1. Developmental Biology
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Correct setup of the substantia nigra requires Reelin-mediated fast, laterally-directed migration of dopaminergic neurons

  1. Ankita Ravi Vaswani
  2. Beatrice Weykopf
  3. Cathleen Hagemann
  4. Hans-Ulrich Fried
  5. Oliver Brüstle
  6. Sandra Blaess  Is a corresponding author
  1. University of Bonn School of Medicine & University Hospital Bonn, Germany
  2. German Center for Neurodegenerative Diseases, Germany
Research Article
Cite this article as: eLife 2019;8:e41623 doi: 10.7554/eLife.41623
13 figures, 3 videos, 2 tables and 2 additional files

Figures

Figure 1 with 4 supplements
Direct role of Reelin signalling in tangential migration of mDA neurons.

(A) Schematic showing Cre-mediated inactivation of Dab1 in mDA neurons. (B) Schematic representing the anteroposterior level of coronal sections used for the analysis, and the mediolateral grid used to quantify distribution of TH+ (Tyrosine Hydroxylase) neurons. (C–J) Immunostaining for TH and quantification of cell distribution for control, Dab1 CKO, and Dab1-/- midbrain regions at E18.5 (C–F) and at P21-P30 (G–J). White arrowheads indicate differences in the mediolateral distribution of TH+ cells. Yellow arrowheads point to cells in the substantia nigra pars lateralis used as a landmark for the most lateral position in the mediolateral grids. (F,J) Quantification of mediolateral distribution of TH+ cells for control, Dab1 CKO and Dab1-/- brains at E18.5 (<styled-content style="background-color: #ffffff;"><styled-content>F</styled-content>,</styled-content> n = 4 for each genotype) and at P21-P30 (J, n = 6 for each genotype). Data is represented as mean + s.e.m. **** indicates significant difference p<0.0001, *** indicates significant difference p<0.001, * indicates significant difference p<0.05 as assessed by two-way ANOVA with Tukey’s multiple comparison correction. Scale bars: (C–E) 100 μm, (G–I) 500 μm.

https://doi.org/10.7554/eLife.41623.002
Figure 1—figure supplement 1
Scl6a3Cre mediated recombination pattern.

(A–C) Analysis of Cre-mediated recombination in Scl6a3Cre/+,Rosa26lox-stop-lox-EYFP/+ mice. Immunostaining for TH and YFP at E13.5 shows Cre-mediated recombination in the lateral mDA regions (arrowhead). (D–F) Immunostaining for TH and YFP at E14.5 shows almost complete recombination (YFP expression) in mDA neurons of the SN (arrowhead). (G–I) By E15.5, TH+ mDA neurons of the SN and lateral VTA are YFP+ (arrowhead). Scale bar: 50 μm.

https://doi.org/10.7554/eLife.41623.003
Figure 1—figure supplement 2
DAB1 expression in the embryonic midbrain and specific loss of DAB1 protein in mDA neurons of Dab1 CKO brains.

(A–C) Analysis of DAB1 protein expression in control embryos at E13.5. DAB1 expression (B) is colocalized with TH (A) in lateral mDA neurons (yellow arrowheads in C) but not in medially located mDA neurons (white arrowheads). (D–F) Immunostaining for TH and DAB1 at E15.5 in control brains reveals DAB1 expression in the region of the SN and lateral VTA (yellow arrowheads in F) and in non-dopaminergic regions lateral to mDA neurons (white arrow). (G–I) Higher zoom of region indicated by dashed square in (D) shows colocalization (indicated by yellow arrowheads in F) of TH and DAB1 in SN-mDA neurons. (J–L) DAB1 expression is absent in Dab1 CKO mice at E15.5 in mDA neurons in the SN and lateral VTA (white arrowhead in L), while DAB1 expression persists in lateral non-dopaminergic regions (white arrow). (M–O) Higher zoom of region indicated by dashed square in (J) shows that DAB1 is not expressed in TH-positive mDA neurons in Dab1 CKO mice. Scale bars: 50 μm.

https://doi.org/10.7554/eLife.41623.004
Figure 1—figure supplement 3
Mediolateral distribution of mDA neurons at E15.5 in absence of Reelin signaling.

