Journal: Cell reports

Article Title: Netrin1 patterns the dorsal spinal cord through modulation of Bmp signaling.

doi: 10.1016/j.celrep.2024.114954

Figure Lengend Snippet: Figure 2. Overexpression of ntrin1 in chicken embryos results in the loss of dorsal interneurons (A–L) Chicken spinal cords were electroporated at HH stage 14 with Gfp (A–C and G–I) or different concentrations of netrin1 (50 ng, 500 ng, 1 mg) (D–F and J–L) under the control of the CAG enhancer and incubated until HH stage 24. Thoracic transverse sections were labeled with antibodies against Sox2 (red, A, B, D, and E), p27 (blue, A, C, D, and F), Lhx2 (red, G and J), Isl (red, H and K), Lhx1/5 (blue/green G, I, J, and L) and Pax2 (red, I and L). The dotted box (G–L) indicates the magnified region in the adjacent panel(s). (M) Schematic spinal cord, showing the position of the dorsal progenitor (dP) domains and post-mitotic dorsal interneurons (dIs). (N and O) Ectopic Gfp expression had no significant effect on cell fate specification. In contrast the total area occupied by Sox2+ (progenitors) or p27+ (neurons) cells was significantly reduced at all concentrations of netrin1 tested (N). The dorsal spinal cord was more profoundly affected at lower concentrations of netrin1 than the ventral spinal cord (O). n > 20 sections from four embryos from each experimental condition (i.e., GFP, 50 ng, 500 ng, and 1 mg netrin), Student’s t test. (P) The different classes of dIs can be identified by specific combinations of transcription factors.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti neurofilament Cell Signaling Technology Cat#C28E10; RRID: AB_10828120 Mouse anti tubulin b3 (Tubb3)/Tuj1 BioLegend Cat#801202; RRID:AB_10063408 Rabbit anti laminin Abcam Cat#11575; RRID: AB_298179 Mouse anti chicken transitin/nestin Developmental Studies Hybridoma bank Cat#EAP3; RRID: AB_2282449 Mouse anti myc tag Abcam Cat#ab32; RRID: AB_303599 Mouse anti GFP Invitrogen Cat#A-11120 Goat anti mouse netrin1 R&D Systems Cat#AF1109; RRID: AB_2298775 Goat anti chicken netrin1 R&D Systems Cat#AF128; RRID:AB_354716 Goat anti chicken netrin2 R&D Systems Cat#AF127; RR1D: AB_2154709 Goat anti Sox2 Santa Cruz Biotechnology Cat# sc-17320; RRID:AB_2286684 Mouse anti P27 BD Biosciences Cat#610241; RRID:AB_397636 Rabbit anti cleaved caspase 3 BD Biosciences Cat#559565; RRID:AB_397274 Rabbit anti phospho histone H3 Cell signaling Technology Cat#9701; RRID:AB_33153 Goat anti Lhx2 Santa Cruz Biotechnology Cat#Sc-19344; RRID:AB_2135660 Rabbit anti Lhx2 Gift from Tom Jessell; Liem et al.13 N/A Mouse anti Lhx1/5 Developmental Studies Hybridoma bank Cat#4F2; RRID:AB_531784 Guinea Pig anti Tlx3 Gift from Thomas Muller; Muller et al.76 N/A Mouse anti Isl1/2 Developmental Studies Hybridoma bank Cat#39.4DS Goat anti Isl1/2 R&D Systems Cat#AF1837, RRID:AB_2126324 Rabbit anti Pax2 Invitrogen Cat#71-6000 Guinea Pig anti Olig2 Gift from Bennett Novitch; Novitch et al.62 N/A Rabbit anti Math1/Atoh1 Helms and Johnson77 N/A Goat anti Ascl1/Mash1 R&D Systems Cat#AF2567; RRID:AB_2059505 Guinea Pig anti Foxd3 sera Gift from Thomas Muller; Muller et al.76 N/A Rabbit anti Gapdh Protientech Cat# 10494-1-AP; RRID:AB_2263076 Rabbit anti Phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser465/467) (D5B10) Cell Signaling Technology Cat#11971; RRID:AB_2797785 Chemicals, peptides, and recombinant proteins Mouse Recombinant netrin1 R&D Ca#1109-N1-025 Human Recombinant Bmp4 Thermo Fisher Scientific Cat#PHC9534 Retinoic acid Sigma Aldrich Cat#R2625 Human basic FGF (bFGF) Thermo Fisher Scientific Cat#PHG0023 Neurobasal medium Thermo Fisher Scientific Cat#21103049 B27 supplement Fisher Scientific Cat#17-504-044 N2 supplement Thermo Fisher Scientific Cat#17502048 CHIR99021 Tocris Cat#4423 (Continued on next page) 18 Cell Reports --, 114954, --, 2024 OPEN ACCESS

Techniques: Over Expression, Control, Incubation, Labeling, Expressing