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

Generation and Characterization of an <i>Nse</i>-<i>CreER^T2</i> Transgenic Line Suitable for Inducible Gene Manipulation in Cerebellar Granule Cells

<div><p>We created an <i>Nse</i>-<i>CreER^T2</i> mouse line expressing the tamoxifen-inducible CreER^T2 recombinase under the control of the neuron-specific enolase (<i>Nse</i>) promoter. By using Cre reporter lines we could show that this <i>Nse</i>-<i>CreER^T2</i> line has recombination activity in the granule cells of all cerebellar lobules as well as in postmitotic granule cell precursors in the external granular layer of the developing cerebellum. A few hippocampal dentate gyrus granule cells showed Cre-mediated recombination as well. Cre activity could be induced in both the developing and adult mouse brain. The established mouse line constitutes a valuable tool to study the function of genes expressed by cerebellar granule cells in the developing and adult brain. In combination with reporter lines it is a useful model to analyze the development and maintenance of the cerebellar architecture including granule cell distribution, migration, and the extension of granule cell fibers <i>in vivo</i>.</p></div

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

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

Cerebellar lobules of tamoxifen-injected <i>Nse-CreER^T2;mTmG</i> mice labeled for different neuronal marker proteins.

<p>A-E: mGFP-immunoreactivity is shown in green (mGFP), immunoreactivity for different cell type specific markers is shown in the red channel (labeled with blue fluorescence). Arrowheads point to the fissure between two lobules. A: NeuN (in the cerebellum a GC-specific marker), B: GFAP (produced by cells of glial lineage), C: Calbindin (CB, produced by Purkinje cells), D: MAP2 (located in neuronal dendrites), and E: Parvalbumin (PV, expressed by Purkinje cells and ML interneurons). NeuN was mainly found in the GCL and cells were co-immunoreactive for GFP. GFAP-labeled structures showed no GFP-signal, nor did Purkinje cells or the interneurons of the ML. MAP2-positive structures were densely immunostained in the GCL, representing the dendrites of GCs. Scale bar  = 50 µm.</p

Tamoxifen-induced CreER^T2-activity in the juvenile and mature brain of P3- and adult-injected <i>Nse-CreER^T2;mTmG</i> mice.

<p>In control mice not carrying a <i>CreER^T2</i>-tansgene (A) or in <i>CreER^T2</i>-transgenic mice without tamoxifen injection (B), the mGFP reporter was not labeled. C-E: Tamoxifen injection of P3 (C, D) and adult (E, section more lateral, compared to A-D) mice led to a strikingly similar fluorescence pattern of mGFP in the juvenile (P23 in C) or mature (D and E) brain. mGFP fluorescence was found to be most intense in the cerebellum. Less intense staining of cells and fibers was observed in the hippocampus, and some fibers in nuclei of the midbrain and pons showed mGFP-immunoreactivity.</p

mGFP-labeled, postmitotic migrating GCPs in the EGL with trailing and leading processes at P8.

<p>Left: P3-injected P8 cerebellum, mGFP  =  cells after recombination, mTomato  =  cells without recombination. Frames A-C are shown enlarged, A′-C′  =  further enlargement to show mGFP-immunoreactive GCPs in the inner EGL, A″-C″  =  mTomato. Arrowheads in A-C point to the fissure between two lobules. Arrows presumable point to tangentially (A′, with leading and trailing processes) or radially (B′, C′, with leading and/or trailing processes) migrating GCPs in the EGL.</p

Development of the GCL and ML in the cerebellum of <i>Nse-CreER^T2;mTmG</i> mice after tamoxifen-induction.

<p>A-C: P3-injected mice were killed at P8 (A), P23 (B), or P75 (C). Pictures show the EGLs (A) or MLs (B-D) next to a fissure (arrowheads in A′-D′) of two lobules. A: At P8 some mGFP-postive GCPs are found in the EGL (arrows in A″) and a substantial amount of GCs are labeled in the IGL. Double arrowhead points to a presumably migrating cell with leading/trailing processes. Structures in the ML are immunoreactive as well. B: At P23 cells are labeled in the GCL, and structures are labeled in the ML. C: At P75, the labeling of GCs in the GCL is comparable to P8 and P23, but the structures in the ML show a more brightly immunolabeling, filling the entire ML. D: The mGFP labeling in the GCL and ML of an adult-injected mouse is strikingly similar to that of the P3-injected adult mouse in C. A-D  =  merge, A′-D′  =  DAPI, A″-D″  =  mGFP. Scale bar  = 100 µm.</p

CreER^T2-activity in <i>Nse-CreER^T2;mTmG</i> after tamoxifen injection.

<p>Sagittal section showing mGFP-immunoreactive cells (green, A) indicating that recombination has occurred (mGFP/single channel in the middle) and DAPI-stained nuclei (blue/single channel at the bottom). Enlargements of the framed rectangles in sections B to E are shown on the right: (B) fibers in a region of the colliculus superior in the midbrain, (C) layers of the cerebellum, (D) hippocampal granule cells and their fibers, and (E) fibers in the medial vestibular nuclei (medulla) show green fluorescence. Red (mTomato) fluorescence in 1–4 shows cells where no recombination occurred.</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