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1.
Figure 5

Figure 5. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

The distribution of MbDA neurons in adult mouse. A: Each individual MbDA neuron was analyzed in forty horizontal sections that contain all adult MbDA neurons. On each 40 μm thick section, a dot was placed on immunolabeled (TH+) MbDA neuron and reconstructed in three dimensions using Volocity 3D module to show the full distribution of MbDA neurons; the VTA and SNc are indicated. B–D: Adjacent sections were immunolabeled for TH (green) and the indicated marker (red) to label MbDA neuron subtypes. E: The three dimensional distribution of MbDA neuron subtypes. Scale bar = 32μm (B–D).

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
2.
Figure 6

Figure 6. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

MbDA neurons in the VTA, RRF, and SNc domains were derived from Wnt1-expressing progenitors over a prolonged time period. MbDA neurons in adult Wnt1-CreERT;TaumGFP mice marked by tamoxifen administration at distinct 24 hour time points between E7.5–E13.5. A–G: VTA (medial) MbDA neurons. H–N: A8/RRF (retrorubral field) MbDA neurons. O–V: SNc (lateral) MbDA neurons. MbDA neurons are indicated by TH immunolabeling (green) and the Wnt1 lineage is indicated by nuclear β-gal immunolabeling (red). Arrows show examples of neurons that expressed both TH and β-gal. Scale Bar = 32μm.

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
3.
Figure 14

Figure 14. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

The relative contribution of Wnt1-expressing cells to dorsal, intermediate, and ventral MbDA neurons. A–C: The percentage of MbDA neurons in the dorsal (A), intermediate (B), and ventral (C) spatial domains marked by GIFM with tamoxifen administration between E7.5–E13.5. The counting frames that were used for group analysis are shown in Fig. 9 (dorsal: all counting frames in H and I; intermediate: all counting frames in J, ventral: all counting frames in K–L). The contribution of the Wnt1 lineage to dorsal and intermediate MbDA neurons follows a regression model (black trend line) similar to the model for the contribution of the Wnt1 lineage to MbDA neurons as a whole. However, the Wnt1 lineage contribution to ventral MbDA neurons does not follow this pattern. A–C, insets. Orthogonal linear contrast models showing that the Wnt1 lineage contribution to the dorsal, intermediate, and ventral MbDA neurons at E10.5 was significantly less than at E9.5 or E11.5; * indicates p<0.0001.

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
4.
Figure 13

Figure 13. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

The relative contribution of Wnt1-expressing cells to rostral, intermediate, and caudal MbDA neurons. A–C: The percentage of MbDA neurons in the rostral (A), intermediate (B), and caudal (C) spatial domains marked by GIFM with tamoxifen administration between E7.5–E13.5. The counting frames that were used for group analysis are shown in Fig. 9 and were applied in the following manner: (rostral: Fig. 9H1–2, I4, J3, J7, K4, K6–8, L1–2; intermediate: Fig. 9I1, J2, J6, K3, K5; caudal: Fig. 9I2-3, J1, J4–5, K1–2). The contribution of the Wnt1 lineage to rostral, intermediate, and caudal MbDA neurons follows a regression model (black trend line) similar to the model for the contribution of the Wnt1 lineage to MbDA neurons as a whole. A–C, insets. Orthogonal linear contrast models showing that the Wnt1 lineage contribution to the rostral, intermediate, and caudal MbDA neurons at E10.5 was significantly less than at E9.5 or E11.5; * indicates p<0.0001.

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
5.
Figure 12

Figure 12. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

The relative contribution of Wnt1-expressing cells to VTA, RRF, and SNc MbDA neurons. A–C: The percentage of MbDA neurons marked by GIFM compared to the total number of MbDA neurons counted in the VTA (A), RRF (B), and SNc (C) when tamoxifen was administered between E7.5–E13.5. The counting frames that were used for group analysis are shown in Fig. 9 and were applied in the following manner: The VTA (medial) MbDA neurons are from Fig. 9H1, I1, J1–3, K2–4; The RRF (intermediate) MbDA neurons from Fig. 9H2, I2; The SNc (lateral) MbDA neurons from Fig. 9 I3–4, J4–7, K1, K5–8; L1–2. The contribution of the Wnt1 lineage to VTA, RRF, and SNc follows a regression model (black trend line) similar to the model for the contribution of the Wnt1 lineage to MbDA neurons as a whole. A–C, insets. Orthogonal linear contrast models showing that the Wnt1 lineage contribution to the VTA, RRF, and SNc at E10.5 was significantly less than at E9.5 or E11.5; * indicates p<0.0001.

