Journal: The Journal of Neuroscience
Article Title: WT1 -Expressing Interneurons Regulate Left–Right Alternation during Mammalian Locomotor Activity
Figure Lengend Snippet: WT1-expressing neurons terminate in close proximity to populations of commissurally projecting interneurons. A, A 20-μm-thick section of a spinal cord 46 h after PRV-152 injection into the GS muscle on the left side and stained with antibodies to GFP (green) and WT1 (blue). At this time point, no WT1-expressing neurons on either side of the spinal cord have taken up the tracer. Contralateral region containing WT1-expressing cells (dashed box) is expanded to the right. B, To identify presumptive synapses on genetically defined interneuronal subtypes, we inspected the synaptotagmin+ “halo” (indicated by white arrows) surrounding each labeled nuclei for WT1+/synaptotagmin+ terminals. C–F, The 20-μm-thick sections cut from a P0 WT1CreERROSA26tdTomato spinal cord and stained with antibodies to tdTomato (red), the synaptic marker synatotagmin (blue), as well as (green) a nuclear marker of V2a cells (Chx10-, C), V1 cells (En1-, D), V0V cells (Evx1-, E), or DMRT3-expressing dI6 cells (F). WT1-expressing terminals (tdTomato+/synaptotagmin+ processes) were rare or absent nearby Chx10- or En1-expressing cells but were commonly seen in close proximity to Evx1+ and DMRT3+ neurons. C, D, Dashed boxes are expanded to the right. E, F, Arrow in the low-magnification image indicates the specific WT1 cell of interest in the panel to the right. In magnified images, arrows indicate presumptive WT1+ axon terminals. Double labeling of these processes is confirmed in orthogonal views to the right of E, F. Scale bars: Low-magnification images, 100 μm; High-magnification images: A, C, D, 20 μm; B, E, F, 5 μm.
Article Snippet: Primary antibodies used were as follows: WT1 (rabbit, 1:100, Santa Cruz Biotechnology, RRID: AB_632611 ), GFP (goat, 1:5000, gift from Eusera), glycine (rat, 1:1000, Immunosolutions, RRID: AB_10013222 ), En1 (gift from Jessel laboratory, Columbia University, guinea pig, 1:1000), Chx10 (mouse, 1:200, Santa Cruz Biotechnology, RRID: AB_10842442 ), DMRT3 (goat, 1:100, Santa Cruz Biotechnology, RRID: AB_2091664 ), synaptotagmin (rabbit, 1:200, Alomone Labs), Evx1 (mouse, 1:100, DSHB, RRID: AB_2246711 ), and NeuN (mouse, 1:500, Millipore, RRID: AB_177621 ).
Techniques: Expressing, Injection, Staining, Labeling, Marker