Expression of <i>Gli1</i>-eGFP and TH does not co-localize in the midbrain in the <i>Gli1</i>-eGFP-transgenic mouse line.

<p>(<b>A</b>) Model of the BAC-transgenic mouse line. This BAC-transgene contains several (minimum of 5) copies of eGFP which is transcribed under control of the <i>Gli1</i> promoter and immediately upstream of the <i>Gli1</i> sequence. When GLI1 expression is initiated by SHH-signaling, these cells start expressing eGFP. eGFP persists in the cell long after GLI1 has been broken down. Cells that expressed GLI1 can therefore be traced up to 2 days after expression of GLI1 has stopped. (<b>B</b>) Expression of <i>Gli1</i>-eGFP and TH does not co-localize at E11.5-E13.5 in the BAC-transgenic mouse-line. A strong separation between cells expressing <i>Gli1</i>-eGFP and TH⁺cells can be detected at all stages, but is most apparent at E11.5 and E12.5 (<b>1–4</b>). At E13.5 a few <i>Gli1</i>-eGFP expressing cells can be detected in the TH⁺ area. However, no co-localization can be detected (<b>5–6</b>). A: Anterior; P: Posterior.</p

Cells of the mdDA system seem to originate in the FP and at the FP-BP boundary and are positioned along-side and perpendicular to radial glia.

<p>Radial positioned neurons can be detected from E11.5 onwards. TH⁺ neurons appear to originate in the FP and at the FP-BP boundary (white arrowheads in overview) and are positioned along-side radial glia (white arrowheads) (1–2). At E11.5 some radial positioned neurons can be detected rostrally, but most are present in the caudal part of the midbrain. When TH⁺ neurons reach the ventral part of the midbrain, most neurons are positioned tangential (white arrows), suggesting migration to more lateral and rostral regions (3–4 and 3′, 3", and 4′). A: Anterior; P: Posterior.</p

Analysis of TH and GFP expression in multiple En1-/Pitx3-mutants at E14.5.

<p>(A) Immunohistochemistry of GFP and TH in different sections from medial to lateral encompassing the mdDA neuronal pool in Pitx3GPF/+ animals. (B-D) Same setup as described for A for (B) En1WT;<i>Pitx3</i>GFP/GFP, (C) En1Het;<i>Pitx3</i>GFP/GFP, and (D) En1KO;<i>Pitx3</i>GFP/GFP animals (matching sections with A). (D) Asterisk indicate the loss of TH immunoreactivity in more medial sections. Arrowheads indicate the presence of ectopic mdDA neurons.</p

Quantitative PCR analysis of mdDA neurons in double En1KO;Pitx3GFP/GFP animals at E14.5.

<p>(A) Quantitative PCR demonstrates no further loss of <i>Ahd2</i> expression in En1KO;Pitx3GFP/GFP animals compared to En1WT;Pitx3GFP/GFP (P>0.05, n = 3/4). Significant loss of <i>Cck</i> expression in both the En1Het;Pitx3GFP/GFP (P<0.05, n = 4) and En1KO;Pitx3GFP/GFP animals (P<0.01, n = 3/4), compared to En1WT;Pitx3GFP/GFP. (B) Quantitative PCR demonstrates no changes in <i>Pbx1</i>, <i>Tle3</i>, <i>Tle4</i> and <i>Otx2</i> expression in En1KO;Pitx3GFP/GFP animals compared to En1WT;Pitx3GFP/GFP (P>0.05, n = 3/4). Significantly increased expression of <i>Pbx3</i> in En1KO;Pitx3GFP/GFP animals, compared to both the En1Het;Pitx3GFP/GFP and En1WT;Pitx3GFP/GFP animals (*** = P<0.01, n = 3/4).</p

SHH expression does not overlap with the majority of the TH⁺ area of the midbrain.

<p>(<b>A</b>) <i>In situ</i> hybridization of <i>Shh</i> in comparison to TH-DAB shows expression in the ventricular zone at E12.5. However, expression of <i>Shh</i> is stronger in the BP than in the FP of the embryonic midbrain. <i>Shh</i> expression seems to end at the border with the TH expressing area (<b>1</b>). (<b>B</b>) At E11.5 and E12.5 SHH expression does not overlap with most of the TH⁺ cells in both rostral and caudal regions of the TH⁺ area. However, at the most lateral parts of the TH expressing area some overlap seems to exist with SHH (<b>1–2</b>).</p

Breeding schedule and outcome of the generation of En1KO;Pitx3GFP/GFP animals.