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
6.
Figure 4

Figure 4. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

Molecular identity of Wnt1-expressing progenitors in late vMes. A: An E12.5 whole mount embryo indicates the vMes. Illustration shows region of analysis. B–E: Medial and off-midline sagittal sections from E12.5 Wnt1-Venus embryos showing Wnt1(GFP)+ progenitors (green) in the mes and the indicated markers (red); hoechst staining (blue) shows tissue morphology. Black and white insets show single channel of each marker. F–G: Transverse sections showing the morphology and domains of interest in H–L. H–L: Transverse sections at rostral (r), intermediate (i), and caudal (c) levels showing Wnt1(GFP)+ cells (green) and indicated markers (magenta). K: Summary schematics showing domains of expression of Wnt1(GFP) (green), Otx2 (red), Lmx1a (blue), and TH (yellow). Note that intermingling of markers indicates overlapping domains versus exclusive expression domains. L–N: Horizontal planes showing Otx2 (green) and Lmx1a (magenta) at dorsal (d), intermediate (i), and ventral levels (v).

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
7.
Figure 10

Figure 10. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

Sampling method for quantifying MbDA neurons derived from the Wnt1 lineage. A,B: Hemi-horizontal sections from ventral location (See Fig. 9F,K) at low magnification and representative examples of MbDA neurons (TH+ green) and the Wnt1 lineage (β-gal+, red). Coincident labeling indicates Wnt1 derived MbDA neurons. Counting frames were overlaid on MbDA neurons and allowed for the reproducible partitioning of medial to lateral or rostral to caudal populations. For each counting frame, a 40x z-series was collected and neurons expressing both TH and β-gal were counted (see methods). We provided two examples of how counting frames were applied to sections from Wnt1-CreERT;TaumGFP adult mice that were marked by tamoxifen administration at E9.5 (A) or E10.5 (B). Examples of 1 μm thick optical sections from 40x z-series stacks; each number corresponds to the assigned counting frames shown in the low magnification images. Scale Bar = 520 μm (A,B), 31 μm (40x z-series images).

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
8.
Figure 7

Figure 7. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

Biochemically distinct MbDA neuron subtypes were derived from progenitors expressing Wnt1 over a prolonged time period. MbDA neurons in adult Wnt1-CreERT;TaumGFP mice were marked by tamoxifen administration at distinct 24 hour time points between E7.5–E13.5. A–G: Wnt1-derived MbDA neurons from VTA that expressed calbindin. H–N: Wnt1-derived MbDA neurons from SNc that expressed GIRK2. O–V: Wnt1-derived MbDA neurons that expressed calretinin. The panels show a single optical plane (1μm thick) after iterative restoration using Volocity software to deconvolve collected Z-series image stacks. MbDA neurons (TH+, green), the Wnt1 lineage (β-gal+, blue), and the expression of distinct molecular markers (calbindin, GIRK2, or calretinin as indicated, green). Arrows show examples of triple immunolabeled neurons that expressed TH, β-gal, and molecular markers in the XY plane. The XZ and YZ planes are shown to further confirm the overlap of the three markers. Scale Bar = 32μm.

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
9.
Figure 3

Figure 3. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

Molecular identity of Wnt1-expressing progenitors in vMes at an intermediate developmental stage. A: An E10.5 whole mount embryo indicates the location of vMes, which was dissected for clarity; the plane of sagittal sections are shown. B: Illustration shows transverse plane of analysis. C–E: Coronal sections at rostral (C) and intermediate (D,E) levels analyzed for Lmx1a and Wnt1 expression. F–G: Transverse sections at intermediate (F) and caudal (G) levels analyzed for Otx2 and Wnt1 expression. Brackets show medial (1) and off-midline (2) domains. H–J: Sagittal sections at medial and off-midline planes showing Wnt1(GFP)+ progenitors (green) in the vMes and the indicated markers (red); hoechst staining (blue) shows tissue morphology. Black and white insets show single channel of each marker. K–M: Sections from E10.5 Wnt1-Venus;TOPGAL embryo showing Wnt1(GFP)+ progenitors (green) in the mes and the indicated markers (magenta). K: Rostral Wnt1(GFP)+ progenitors were largely not mitotic (PHH3 negative), expressed LMX1a, and overlapped with OTX2. L: Intermediate Wnt1(GFP)+ progenitors were also largely not mitotic, did not express LMX1a, and overlapped with OTX2. M: Caudal Wnt1(GFP)+ progenitors were also largely not mitotic, expressed LMX1a, and overlapped with OTX2. Scale bar = 16μm (B–J).

