Figure S2.
Rbp4-Cre neurons are located in the region from the subplate to the surface of cortex, are distinct from Cajal-Retzius cells and subplate neurons, and express layer 5 markers, related to Figure 2
(A) Within the UMAP embedding of all 25681 sequenced excitatory neuron transcriptomes (gray), cells expressing a number of genes previously associated with Cajal-Retzius cells66,68,79 overlap in a location (green outline) distinct from positively identified Rbp4-Cre neurons (as in Figure S1) (red outline; Rbp4-Cre neurons) (labeled in bottom right). Clusters of Rbp4-Cre neurons are additionally labeled based on the cluster identification from Figure 1E.
(B) Within the UMAP embedding of all 25681 sequenced excitatory neuron transcriptomes (gray), cells expressing genes previously associated with subplate neurons overlap in a location (green outline) distinct from positively-identified Rbp4-Cre neurons (as in Figure S1) (red outline; Cre neurons) (labeled in the bottom right). Clusters of Rbp4-Cre neurons are additionally labeled based on the cluster identification from Figure 1E.
(C) Rbp4-Cre neurons (green), counterstained with Hoechst (blue), show that neurons in the deep layer lie physically within the subplate (SP; dotted line), as localized by the expression of a common subplate marker, Nr4a2 (Nurr1) (magenta).
(D) Only a small fraction of Rbp4-Cre neurons express Nr4a2 from E13.5 to E18.5.
(E) Rbp4-Cre neurons are located in the region from the subplate to the surface of cortex. Rbp4-Cre neurons (stained using GFP antibody labeling GCaMP6s, green) in both spatial configurations (on E14.5, E16.5, and E18.5) co-labeled with antibodies labeling different zones in the developing cortical wall (Pax6 (first row), Tbr2 (second row), Tbr1 (third row), Satb2 (fourth row), red), counterstained with Hoechst (blue). Dotted line outlines the area from subplate to surface of cortex.
(F) Rbp4-Cre neurons (stained using GFP antibody, green), counterstained with Hoechst (blue), show that neurons within both the superficial and deep layers at E14.5, as well as neurons within both the intermediate and deep layers at E18.5, all colocalize with Bcl11b (red), the expression of which is restricted in the adult cortex to layer 5.69 Arrows: example Rbp4-Cre neurons.
(G) Genes selective for each embryonic layer 5 type. Transcript counts for each gene shown as colored circles in all types at all ages. Radius of circle: fraction of cells expressing the gene; color of circle: mean normalized transcripts per cell (log2). Bold text: genes used for in situ hybridization, shown in Figure 2C.
(H) Spatial distribution of Rbp4-Cre neurons into layers is indistinguishable when expressing GCaMP6s compared to tdTomato on both E14.5 and E18.5, and Rbp4-Cre neurons. Quantifying the distribution of Rbp4-Cre neurons into layers at E14.5 (top) and E15.5 (bottom) in embryos generated by using the GCaMP6s-tTA2 reporter line, or a tdTomato reporter line. Probability: χ2 test comparing the fraction of Rbp4-Cre neurons in each layer for the two different reporter lines, p = 0.05. Scale bars: 20 μm (C, E, F).