Analysis of mediolateral distribution of TH+ cells in control (A) and Dab1 CKO mice (B) at E15.5 shows a medial accumulation of cells in Dab1 CKO mice (C). Data are represented as mean +s.e.m for n = 3 brains. * indicates p<0.05 by Student’s t-test corrected for multiple comparisons (Holm-Sidak method). Scale bars: 50 μm.

https://doi.org/10.7554/eLife.41623.005
Figure 1—figure supplement 4
Characterization of mDA distribution at different rostrocaudal levels in Dab1 CKO and Dab1-/- brains.

(A–C) SN-mDA neurons are disorganized at rostral levels (level 1) in Dab1 CKO and Dab1-/- postnatal brains (P21-P30). Arrowheads: disorganized areas. (D–I, K,M) Rostral sections (Level 2) or caudal sections (level 4) containing SN and VTA-mDA neurons were analyzed in control (D, G), Dab1 CKO (E, H) and Dab1-/- (F, I) postnatal brains. The SN was disorganized, (arrowheads), but no significant difference was observed in the relative fractions of mDA neurons located in the SN or the VTA (K, M) (Student’s t-test). (L) Quantification of the relative fractions of mDA neurons located in the SN or the VTA at intermediate rostrocaudal levels (TH immunostaining shown in Figure 1) revealed a significant decrease in mDA neurons in the SN and a corresponding increase in the region of the VTA (p<0.0001, Student’s t-test) in both Dab1 CKO and Dab1-/- brains compared to controls. Student’s t-test, n = 5 animals/genotype for all levels. Scale bars: 100 μm.

https://doi.org/10.7554/eLife.41623.006
Reelin protein in the ventral midbrain at E13.5 and E14.5.

(A–C) Double immunolabeling for TH and Reelin shows Reelin protein in the region of the red nucleus (RN, dashed outline) and in the lateral TH+ mDA domain (arrowhead) at E13.5. (D–G) Double immunolabeling for TH and Reelin (D–F) and RNA in situ hybridization for Reelin mRNA (G) at E14.5. Reelin mRNA and Reelin protein are strongly expressed in the RN. Reelin protein is also localized ventral and lateral to the RN, overlapping with the lateral mDA domain (arrowheads). Note that a brightfield image was acquired for the in situ hybridization signal of Reelin. The brightfield image was inverted to obtain the image shown in (G). Asterisks indicate ventral midline. Scale bar: 50 μm.

https://doi.org/10.7554/eLife.41623.007
Figure 3 with 1 supplement
SN-mDA neurons do not completely segregate from VTA-mDA neurons at the SN-lateral VTA border in the absence of Reelin signaling.

(A–C) Immunostaining for TH and GIRK2 in P30 Dab1 CKO mice. Arrowheads: medial accumulation of TH+, GIRK2+ cells in Dab1 CKO (B) compared to control littermates (A). TH+, GIRK2+ cells are shifted medially in Dab1 CKO brains compared to controls (C). * indicates p<0.05 as assessed by Student’s t-test corrected for multiple comparisons (Holm-Sidak method) for n = 3 brains/genotype. (D,E) Triple immunostaining for SOX6 (magenta), OTX2 (green) and TH (blue) on E18.5 control and Dab1 CKO brains. Arrowheads indicate altered distribution of TH+, SOX6+ cells in Dab1 CKO mice. Boxed areas indicate regions presented in F and G. (F,G) Higher zoom of TH+ lateral VTA region shown in D and E. In controls, SOX6+ cells (dashed magenta line) and OTX2+ cells (dashed green line) are largely localized to separate regions (F). In Dab1 CKO mice, SOX6+ cells accumulate medially and are partially intermingled with OTX2+ cells at the SN-lateral VTA border (arrowheads) (G). (H,I) Immunostaining for TH and RNA in situ hybridyzation for Lmo3 shows ectopic localization of TH+, Lmo3+ cells in Dab1 CKO mice (arrowheads in I). Note that the signal for Lmo3 expression was inverted and then false-colored in green. Cells, in which Lmo3 was detected, show weak TH immunostaining, since the strong RNA in situ hybridization signal interferes with antibody binding. Scale bars: (A,B) 200 μm, (D,E,H,I) 50 μm, (F,G) 25 μm.

https://doi.org/10.7554/eLife.41623.008
Figure 3—figure supplement 1
Characterization of mDA projections in the absence of Reelin signaling.

