hmx2 and hmx3a are expressed in V1 and dI2 interneurons. (A–F) Lateral views of spinal cord at 27 hpf. Rostral, left; Dorsal, up. (A) hmx2 and hmx3a are coexpressed in V1 and dI2 interneurons. (B–F) V1 interneurons are inhibitory (slc32a1-expressing) (Jellali et al. 2002; Goulding et al. 2014) (B) and express en1b (C). dI2 interneurons are glutamatergic (excitatory, express slc17a6a/b) (Alaynick et al. 2011; Serrano-Saiz et al. 2013) (D) and express lhx1a (E), but not evx1 (F). Asterisks indicate double-labeled and white crosses indicate single-labeled hmx3a-expressing cells (green). Expression of other genes is red. Bar, 20 µm. (G) Heatmap analysis of gene expression profiling of V1 interneurons. A three-class ANOVA analysis of differential expression was performed on different FAC-sorted populations of cells. Class 1: All postmitotic spinal neurons. Class 2: V1 interneurons. Class 3: All trunk cells. Each column is a different biological replicate. Rows show relative expression levels for a single transcription factor gene as normalized data transformed to a mean of 0, with standard deviation of +1 (highly expressed) or −1 (weakly/not expressed) sigma units. Adjusted P-values corrected for multiple testing are <0.000001 for all genes shown. For more information on these experiments see (Cerda et al. 2009). Expression profiles are included for positive control genes (green), en1b and pax2a, that are expressed by V1 cells; and negative control genes (purple), gata3 and vsx1, that are expressed by other spinal cord interneurons, but not V1 cells. Data for lhx1a, lhx5, and hmx3a shows that they are coexpressed in V1 interneurons. (H) Schematic showing neurotransmitter and postmitotic transcription factor phenotypes of spinal cord interneurons found in the dorsal-ventral spinal cord region shown in panels A–F. It is currently unclear whether V0v interneurons express Lhx1/5 (*, see Results).
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