Figure 1.
Expression of Lbx1, MafA, c-Maf, RORα, RORβ, Pax2, Lmx1b, Gbx1 and Tlx3 in the postnatal and adult spinal cord.
Immunostaining of trancription factors that are enriched in the dorsal horn at P4 (A–J) and adult (K–P) stages. Lbx1, Lmx1b, Tlx3, Gbx1, MafA and c-Maf expression is maintained in the spinal cords of five month old mice (K–P). RORα* denotes RORα-Cre; R26floxstop-GFP in all figures. The lines in A indicate the border of lamina III–IV.
Figure 2.
Lmx1b and Pax2 are postnatal markers of excitatory and inhibitory neurons.
Analysis of Gbx1, Pax2 and Lmx1b expression in Pax2-Cre; R26floxstop-lacZ mice at P10 (A–D′,G) and P1 (E–F). The Pax2-derived β-gal reporter is localized to GAD67-GFP+ neurons in the dorsal horn (A,A′). A large fraction of the Pax2-derived β-gal cells express the Pax2 transcription factor (C,C′). GAD67-GFP and Pax2+ cells express the Gbx1 transcription factor (B,B′,E,E′). Lmx1b is not expressed in Gbx1+ neurons (F) or Pax2-derived neurons in the Pax2-Cre; R26floxstop-lacZ spinal cord (G). The lines in A indicate the border of lamina III–IV.
Figure 3.
Comparative expression of transcription factors that are co-localized with the excitatory marker Lmx1b.
Lbx1, RORα, RORβ, MafA, MafB and c-Maf all show overlapping expression with Lmx1b in excitatory neurons, albeit at low levels in some neurons. Analyses were performed at P0 (C–D, K–L), P7 (I–J, G–H), P8 (E–F) and P10 (A–B). Neurons were assigned a cell type number (1–9) according to their expression profile, with profiles 1–5 being classified as inhibitory neurons, while profiles 6–9 are excitatory neurons (see Table 2).
Table 1.
Summary of the expression analyses used to define the inhibitory or excitatory phneotype of postnatal neurons that express Pax2, Gbx1, Lmx1b, RORβ, RORα, Lbx1, MafA, MafB and c-Maf.
Figure 4.
Comparison of transcription factor expression with inhibitory neuronal markers.
Lbx1 (A–D), RORβ (E–H), MafB (I–L) and c-Maf (M–P) are expressed in many neurons that express the inhibitory markers Gbx1 (C–D, G–H, K–L, O–P, V–W), GAD67-GFP (A–B, M–N) and Pax2-Cre; R26floxstop-GFP (E–F, I–J). In RORα-Cre; R26floxstop-Tomato mice (V–W), we observed a few Tomato+ neurons that expressed Gbx1. MafA is the only transcription factor that did not co-localize with Gbx1 (S–T) or GFP in GAD67-GFP and Pax2-Cre; R26floxstop-GFP mice (Q-R, data not shown). Pax2-Cre; R26GFP denotes GFP+ cells in Pax2-Cre; R26floxstop-GFPmice. Spinal cords were analyzed at P0 (E–F), P1 (C–D, I–J, S–T), P2 (G–H), P7 (A–B, M–N, Q–R), P8 (V–W), and P10 (K–L, O–P). Examples of the nine different cell types (numbered 1–9) are shown. See Table 2 for further details.
Figure 5.
Relative expression of the transcription factors that define the transcription factor code for the different neuron populations.
The RORβ inhibitory neurons that expressed Gbx1 did not express GFP in MafB-GFP mice but not all Gbx1-positive neurons expressed GFP (A–A′). Therefore RORβ/Gbx1 double-positive neurons do not express MafB and not all Gbx1 neurons express MafB. Tomato-positive neurons that expressed Gbx1 in RORα-Cre; R26floxstop-Tomato mice also expressed MafB (B), therefore all RORα+/Gbx1+ cells express MafB. No c-Maf-positive neurons expressed GFP in MafB-GFP mice (C) and consequently, c-Maf and MafB do not co-localize in MafB inhibitory neurons. All RORβ-positive neurons that expressed Lmx1b also expressed c-Maf (D–D”). Many c-Maf-positive neurons expressed Gbx1 (E). Many RORβ-positive neurons expressed MafA, as MafA is an excitatory marker (F). Many RORβ-positive neurons expressed Tomato in RORα-Cre; R26floxstop-Tomato mice (G). RORβ+ neurons that express MafB are Lmx1b+ excitatory neurons (H–H′). Most MafA expressed C-Maf although there was a significant number of cells that only expressed MafA single or c-Maf single-positive (I). All MafA-positive neurons expressed Lbx1, although some of them at very low levels like (J). All MafB-positive neurons that expressed Lmx1b also expressed MafA (K–K”). See Table 2 for number designations.
Table 2.
A combinatorial transcription factor code defines nine different postnatal populations of interneurons in laminae III–IV.
Figure 6.
GFP expression in the dorsal horn of MafB-GFP mice is restricted to inhibitory neurons.
(A) GFP is not expressed in any Lmx1b-positive neurons. (B) Whereas all GFP+ neurons are MafB+, many MafB neurons do not express GFP. (C) MafB+ neurons that are Lmx1b+ do not express GFP.