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
10.
Figure 2

Figure 2. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

Molecular identity of Wnt1-expressing progenitors in early vMes. AB: Whole mount E8.5 embryos indicate the location of sections that are shown. A: sagittal view. B: dorsal view. Illustration shows region of analysis. CJ: Sections from E8.5 Wnt1-Venus;TOPGAL embryo showing Wnt1(GFP)+ progenitors (green) in the mes and the indicated markers (magenta); note that Wnt1(GFP) was not expressed in rhombomere 1 (r1) or in the prosencephalon (pros). White arrowheads with asterisk show co-localization of Wnt1(GFP)+ progenitors and marker; white arrowheads show examples of Wnt1(GFP)+ only cells; white arrows show cells expressing only the marker. C,G: Wnt1(GFP)+ progenitors that were mitotic (pHH3+) were located in mitotic zone at the periphery of the tissue. D,H: LMX1a was co-localized with medial, but not lateral Wnt1(GFP)+ progenitors. E,I: OTX2 was co-localized with Wnt1(GFP)+ progenitors both medially and laterally; in medial sections the OTX2+/Wnt1(GFP)− prosencephalon (red) was apparent. F,J: There were Wnt1(GFP)+ cells that responded to canonical WNT signaling in the medial domain as evident by overlap with β-gal from the TOPGAL reporter (Fig 2F). However, fewer Wnt1(GFP)+ cells responded to canonical WNT signaling in the lateral domain (Fig. 2J). Scale bar = 32μm (C–J).

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
11.
Figure 11

Figure 11. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

Quantitative analysis of the Wnt1 lineage contribution to total MbDA neurons. The data shows the percentage of MbDA neurons marked by GIFM at indicated tamoxifen administration time points from E7.5–E13.5 from three adult brains sampled as described in Figs. 9 and 10. The contribution of Wnt1-expressing cells compared to the total number of MbDA neurons counted. The Wnt1 lineage gives rise to a relatively moderate level of MbDA neurons early (E7.5–E8.5). Overall, the E9.5 marking period resulted in the largest contribution to MbDA neurons. From E9.5 to E12.5, there is a significant decrease in Wnt1 lineage contribution. E13.5 represents the cessation of the Wnt1 lineage contribution to MbDA neurons. Statistical analysis showed the data fit a positive linear regression from E7.5 to 9.5, followed by a negative quadratic regression from E9.5 to E13.5. (p<0.0001 for each component). A trend line representing this regression is shown in gray. The contribution of the Wnt1 lineage marked at E10.5 appeared less than when marked at E9.5 and E11.5. A, inset: The comparison between E9.5, E10.5, and E11.5 was evaluated in a statistical model using orthogonal linear contrasts, which showed that the lineage contribution at E10.5 was significantly less than at E9.5 or E11.5 (p<0.0001, each time point) indicating that there was a significant decrease in Wnt1 lineage contribution from E9.5 to E10.5 and a significant increase from E10.5 to E11.5.

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
12.
Figure 1

Figure 1. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

Transgenic mice used in this study. A: Illustrations of the alleles contained in the mouse lines used in this study: Wnt1-CreERT, TaumGFP, Wnt1-Venus, and TOPGAL. Details of the mouse lines are provided in the “Mice” section of Materials and Methods. Yellow and blue boxes indicate Wnt1 (Wnt1-CreERT and Wnt1-Venus) or Tau (TaumGFP) translated and nontranslated exons. tag indicates a short sequence of LacZ in Wnt1-CreERT and Wnt1 Venus; Z indicates a lacZ gene (TaumGFP and TOPGAL); L indicates a consensus binding motif for LEF1/TCF (TOPGAL); white arrowheads indicate loxP sites (TaumGFP). BE: Venus expression in Wnt1-Venus embryos mimics endogenous Wnt1 expression. B: Sagittal view of a Wnt1-Venus E10.5 embryo indicating the location of the ventral mes, the region of interest in this study. C–D: In-situ hybridization in E10.5 Wnt1-Venus embryos for with probes recognizing Wnt1 (C) and YFP (D, identifying the expression of the Wnt1-Venus transgene). The expression of Wnt1 and YFP overlaps in a very similar domain in the ventral mes, indicating that the Wnt1-Venus transgene is not expressed ectopically and that it is a true readout of endogenous Wnt1 expression. E: Immunolabeling for GFP protein in an E10.5 Wnt1-Venus embryo. GFP protein expression also overlaps in a very similar domain of expression to the Wnt1 gene, also indicating that the Wnt1 transgene is not expressing ectopically. Scale bar = 63μm (BE).