(A,B) No gross morphological difference are observed in the axonal paths of TH+ projections in Dab1-/- E13.5 whole-mount brains (B) compared to controls (A). (C,D) Higher magnification images of TH+ projections at the level indicated by the dashed line in (A,B) do not reveal significant differences in their number or density in Dab1-/- brains (D) compared to control brains (C). (E–G) Normalized density of TH+ innervation to the striatum is similar in postnatal brains of controls and Dab1-/- mice as assessed by Student’s t-test. n = 6 animals/genotype. Scale bars: (C, D) 25 μm; (E, F) 500 μm.

https://doi.org/10.7554/eLife.41623.009
Figure 4 with 1 supplement
Visualizing mDA tangential migration with 2-photon excitation microscopy.

(A) Schematic of the inducible genetic fate mapping system used to mosaically label mDA neurons by administering tamoxifen (TM) at E8.5. Shh: Sonic Hedgehog; YFP: yellow fluorescent protein. CreER: gene encoding a CRE-Estrogen Receptor fusion protein. (B) Schematic of horizontal organotypic slice culture preparations. Green regions represent location of mDA neurons in the embryonic brains (left) and in horizontal slices (right). Red dashed line indicates dorsoventral level of slices. Black arrows indicate direction of tangential mDA migration. (C) Maximum intensity projection of an image of a control slice at t = 0. Orange rectangle indicates location of cell shown in E. (D) Trajectories of tracked neurons in slice shown in (C) after imaging for 270 min. Trajectory in orange represents trajectory of neuron in (E). (E) Maximum intensity projection frames of time-lapse imaging show soma and processes of a tangentially migrating cell. Track overlay frames show the position of the soma (green circle) and trajectory of the cell (orange line) analyzed with the semi-automated tracking plugin TrackMate in Fiji. Magenta dots and circles represent tracked soma of close-by cells at different z-levels. (F) Speed profile of cell in (E) shows large variations in speed over time, with a maximum speed (dashed arrow) that is much higher than the average speed (dashed horizontal line). Rest phase (soma speed less than 10 μm/hr) is indicated in red. Scale bars: (C,D) 50 μm, (E) 20 μm.

https://doi.org/10.7554/eLife.41623.010
Figure 4—figure supplement 1
Average speed distributions and speed profiles of SN-mDA neurons.

(A) Average speed distributions across three control slices are not significantly different (p=0.1065, Kruskal-Wallis test for non-parametric distributions). (C) Average speed distributions across 3 Dab1-/- slices are not significantly different (p=0.2279, Kruskal-Wallis test for non-parametric distributions). (C,D) To demonstrate the extent of max-speed variation in control and Dab1-/- mDA neurons from their mean speeds, each neuron in the dataset was ranked by its max-speed (green curve for the population). For each neuron, the mean and standard deviation around the mean were also plotted: grey and black curves represent variation of soma speed (grey) around average speed (black) for all mDA neurons in the dataset.

https://doi.org/10.7554/eLife.41623.011
Figure 5 with 2 supplements
Reelin promotes infrequent, fast movements in mDA neurons.

(A) The distribution of average speeds for the entire population of tracked control and Dab1-/- mDA neurons. Overall distribution of average soma speeds of mDA neurons is not significantly altered in Dab1-/- slices compared to controls. Note that entire distribution of control (solid black line) and Dab1-/- mDA neurons (dashed black line) were compared (p=0.0657, Mann-Whitney’s test, n = 806 control, 844 Dab1-/- mDA neurons). (B) The same analysis as shown in (A) was carried out for the distributions of maximum (max) speed. At the population level (distributions were compared), max-speeds of Dab1-/- mDA neurons were shifted significantly towards slower speeds compared to controls (p<0.0001, Mann-Whitney’s test, n = 806 control, 844 Dab1-/- mDA neurons). Non-migratory (max-speed 0–10 μm/hr), slow (30–10 μm/hr), moderate (60–30 μm/hr) and fast cells (>60 μm/hr) are indicated by dark red, light red, yellow and light green background colors, respectively. (C,D) Total displacement (3D) of mDA neurons is significantly higher in moderate compared to slow mDA neurons, and higher in fast mDA neurons compared to moderate neurons in both control (C) and Dab1-/- (D) brains (****p<0.0001, **p<0.01, Kruskal-Wallis test; n = 680 control; n = 639 Dab1-/- cells, three slices/genotype). (E,F) Directionality (defined as ratio of total displacement to path length) in control and Dab1-/- slices is the least in slow mDA neurons, higher in moderate mDA neurons and the highest in fast mDA neurons (*p<0.05, **p<0.01, ****p<0.0001, Kruskal-Wallis test; n = 680 control; n = 639 Dab1-/- cells, three slices/genotype).

