification of the NCAM1 and CNTN2 levels in control and ulk1/2-DKO cortex and striatum. Abbreviations: Cnt, controls; NCAM1, neuronal cell adhesion molecule 1; ns, not significant.*P < 0.05. Scale bars: 200 µm.

Article Snippet: After incubation with a 5% skim-milk block, blots were probed with antibodies directed against the following targets: ULK1 (Sigma Aldrich, A7481), SQSTM1 (Sigma Aldrich, P0067), LC3B (Novus Biologicals, NB100–2220), ATG13 (Sigma Aldrich, SAB4200100), RB1CC1 (Cell Signaling Technology, 12436), ATG14 (MBL, PD026), CNTN2 (R&D, AF4439), NCAM1 (Developmental Studies Hybridoma Bank, 5B8), and GAPDH (Sigma Aldrich, G9545).

Techniques: Immunostaining, Western Blot, Expressing