Fig 2: Lamination defects in the Rb/Bax DKO retina.a Retinal sections of indicated genotypes and ages were stained for nuclei (DAPI, blue), synaptic vesicles (SV2a, red; Syt1, green), cones (M opsin, red), and horizontal cells (OC2, blue). b RT-PCR analysis of the indicated genes and ages (n = 3). c Retinal sections of indicated genotypes and ages were stained for nuclei (DAPI, blue), division (Ki67, green), and synaptic vesicles (SV2a, red). Arrows indicate ectopically dividing cells disrupting the SV2+ OPL. d Retinal sections of indicated genotypes and ages were stained for nuclei (DAPI, blue), cyclin E (red), Cdk5 (red), and Dcx (red). e P18 Rb/Bax DKO retinal sections were stained for nuclei (DAPI, blue), cone (cone arrestin, red), horizontal (D28K, green), Müller (GS, green), or amacrine cells (Calretinin, red; Ap2a, red). Arrowheads highlight the OMPL separating the INL. f Retinal thickness for the indicated genotypes and ages. g Cell counts of cone (cone arrestin+), horizontal (D28K+), amacrine (Ap2a+), and Müller cells (GS+) of the indicated genotypes at P18. h P18 Rb/Bax DKO retinal sections were stained for nuclei (DAPI, blue), division (Ki67, green), and amacrine cells (Calretinin, red). Dendrites of Calretinin+ amacrine cell stratify in the IPL, as usual, but also the OMPL (arrow). Data are mean ± SD. Asterisks indicate significant difference between WT and other genotypes, or between RbKO and Rb/Bax DKO as indicated by square brackets (*p < 0.05, one-way ANOVA followed by Bonferroni correction). ONL outer nuclear layer, OMPL outer misplaced plexiform layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer. Scale bar is 50 µm

Fig 3: Sustaining Foxp1 in RPCs increases in early-born and decreases in late-born retinal cells(A and B) Foxp1 expression in P0 Foxp1tg/+ (control) and Six3-Cre; Foxp1tg/+ (Foxp1 cTG) littermates.(C–H and K–M) Foxp1 cTG showed increased (C, D, and K) Brn3a-labeled RGCs (E, F, and L) Onecut2-labeled horizontal cells, and (G, H, and M) Rxr?-labeled cone photoreceptors at P0.(I and J) Foxp1 expression and Brn3a-labeled RGCs in P0 Six3-Cre; Foxp1tg/tg retina. Foxp1 cTG at P10 showed (N, O, and T) decreased Vsx2-labeled bipolar cells and (P, Q, and U) reduced thickness of Rhodospin-labeled rod photoreceptor layer. Arrowheads in (P) and (Q) indicate abnormal structure in the subretinal region.(R, S, and V) Foxp1 cTG had reduced Lhx2 and Cralbp-colabeled Müller glia in the INL and (V') increased Müller glia in the ONL and OPL. Arrowheads in (S, S’, and S'') indicate mislocated Müller glia in the ONL and OPL.(Y) Foxp1 cTG administered EdU at E18.5 and analyzed at P0 (W–Z) showed increased EdU+Brn3a+ RGCs. Zoomed-in images were from a single imaging plane. (AA) Foxp1 cTG retina showed increased cell-cycle exit of progenitors. NBL, neuroblastic layer; GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer. Graphs represent mean ± SEM, n = 6. *p < 0.05, **p < 0.01, ***p < 0.001, by Student’s t test. Scale bars, 100 µm. See also Figure S7 and Table S6 for statistical details.

Fig 4: The OC factors moderate Pou2f2 expression in dI3 interneurons. (A–F) Immunodetection of Pou2f2 (red) in Isl1+ (green) dI3 interneurons on transverse sections of thoracic spinal cord at e10.5 (A,B), at e11.5 (C,D) and e12.5 (E,F). Pou2f2 is detected in the ventral part of the post-mitotic Isl1+ dI3 interneuron population from e10.5. Pou2f2 signal is stronger in Isl1+ dI3 interneurons in Hnf6/Oc2-/- spinal cord at all studied developmental stages. (G–I) Relative quantification of Pou2f2 positive dI3 neurons at e10.5 (G), e11.5 (H) and e12.5 (I). The proportion of Pou2f2+ dI3 is not significantly different in Hnf6/Oc2-/- spinal cords. The pictures show part of right hemisections as indicated on the schemes to the left. Solid lines delineate the spinal cord. Mean values ± SEM, n = 3. Scale bars = 100 µm.

Fig 5: RNA levels and fold-change for TFs in LVST versus cMVST neurons, normalized to control tissue. Top row (A) shows mouse and bottom row (B) shows chicken data. A, B, Left, Names of genes with significant differential expression (FDR < 0.01, ANOVA-like analysis) and high fold-change between groups. Gene names of those TFs subsequently validated by immunohistofluorescence indicated in red (except for chicken ONECUT2, ONECUT3, and EVX2, for which antibodies were only functional in mouse). Right, Only the latter gene names. Chicken genes prefixed with ENSGALG and leading zeros have been shortened to non-zero digits only. Immunohistofluorescence for the TFs marked with an asterisk (Evx1*, Casz1*, and Myc*) stained most hindbrain cells above background level, and these genes were therefore not analyzed further. The x- and y-axes show, respectively, increasing levels of expression in the LVST and cMVST groups relative to control. The red diagonal indicates unity, and the color scale represents log2(TPM + 1) for cMVST group for points above the red diagonal, and for LVST group below the red diagonal, i.e., the VS group with the highest TPM value for each gene determines the point color. Genes above the upper blue diagonal have a >4-fold increase of TPM + 1 values for cMVST versus LVST group, and those below the lower blue diagonal have a >4-fold increase of TPM + 1 values for LVST versus cMVST group. For mouse, n = 2529 TFs; for chicken, n = 620 TFs. Figure 3-1, RNA level comparison of identified mouse/chicken TF orthologs.