https://doi.org/10.7554/eLife.41623.013
Figure 5—figure supplement 1
Variation in instantaneous soma speed of mDA neurons.

(A–C) Instantaneous soma speeds of 20 fast, moderate or slow cells in control slices as a heat map across 20 consecutive time frames. Each cell is one row in the heat-map, each column is a time frame. Soma speeds range from 0 to 150 μm/hr. (D–F) Individual fast, moderate and slow cells spend comparable periods of time in fast (light green), moderate (yellow), slow (light red) and rest phases (red) of migration in Dab1-/- slices compared to control slices. Slow cells: 355/806 (control), 430/844 (Dab1-/-); moderate cells: 186/806 (control), 139/844 (Dab1-/-); fast cells : 139/806 (control), 70/844 (Dab1-/-).

https://doi.org/10.7554/eLife.41623.014
Figure 5—figure supplement 2
Individual fast, moderate and slow mDA neurons from Dab1-/- slices have similar directionality and displacement profiles as mDA neurons in control slices.

(A,B) Total displacement (3D) increases with increasing max-speed of mDA neurons in control and Dab1-/- slices. (C,D) Directionality of mDA neurons increases with increasing max-speed in control and Dab1-/- slices. Note that inverse values of directionality are plotted against the max-speeds of migratory mDA neurons. Slow cells: 355/806 (control), 430/844 (Dab1-/-); moderate cells: 186/806 (control), 139/844 (Dab1-/-); fast cells : 139/806 (control),70/844 (Dab1-/-).

https://doi.org/10.7554/eLife.41623.015
Lateral migration of mDA neurons is reduced in the absence of Reelin signaling.

(A) Schematic showing trajectory angle measurement. (B) Trajectories of control (green) and Dab1-/- (red) mDA neurons (from 1 control and 1 Dab1-/- slice imaged over the same duration), plotted relative to their starting point show loss of lateral directionality in Dab1-/- mDA neuron trajectories. (C) Polar histogram for angle of the mDA trajectories to the midline (0°) for all control (green) and Dab1-/- (red) mDA neurons analyzed. (D) Circular statistical analysis for angular distributions in (C) of control (green) and Dab1-/- (red) mDA neurons shows significant decrease in lateral anisotropy for Dab1-/- slices (****p<0.0001, Kuiper’s test for circular variables; n = 680 control, n = 639 Dab1-/- mDA neurons). CDF: cumulative distribution function.

https://doi.org/10.7554/eLife.41623.016
Figure 7 with 1 supplement
Reelin promotes preference for lateral migration in slow mDA neurons.

Polar histogram for the angle of mDA trajectories to the midline. Radial axes represent the percentage of cells that migrate with various angles to the midline. (A,B) shows that slow cells have the least preference for lateral migratory direction in both control (A) and Dab1-/- (B) slices. (C) Circular statistical analysis for angular distributions of slow mDA neurons shows significant loss of preference for lateral migration in slow Dab1-/- mDA neurons compared to controls (****p<0.0001, Kuiper’s test for circular variables; n = 355 control, 480 Dab1-/- mDA neurons). (D–I) Moderate (D,E) and fast mDA neurons (G,H) show high preference for lateral migration. Moderate (F; n = 186 control, n = 139 Dab1-/- mDA neurons) and fast mDA neurons (I; n = 139 control, n = 70 Dab1/- mDA neurons) are laterally directed and their angular cumulative distribution functions (CDF) are comparable in control and Dab1-/- slices. Red (A,B), yellow (D,E) and green (G,H) lines represent mean angular direction for slow, moderate and fast populations, respectively.

https://doi.org/10.7554/eLife.41623.017
Figure 7—figure supplement 1
Lateral migration occurs predominantly during moderate and fast migratory phases of mDA neurons.

