ZFIN Image: Huang et al., 2022, Fig. 5

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Figure Caption

Fig. 5

Figure 5. Genetic ablation of calb2b impaired axon regrowth of fast propriospinal V2a interneurons and restoration of fast-speed locomotion

(A) Illustration of bulk RNA sequencing procedures.

(B) Heatmap of mean FPKM values showing differentially expressed genes associated with axon regrowth and neuron projection. Differential expression analysis was performed either between V2a interneurons with regrown axons (GFP⁺/RD⁺) and without regrown axon (GFP⁺/RD⁻) at the same time point or between any two SCI time points of GFP⁺/RD⁺ using the DESeq2 package (p < 0.05). n = 20 fish for each library, N = 5–6 libraries for each group.

(C) Mean FPKM value of calb2b in six groups. ^∗p < 0.05.

(D) Immunostaining for calretinin in RD retrogradely labeled V2a interneurons in cross-sections of an uninjured Tg(Chx10:GFP) animal. The enlarged cells (dished box), central canal (dashed semicircle), and Mauthner axon (line circle) are indicated.

(E) Left: immunostaining of calretinin in V2a interneurons with regrown axons in cross-sections rostral to the lesion at 3 wpi. Right: the somata of V2a interneurons with regrown axons co-labeled with calretinin (GFP⁺/RD⁺/calretinin⁺) displayed a dorsomedial position of the ventral cord. One dot equals one neuron. N = 6 fish and the neurons within the first spinal segment rostral to the lesion were calculated in each fish.

(F) Left and middle: co-labeling and distribution of GFP⁺/RD⁺/calretinin⁺ neurons at the dorsomedial position of the ventral cord at 8 wpi. Right: image showing no GFP⁺/RD⁺/calretinin⁺ neurons at the ventrolateral position at 8 wpi.

(G–I) Left: RD labeling of propriospinal V2a interneurons with descending axon regrowing over the injury site in calb2b^+/+ and calb2b^−/− SCI fish of Tg(Chx10:GFP) at 2, 4, and 8 wpi. Right: soma spatial distribution of axon regrown V2a interneuron in calb2b^+/+ and calb2b^−/− SCI fish at 2, 4, and 8 wpi.

(J) Quantification of the dorsomedial (fast) and ventrolateral (slow) subpopulations of V2a interneurons with regrown axons in calb2b^+/+ and calb2b^−/− SCI fish at 2 or 8 wpi.

(K) Quantification of numbers of V2a interneurons with regrown axons (top), and free-swimming speed (middle) and maximum speed (bottom) in calb2b^+/+ and calb2b^−/− SCI fish at 2, 4, and 8 wpi. One dot equals one fish. All data are presented as mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, significant difference. See also Figures S5 and S6.

Acknowledgments

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