Fig. 8

evx1;evx2 Mutant Group 3 cells mis-express inhibitory spinal interneuron, or motoneuron genes. (A-R) 2D UMAP plots of differential gene expression between cell clusters in the 48 h post-mitotic V0v spinal interneuron single-cell RNA-seq atlas. For cell cluster identities, see Fig. 6A. Black shows high levels of expression, light grey shows low levels of expression. Inset panels in A-R show high-magnification views of Mutant Group 3 cells. For the number of cells expressing each gene see Table 3. (T-X) High magnification views of Mutant Group 3 cells showing three-way differential gene expression (T-W) or different cell fates (X). Panel (S) indicates the color-coding for panels (T-W). (T-W) Cells expressing only gene 1 are green. Cells expressing only gene 2 are red. Cells expressing only gene 3 are blue. Cells are yellow, pink, or turquoise if they co-express genes 1 and 2, genes 2 and 3, and genes 1 and 3 respectively. Cells expressing all three genes are white. All expression data have been normalized (see Methods). (A-G) Distinct subsets of Mutant Group 3 cells express markers of inhibitory spinal neurons, including (A) gata2a, (B) gata3, and (C) tal1 (usually expressed by KA’, KA’’ and V2b inhibitory interneurons), (D) sst1.1 (usually expressed by KA’ inhibitory interneurons), (E) en1b (usually expressed by V1 inhibitory interneurons), (F) dmrt3a (usually expressed by dI6 inhibitory interneurons), and (G) lbx1a (usually expressed by dI4 and dI6 inhibitory interneurons, although it is also expressed in dI5 excitatory interneurons). (H-M) A further subset of Mutant Group 3 cells co-express markers of acetylcholinergic motoneuron cells, including (H) isl1a, (I) isl2a, (J) isl2b, (K) mnx1, (L) mnx2a, and (M) mnx2b. (N-R) In contrast, Mutant Group 3 cells do not strongly express markers of other excitatory spinal neurons, such as (N) sim1a (usually expressed by V3 excitatory interneurons), (O) vsx2 (usually expressed by V2a excitatory interneurons), (P) tlx3b (usually expressed by dI3 and dI5 excitatory interneurons), (Q) foxp2 (usually expressed by dI2 excitatory interneurons, although it is also expressed in V1 inhibitory interneurons), and (R) barhl2 (usually expressed by dI1 excitatory interneurons). (T) KA’, KA’’ and V2b genes, gata2a, gata3 and tal1 are co-expressed in a distinct subset of Mutant Group 3 cells. (U-V) Adjacent and to the left of this KA/V2b-like subset are two distinct subsets of cells expressing lbx1a (green cells in U and V) and/or dmrt3a (blue and turquoise cells in V), or en1b (red cells in U-V), which are expressed by dI4 (lbx1a), dI6 (lbx1a + dmrt3a) and V1 (en1b) interneurons respectively. (W) Adjacent and to the right of the KA/V2b-like subset of Mutant Group 3 cells shown in T, is a subset of cells co-expressing the motoneuron genes isl1a, mnx1, and mnx2b. (X) Sub-clusters are color-coded by cell identity assigned based on the differential expression profiles shown in A-M and T-W: Motoneurons (pink), V2b + KA neurons (yellow), V1 neurons (red), dI6 neurons (blue), and dI4 neurons (green)