Polar histogram for the angle of individual mDA phases to the midline. Radial axes represent the fraction of mDA slow, moderate and fast phases that have various angles to the midline. (A,B) Polar histogram for angle of slow mDA trajectories to the midline shows that slow migratory movements have the least preference for lateral migratory direction in both control (A) and Dab1-/- (B) slices (n = 3506 movements in control, n = 2622 movements in Dab1-/- mDA neurons). (C–F) Moderate (C,D; n = 506 movements in control, n = 298 movements in Dab1-/- mDA neurons) and fast movements (E,F; n = 184 movements in control, n = 96 movements in Dab1/- mDA neurons) are laterally directed and comparable in control and Dab1-/- slices. Thick black line represents mean angular direction for slow, moderate and fast populations, respectively.

https://doi.org/10.7554/eLife.41623.018
Figure 8 with 4 supplements
Reelin promotes stable morphology of migrating SN-mDA neurons.

(A) Control mDA neuron displaying a ‘bipolar-unbranched’ morphology at all analyzed time-points. (B) Soma speed profile of mDA neuron shown in (A). (C) Control mDA neuron transitioning between bipolar and multipolar morphology. At t = 0 min, the cell has a branched leading process (LP), the cell soma moves along the LP to reach the branch-point and takes up a multipolar morphology (t = 20 min). The cell remains multipolar until t = 90 min, after which one process is retracted (t = 120 min) and the cell resumes a bipolar morphology (t = 150 min). Bipolar phase: one or two processes arise directly from the soma. Multipolar phase: more than two processes arise directly from the soma. (A,C) Colored circles: soma as defined by the tracking process. Scale bar: 25 μm. (D) Soma speed for the neuron in (C) is higher during its bipolar phase. (E) Relative proportion of constantly bipolar mDA neurons are decreased, while transitionary mDA neurons are increased in Dab1-/- slices. (F) Frequency of transitions from multipolar to bipolar phase (and vice versa) are not significantly altered in the absence of Reelin signalling (p=0.6922; Mann-Whitney’s test). (G) Bipolar and multipolar phase of a Dab1-/- transitionary mDA neuron. In this example, the bipolar phase lasts from t = 0 min to t = 60 min. In the multipolar phase (starting at t = 80 min) many unstable protrusions form. Scale bar: 25 μm. See Figure 8—figure supplement 3 for more detail. (H) Quantification of appearance and disappearance of branches (defined as branch transitions per hour) in control and Dab1-/- mDA transitionary neurons shows a significant increase in branch transitions in mDA neurons in Dab1-/- slices (****p<0.0001; Mann-Whitney’s test).

https://doi.org/10.7554/eLife.41623.019
Figure 8—figure supplement 1
Morphological characterization of mDA neurons in control slices.

(A) Control bipolar mDA neuron displaying bipolar-unbranched morphology at all analyzed time-points. (B) Bipolar mDA neuron that switches between a branched and unbranched leading process. (C) Transitionary mDA neuron that switches between bipolar and multipolar phases twice in the duration of analysis. (D) mDA neuron that remains multipolar during the entire duration of analysis.

https://doi.org/10.7554/eLife.41623.020
Figure 8—figure supplement 2
mDA neurons are predominantly associated with bipolar morphology during moderate-to-fast phases of migration.

Bipolar unbranched and bipolar branched morphologies are the predominant morphologies of mDA neurons during fast and moderate phases of migration (A,B). mDA neurons during their slow migration phase show a higher proportion of multipolar morphologies in both control and Dab1-/- mDA neurons (C) (*p<0.05, **p<0.005, ***p<0.001; two-way ANOVA). Total number of data points in fast phase (A): Control: 126, Dab1-/-: 81; in moderate phase (B): Control: 156, Dab1-/-: 99; in slow phase (C): Control: 408, Dab1-/-: 626.

https://doi.org/10.7554/eLife.41623.021
Figure 8—figure supplement 3
Absence of Reelin signaling results in the formation of unstable protrusions on the soma and leading process of mDA neurons.

Maximum intensity projections of cell shown in Figure 8 for each time point of the analysis. Cell in (A–C) is color-coded for the intensity of YFP in images (Physics LUT in Fiji). Red represents bright structures, green-blue represents weakly labelled structures. White arrowheads indicate appearance of a branch/protrusion, yellow arrowheads indicate disappearance. Scale bar: 25 μm.

https://doi.org/10.7554/eLife.41623.022
Figure 8—figure supplement 4
Greater spread in length of leading process in Dab1-/- mDA neurons.