<p>(A) En1Het;Pitx3GFP/GFP animals were inter-crossed to generate a litter that includes three genotypes: En1WT;Pitx3GFP/GFP, En1Het;Pitx3GFP/GFP, and En1KO;Pitx3GFP/GFP. (B) The presence of double En1KO;Pitx3GFP/GFP animals occurred below (Mendelian) predicted chance level (12.7% versus 25%). (C) DAPI staining at E14.5 reveals that in En1WT;Pitx3GFP/GFP and En1Het;Pitx3GFP/GFP animals the cerebellar <i>anlage</i> is correctly developed (Cb), whilst it is absent in the double En1KO;Pitx3GFP/GFP (*). (Rf: retroflexus, Cb: cerebellum, Cp: choroid plexus).</p

Schematic representation of roles of <i>En1</i> and <i>Pitx3</i> in the programming of the rostral-caudal identity of mdDA neurons.

<p>(A) In wild-type midbrain <i>Nurr1</i> initiates the development of mdDA differentiation, <i>Pitx3</i> promotes <i>Ahd2</i> expression and represses <i>En1</i> in rostral midbrain, whilst <i>En1</i> promotes <i>Pitx3</i> and <i>Cck</i> expression. The mdDA neuronal pool includes rostral-coded and caudal-coded neurons. (B) In <i>Pitx3</i>-ablated animals <i>Nurr1</i> initiates the development of mdDA differentiation, though <i>Ahd2</i> expression is lost, and the inhibition of <i>Pitx3</i> on <i>En1</i> is lifted, thus <i>En1</i> and subsequently <i>Cck</i> are up-regulated. The mdDA neuronal pool includes only caudal-coded neurons. (C) In <i>En1</i>-ablated animals <i>Nurr1</i> initiates the differentiation of mdDA progenitors, though <i>Cck</i> expression is lost, and <i>Pitx3</i> expression in the rostral midbrain is not initiated, thus <i>Ahd2</i> expression is lost as well. The mdDA neuronal pool includes only non-coded neurons. (D) In double En1KO;Pitx3GFP/GFP animals elevated levels <i>Nurr1</i> promotes the differentiation of mdDA progenitors, though <i>Cck</i> and <i>Ahd2</i> are lost.</p

Quantification of the number of GFP-positive neurons present in the midbrains of multiple <i>En1</i>-/<i>Pitx3</i>-mutants at E14.5.

<p>(A) Schematic representation of the isolation of midbrain and R1, and subsequent FAC-sorting setup at E14.5 to be used for quantification of number of GFP-positive neurons. (B) At E14.5, ~15000 GFP-positive mdDA neurons were sorted from control, En1WT;Pitx3GFP/GFP and En1Het;Pitx3GFP/GFP animals. In contrast, only ~5000 GFP-positive mdDA neurons were present in the En1KO;Pitx3GFP/GFP midbrain/R1 (** = P<0.01, n = 3/4).</p

TH⁺ cells in the midbrain co-localize at both E12.5 and E14.5 with β-galactosidase, in a BATGAL expressing transgenic mouse line.

<p>A) Model of the transgenic BATGAL mouse line. β-galactosidase is expressed by binding of stable β-catenin to the TCF/LEF binding sites in the promoter of the <i>LacZ</i> gene. When β-catenin is activated, the <i>LacZ</i> gene in-cooperated in the genome is transcribed and β-galactosidase expressed. (B) At E12.5 β-galactosidase expression overlaps with TH both lateral and medial in the mdDA system (1–4). At E14.5 no β-galactosidase staining is detected lateral. However, medial many mdDA neurons can be detected to express β-galactosidase (5–8). Indicating that canonical WNT-signaling is or has been present in mdDA neurons. A: Anterior; P: Posterior.</p

Breeding constrains in creating double En1KO;Pitx3GFP/GFP animals.

<p>Breeding constrains in creating double En1KO;Pitx3GFP/GFP animals.</p