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
13.
Figure 8

Figure 8. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

The temporal contribution of the Wnt1 lineage to vMb structures. A–B: Cresyl violet staining indicating the anatomical distribution of selected nuclei is shown. The following vMb structures were analyzed: subthalamic nucleus (STh), interfascicular nucleus (IFN), posterior hypothalamic area (PHA), substantia nigra pars reticulata (SNr), red nucleus (RN), edinger-westphal nucleus (EW). The following MbDA neuron containing structures were also analyzed: substantia nigra pars reticulata (SNc), ventral tegmental area (VTA), retrorubral field (RRF). C–D: Representative examples of the Wnt1 lineage contribution to vMb structures in Wnt1-CreERT;TaumGFP mice marked by tamoxifen administration at E9.5. The Wnt1 lineage derived cells (β-gal+, red) and hoechst nuclear counterstaining (blue) are shown in a dorsal (C) and intermediate (D) horizontal section. E–F: The Wnt1 lineage contributes to a few BRN3a+ neurons in EW (E) and rarely to RN (F). Examples of cells co-expressing β-gal and BRN3a are indicated by arrows. G: The relative contribution of neurons derived from the Wnt1 lineage to the indicated vMb structures that were marked from E7.5 to E13.5. “−” no contribution, “+” sparse contribution, “++” moderate contribution, “+++” high contribution. H: Summary schematic showing the progressive restriction of the Wnt1 lineage contribution to vMb structures with the exception of the pattern of contribution to the MbDA neurons. Scale Bar = 250μm (A,B); 15μm (C,D).

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.
14.
Figure 9

Figure 9. From: Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors in vivo.

Anatomical levels used for quantitative GIFM analysis of the Wnt1 lineage contribution to adult MbDA neurons. A: Mid-sagittal schematic of an adult brain showing the relative locations of the five horizontal planes used for analysis. B: Three-dimensional reconstruction of MbDA neuron distribution plotted along the dorsal-ventral axis and showing planes analyzed: dorsal-most (C), dorsal (D), intermediate (E), ventral (F), and ventral-most (G) C–G: Illustrations of MbDA neuron populations (dark gray shading) in context of their projections to the target striatum (gray). The domain encompassing MbDA neurons is smaller in the most dorsal planes (C,D) compared to intermediate (E) and ventral (F) horizontal sections. In the intermediate horizontal section (E), and the ventral horizontal section (F), both the lateral SNc and the medial VTA are prominent. In the ventral-most horizontal section (G), the lateral SNc is a relatively small domain and the medial VTA is absent. Note that retrorubral field (RRF) MbDA neurons are present in the dorsal sections (C,D), but is absent in more ventral sections (E–G). H–L: Images of MbDA neuron populations in the most dorsal (H), dorsal(I), intermediate (J), ventral (K), and most ventral (L) are shown below their respective illustrations. The numbered boxes indicate where counting frames were placed to sample the relative contribution of the Wnt1 lineage to distinct MbDA subdomains in dorsal-ventral planes shown. The counting frames were organized in the following manner to analyze specific domains. Medial to lateral MbDA neurons (analyzed in Fig. 12): medial (H1, I1, J1–3, K2–4); intermediate (H2, I2), lateral (I3–4, J4–7, K1, K5–8, L1–2). Rostral to caudal MbDA neurons (analyzed in Fig. 13 ): rostral (H1–2, I4, J3, J7, K4, K6–8, L1–2), intermediate (I1, J2, J6, K3, K5), caudal (I2–3, J1, J4–5, K1–2). Dorsal to ventral MbDA neurons (analyzed in Fig. 14 ): dorsal (all counting frames in H and I); intermediate (all counting frames in J), ventral (all counting frames in K–L).

Ashly Brown, et al. J Comp Neurol. ;519(15):2978-3000.

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