(A–F) 6 of 20 manually segmented control mDA neurons. (A’–F’) 6 of 20 manually segmented Dab1-/- mDA neurons. (G) Length of cell soma and leading process shows a wider spread in the absence of Reelin signalling. ****p<0.001 as assessed by Mann-Whitney’s test.

https://doi.org/10.7554/eLife.41623.023
Figure 9 with 2 supplements
Phosphorylation levels of proteins in the canonical Reelin signaling pathway are not altered in Dab1-/- ventral midbrain at E14.5.

(A,E) Relative phosphorylation levels of LIMK1 are not significantly altered in Dab1-/- ventral midbrains. p-value=0.5682, Student’s t-test (n = 5 brains/genotype). (B,F) Relative phosphorylation levels of Cofilin1 are not significantly changed in Dab1-/- ventral midbrains. p-value=0.854, Student’s t-test (n = 5 brains/genotype). (C,D) Total protein in the membrane, stained with Amido black, was used as loading control.

https://doi.org/10.7554/eLife.41623.027
Figure 9—figure supplement 1
CDH2 expression levels in the ventral midbrain of control and Dab1-/- brains at E14.5.

(A, B) Immuno blot for ventral midbrain tissue from control and Dab1-/- brains (A) shows no changes in relative protein levels of CDH2 in Dab1-/- tissue compared to controls (B). p-value 0.08 as assessed by Student’s t-test (n = 5 brains/genotype). (C) Total protein in the membrane, stained with Amido black, was used as loading control.

https://doi.org/10.7554/eLife.41623.028
Figure 9—figure supplement 2
Organization of microtubules is not altered in mDA neurons in the absence of Reelin signaling.

Immunostaining for α-tubulin (A–F), acetylated α-tubulin (G–L) and EB3 (M–R) in coronal sections of E13.5 ventral midbrain reveals no differences in expression levels of these markers or the organization of microtubules in TH+ mDA neurons between controls and Dab1-/- embryos at E13.5.

https://doi.org/10.7554/eLife.41623.029
Schematic representation of Reelin regulation in mDA tangential migration.

Reelin directly regulates lateral, tangential migration of mDA neurons and promotes fast, laterally directed mode of migration by regulating small lateral movements and stabilizing morphology of mDA neurons. In absence of Reelin signaling, slow movements in mDA neurons lose their lateral anisotropy, morphologies are less stable during migration and the fraction of neurons undergoing fast, laterally directed migration is reduced. This results in a medial clustering of SN-mDA neurons at late embryonic and postnatal stages.

https://doi.org/10.7554/eLife.41623.030
Author response image 1
α-tubulin in control and Dab1-/- E13.5 brains.

(A-I) TH (A,D,G) α-tubulin (B,E,H) and overlap (C,F,I) in the developing mDA domain of control embryos (n = 3, from 2 litters). (C,F,I) Overlap of TH and α-tubulin in controls. (JR) TH (J,M,P), tubulin (K,N,Q) and overlap panels (L,O,R) in in Dab1-/- midbrains. Selected region is comparable to the one shown in controls (n = 3, across 2 litters). Control Embryo #1, Dab1-/- Embryo #3 is also shown in Figure 9—figure supplement 2. No obvious phenotype could be identified based on this analysis. (S,T) α-tubulin in the cortex for comparison.

https://doi.org/10.7554/eLife.41623.034
Author response image 2
Acetylated α-tubulin in control and Dab1-/- E13.5 brains.

(A-I) TH (A,D,G) and acetylated α-tubulin (B,E,H) in the developing mDA domain of control embryos (n = 3, from 2 litters). (C,F,I) overlap of TH and acetylated α-tubulin in controls. (J-R) TH (J,M,P), acetylated α-tubulin (K,N,Q) and overlap panels (L,O,R) in Dab1-/- midbrains. Selected region is comparable to the one shown in controls (n = 3, across 2 litters). Control Embryo #1, Dab1-/- Embryo #3 is also shown in Figure 9—figure supplement 2. No obvious phenotype could be identified based on this analysis. (S,T) Acetylated α-tubulin in the cortex for comparison.

https://doi.org/10.7554/eLife.41623.035
Author response image 3
EB3 in control and Dab1-/- E13.5 brains.

