Figure 5. Innervation of Spinal Zic2 Neurons by Primary Sensory and Supraspinal Neurons.

(A) Experimental strategy for monosynaptic retrograde tracing of Zic2cre spinal neurons. CAG promoter; loxP, recognition sites for Cre recombinase. An EnvA-pseudotyped G-deleted eGFP virus (ΔG-GFP) was injected intraspinally at L2/L3 or C2 levels in P7 pups (right panel).

(B–F) Sections of P15 lumbar DRGs showing monosynaptic Zic2 input neurons, and immuno-logical analysis of the sensory neuron subtype markers indicated. Arrows indicate double-labeled neurons with marker shown in red; arrowheads indicate double-labeled neurons with marker shown in blue. Ret and TrkA (B), NF200 and CGRP (C), TrkC and CGRP (D), TH (E), and TrkB (F).

(G) Sensory neuron marker abundance among monosynaptic Zic2 input neurons (n = 3/6 sections, 3–7 mice).

(H) Cell body area of monosynaptic DRG neurons (GFP⁺) compared to TrkA⁺ neurons (GFP: 769 ± 50 μm², n = 42 to 115 neurons, 5 mice; TrkA: 439 ± 30 μm², n = 120 to 150 neurons in 3 sections from 5 mice each; two-tailed Student’s unpaired t test, t₍₈₎ = 5.7; ***p = 0.0005).

(I) Cumulative distribution of cell body area of monosynaptic Zic2 input DRG neurons (green) compared to TrkA⁺ neurons (magenta). Dotted lines indicate results of single animals. Solid lines indicate average distribution.

(J) Coronal-section planes with distance from Bregma of images shown in (K) and (L).

(K and L) P15 brain sections showing monosynaptic Zic2 input neurons. MdV, medullary reticular formation ventral part; Ve, Vestibular nucleus; GRN, gigantocellular reticular nucleus; MRN, midbrain reticular nucleus; M1, primary motor cortex. Distribution of supra-spinal monosynaptic inputs of lumbar (K) and cervical (L) Zic2cre spinal neurons.

Scale bars, 50 μm (B–F) and 500 μm (K and L). Data are presented as mean ± SEM.