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.

<p>Pax2 and Gbx1 are inhibitory markers, whereas Lmx1b and MafA are excitatory markers. Lbx1 and RORα predominantly label excitatory neurons, as well as a small number of inhibitory neurons. MafB and c-Maf are expressed by mixed populations of inhibitory and excitatory neurons. Data is expressed as mean±s.d. RORα expression was analyzed using a <i>RORα</i>^Cre; <i>R26</i>^{floxstop-Tomato} reporter. Asterisk indicates 100% by definition.</p

Comparative expression of transcription factors that are co-localized with the excitatory marker Lmx1b.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077928#pone-0077928-t002" target="_blank">Table 2</a>).</p

Comparison of transcription factor expression with inhibitory neuronal markers.

<p>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), <i>GAD67-GFP</i> (A–B, M–N) and <i>Pax2-Cre; R26</i>^floxstop-GFP (E–F, I–J). In <i>RORα-Cre; R26</i>^{floxstop-Tomat<i>o</i>} 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 <i>GAD67-GFP</i> and <i>Pax2-Cre; R26</i>^floxstop-GFP mice (Q-R, data not shown). <i>Pax2-Cre; R26GFP</i> denotes GFP⁺ cells in <i>Pax2-Cre; R26</i>^floxstop-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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077928#pone-0077928-t002" target="_blank">Table 2</a> for further details.</p

Relative expression of the transcription factors that define the transcription factor code for the different neuron populations.

<p>The RORβ inhibitory neurons that expressed Gbx1 did not express GFP in <i>MafB-GFP</i> 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 <i>RORα-Cre; R26</i>^{floxstop-Tomato} mice also expressed MafB (B), therefore all RORα⁺/Gbx1⁺ cells express MafB. No c-Maf-positive neurons expressed GFP in <i>MafB-GFP</i> 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 <i>RORα-Cre; R26</i>^{floxstop-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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077928#pone-0077928-t002" target="_blank">Table 2</a> for number designations.</p

GFP expression in the dorsal horn of <i>MafB-GFP</i> mice is restricted to inhibitory neurons.

<p>(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.</p