(A-I) TH (A,D,G) EB3 (B,E,H) and overlap (C,F,I) in the developing mDA domain of control embryos (n = 3, from 2 litters). (C,F,I) Overlap of TH and EB3 in controls. (J-R) TH (J,M,P), EB3 (K,N,Q) and overlap panels (L,O,R) in Dab1-/- midbrains. Selected region is comparable to the one shown in controls (n = 3, across 2 litters). Control Embryo #1, Dab1-/- Embryo #3 is also shown in Figure 9—figure supplement 2. No obvious phenotype could be identified based on this analysis. (S,T) EB3 in the cortex for comparison.

https://doi.org/10.7554/eLife.41623.036

Videos

Video 1
Time lapse imaging with two photon excitation of ex vivo embryonic slices of the ventral midbrain.

Time-lapse imaging of control (left) and Dab1-/- (right) organotypic slices with mosaic labelling of SN-mDA neurons reveals aberrant orientation and slower migration of Dab1-/- mDA neurons.

https://doi.org/10.7554/eLife.41623.012
Video 2
SN-mDA neurons display dynamic cell morphology.

3D projection of a transitionary mDA neurons at t = 0 min (360° rotation) followed by MIP frames of the same neuron at subsequent time-points. Migratory spurts only occur in bipolar morphology while cell remains stationary or displays slow migration during multipolar phase.

https://doi.org/10.7554/eLife.41623.024
Video 3
Morphology as detected by YFP mosaic labelling is similar to morphology detected by TH antibody.

Example SN-mDA neuron from fixed, cleared whole-mount embryonic brain of the same age as used in time-lapse experiments (E14.5) shows similar morphology with YFP (green) and TH (magenta) immunostaining.

https://doi.org/10.7554/eLife.41623.025

Tables

Table 1
Morphology of mDA neurons in control and Dab1-/- slices.
https://doi.org/10.7554/eLife.41623.026
ControlDab1-/-
Cell typeMorphologySlice 1Slice 2Slice 3Slice 1Slice 2Slice 3
Bipolar15/2712/267/175/166/155/18
FastTransitionary12/2713/2610/179/168/1512/18
Multipolar0/271/260/172/161/151/18
Bipolar8/164/104/144/135/195/8
ModerateTransitionary7/166/1010/149/1314/193/8
Multipolar1/160/100/140/130/190/8
Bipolar3/117/127/175/145/152/11
SlowTransitionary2/111/123/175/145/155/11
Multipolar6/114/127/174/145/154/11
Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (M. musculus)ShhCreERPMID: 15315763RRID: MGI:J:92504Clifford Tabin, Harvard University
Genetic reagent (M. musculus)Slc6a3CrePMID: 17227870RRID: MGI:3702746Nils-Görran Larsson, Max Planck Institute for Biology of Aging, Cologne, Germany
Genetic reagent (M. musculus)Dab1floxPMID: 21315259RRID: MGI:5141401generated by Ulrich Müller, Johns Hopkins University, Baltimore, USA; obtained
from Amparo Acker-Palmer,
University of Frankfurt
Genetic reagent (M. musculus)Dab1-/-PMID:
21315259, recombined flox allele
RRID: MGI:5141401, recombined flox allelegenerated by Ulrich Müller, Johns Hopkins University, Baltimore, USA; obtained from Amparo Acker-Palmer, University of Frankfurt
Genetic reagent (M. musculus)ROSAloxP-STOP-loxP-EYFP PMID: 11299042RRID: MGI:J:80963Frank Constantini, Columbia University
Antibodysheep anti-DIG-AP Fab fragmentsRocheRRID:AB_514497ISH: 1:5000
AntibodyGoat anti-OTX2NeuromicsRRID:AB_2157174IHC: 1:5000
Antibodygoat anti-ReelinR and D systemsRRID:AB_2253745IHC: 1:50
Antibodymouse anti-α-TubulinMerckRRID:AB_477579IHC: 1:500
Antibodymouse anti-α-Tubulin, acetylatedMerckRRID:AB_477585IHC: 1:500
Antibodymouse anti-THMerckRRID:AB_2201528IHC: 1:500
Antibodyrabbit anti-CalbindinSwantRRID:AB_2314067IHC: 1:5000
Antibodyrabbit anti-CDH2AbcamRRID:AB_444317WB: 1:500
Antibodyrabbit anti-DAB1Dr. Brian HowellHowell et al., 1997IHC: 1:5000 (used with TSA kit)
Antibodyrabbit anti-EB3AbcamRRID:AB_880026IHC: 1:250
Antibodyrabbit anti-GIRK2Alamone LabsRRID:AB_2040115IHC: 1:400
Antibodyrabbit anti-GFPThermo FischerRRID:AB_221569IHC:1:400
Antibodyrabbit anti-SOX6AbcamRRID:AB_1143033IHC: 1:500
Antibodyrabbit anti-THMerckRRID:AB_390204IHC: 1:500
Antibodyrat anti-GFPNalacaiRRID:AB_10013361IHC: 1:1500
Antibodyrabbit anti-Cofilin1Kindly provided by Prof. Dr. Walter WittkeWB: 1:5000
Antibodyrabbit anti-p-Cofilin1 (ser3) (77G2)Cell signalingRRID:AB_2080597WB: 1:1000
Antibodyrabbit anti-p-LIMK1 (Thr508)/LIMK2 (thr505)Cell signalingRRID:AB_2136943WB: 1:500
Antibodyrabbit anti-LIMK1Cell signalingRRID:AB_2281332WB: 1:100
Antibodyanti rabbit-HRP-linkedCell signalingRRID:AB_2099233WB: 1:1000
Antibodydonkey anti-
rabbit Alexa 488
Thermo FischerRRID:AB_2535792IHC: 1:500
Antibodydonkey anti-
rabbit Alexa 350
Thermo FischerRRID:AB_2534015IHC: 1:500
Antibodydonkey anti-mouse Alexa 488Thermo FischerRRID:AB_141607IHC: 1:500
Antibodydonkey anti-rat Alexa 488Thermo FischerRRID:AB_2535794IHC: 1:500
Antibodydonkey anti-goat Alexa 488Jackson ImmunoResearchRRID:AB_2336933IHC: 1:500
Antibodydonkey anti-rabbit Cy3Jackson
ImmunoResearch
RRID:AB_2307443IHC: 1:200
Antibodydonkey anti-mouse Cy3Jackson ImmunoResearchRRID:AB_2340813IHC: 1:200
Antibodydonkey anti-
goat Cy3
Jackson ImmunoResearchRRID:AB_2307351IHC: 1:200
Antibodydonkey anti-rabbit BiotinJackson ImmunoResearchRRID:AB_2340593IHC: 1:200
Recombinant
DNA reagent
in situ mRNA probe: RelnpCRII-Topo vector, 0.64 kb(cDNA) RELN inserted fragmentJoachim Herz, UT Southwestern, Dallas, USA
Recombinant
DNA reagent
in situ mRNA probe: Lmo3pCMV-SPORT6 vector,3 kb(partial cDNA) inserted fragmentImage clone: 4913098; accession #BC034128Source Bioscience, Berlin, DE
Commercial
assay or kit
TSA Plus fluorescence kitPerkin ElmerNEL744001KT
Chemical
compound, drug
TamoxifenMerckT5648
Software,
algorithm
Quantity OneBioRADGel documentation
Software,
algorithm
Zen Blue 2012Carl ZeissImage acquisition
Software,
algorithm
Zen Black 2012Carl ZeissImage acquisition
Software,
algorithm
Leica Application Suite X 4.13Leica microsystemsImage acquisition
Software, algorithmFiji/ImageJ 1.51 nWayne Rasband. National Institutes of HealthImage processing
Software,
algorithm
Adobe Photoshop CS3Adobe SystemsImage processing
Software,
algorithm
Imaris 8.3.1BitplaneImage processing
Software,
algorithm
MatLab R2017bMathWorksImage processing and data analysis
Software,
algorithm
Affinity Designer
1.5.5
SerifImage editing
Software,
algorithm
Image Lab 6.0BioRADImmunoblot quantification and analysis
Software,
algorithm
GraphPad Prism 7.0GraphPad SoftwareStatistical analysis

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1,4,5,6,8,9,10.

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